CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 62/781,511 filed Dec. 18, 2018, the contents of which are incorporated by reference in their entirety herein.

BACKGROUND OF THE DISCLOSURE

The disclosure relates generally to the production of commodity and specialty chemicals, and, more specifically, an integrated bioprocess for producing 3-hydroxypropionic acid (3-HP or 3-HPA) and acetyl-CoA derivatives from malonate semialdehyde (MSA).

Derivatives of 3-HP include acrylic acid, 1-propanol, propene, polypropylene, etc. Derivatives of acetyl-CoA include acetone, 2-propanol, propene, polypropylene, etc. Many of these molecules are produced via synthetic chemistry along with considerable amounts of chemical waste.

There exists a need to minimize chemical waste produced from production of these molecules to seek a more environmentally friendly and cost-effective approach for processes and molecules that utilize these 3-HP and acetyl-CoA derivatives.

As set forth herein, the disclosure provides methods and compositions for the fermentative co-production of 3-HP and acetyl-CoA derivatives.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is BRSK_020_01US_ST25.txt. The text file is 906 kb, and was created on Dec. 18, 2019, and is being submitted electronically.

SUMMARY OF THE DISCLOSURE

This disclosure provides a recombinant microorganism capable of co-producing 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof, from a feedstock comprising one or more carbon sources, wherein the recombinant microorganism comprises one or more of the following: (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionic acid dehydrogenase that catalyzes the production of 3-HP from malonate semialdehyde; (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a (i) malonate semialdehyde dehydrogenase that catalyzes the production of acetyl-CoA from malonate semialdehyde, and/or (ii) malonyl-CoA reductase and/or 2-keto acid decarboxylase that catalyzes the conversion of malonate semialdehyde into malonyl-CoA, and malonyl-CoA decarboxylase that catalyzes the production of acetyl-CoA from malonyl-CoA; (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an enzyme that is able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde.

In some embodiments, the recombinant microorganism is capable of producing 1-propanol, wherein the recombinant microorganism comprises one or more of the following: (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a propionyl-CoA synthase that catalyzes the conversion of 3-HP into propionyl-CoA; (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionyl-CoA synthetase/CoA transferase, 3-hydroxypropionyl-CoA dehydratase, and acrylyl-CoA reductase that catalyzes the conversion of 3-HP into propionyl-CoA; (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an alcohol/aldehyde dehydrogenase that catalyzes the conversion of propionyl-CoA from (a) or (b) into 1-propanol, or an aldehyde dehydrogenase (acetylating) and alcohol dehydrogenase that catalyzes together the production of 1-propanol from propionyl-CoA.

In some embodiments, the derivatives of 3-HP are selected from the group consisting of: acrylic acid, 1-propanol, propene, and polypropylene. In some embodiments, the derivatives of acetyl-CoA are selected from the group consisting of: acetone, 2-propanol, propene, and polypropylene. In some embodiments, at least a portion of excess NAD(P)H generated in the production of acetyl-CoA is utilized as a source of reducing equivalents in the production of 3-HP and/or 3-HP derivatives.

In some embodiments, the recombinant microorganism produces 3-hydroxypropionic acid (3-HP), acetyl-CoA, 1-propanol and/or 2-propanol, in an aerobic or anaerobic production process, preferably an anaerobic process.

In some embodiments, the microorganism is selected from a bacterium, a fungus, or a yeast. In some embodiments, the recombinant microorganism is derived from a parental microorganism selected from the group consisting of: Clostridium sp., Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans, Candida sp, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Candida krusei, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillus sp, Corynebacterium sp., Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacter glycolicus. In some embodiments, the recombinant microorganism is a yeast. In some embodiments, the yeast is Saccharomyces cerevisiae.

In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionic acid dehydrogenase encodes an amino acid sequence comprising MCR-Nterm.Cau (SEQ ID NO: 105), ADH.Ae (SEQ ID NO: 106), MMSB.Bce (SEQ ID NO: 107), YDFG-0.Ec (SEQ ID NO: 108), YMR226C (YDF1) (SEQ ID NO: 109), or HPD1 (SEQ ID NO: 110). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionic acid dehydrogenase encodes an amino acid sequence comprising YMR226C (YDF1) (SEQ ID NO: 109). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionic acid dehydrogenase encodes an amino acid sequence comprising HPD1 (SEQ ID NO: 110).

In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a malonate semialdehyde dehydrogenase encodes an amino acid sequence comprising MSD.Pa (SEQ ID NO: 111), MSD.Cal (SEQ ID NO: 112), iolA (SEQ ID NO: 113), iolA (SEQ ID NO: 114), iolA (SEQ ID NO: 115), mmsA (SEQ ID NO: 116), dddC (SEQ ID NO: 117), or iolA (SEQ ID NO: 118). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a malonate semialdehyde dehydrogenase encodes an amino acid sequence comprising MSD.Pa (SEQ ID NO: 111). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a malonate semialdehyde dehydrogenase encodes an amino acid sequence comprising MSD.Cal (SEQ ID NO: 112).

In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a malonyl-CoA reductase and/or 2-keto acid decarboxylase encodes an amino acid sequence comprising mcr (SEQ ID NO: 278), matA, MLYCD (SEQ ID NO: 279), kivD (SEQ ID NO: 280), kdcA (SEQ ID NO: 281), ARO10 (SEQ ID NO: 282). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a malonyl-CoA reductase and/or 2-keto acid decarboxylase encodes an amino acid sequence comprising mcr (SEQ ID NO: 278). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a malonyl-CoA reductase and/or 2-keto acid decarboxylase encodes an amino acid sequence comprising kivD (SEQ ID NO: 280). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a malonyl-CoA reductase and/or 2-keto acid decarboxylase encodes an amino acid sequence comprising ARO10 (SEQ ID NO: 282).

In some embodiments, the at least one endogenous and/or exogenous nucleic acid molecule encoding an enzyme that is able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde encodes an amino acid sequence comprising (SEQ ID NO: 1) or (SEQ ID NO: 274). In some embodiments, the at least one endogenous and/or exogenous nucleic acid molecule encoding an enzyme that is able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde encodes an amino acid sequence comprising (SEQ ID NO: 1). In some embodiments, the at least one endogenous and/or exogenous nucleic acid molecule encoding an enzyme that is able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde encodes an amino acid sequence comprising (SEQ ID NO: 274).

In some embodiments, the recombinant microorganism comprises one or more of the following: (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase that catalyzes the production of acetoacetyl-CoA from acetyl-CoA; (b) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA synthase that catalyzes the production of acetoacetyl-CoA from malonyl-CoA; (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA transferase that catalyzes the production of acetoacetate from acetoacetyl-CoA; (d) at least one endogenous and/or exogenous nucleic acid molecule encoding a (i) hydroxymethylglutaryl-CoA synthase that catalyzes the production of HMG-CoA from acetoacetyl-CoA, and (ii) hydroxymethylglutaryl-CoA lyase that catalyzes the production of acetoacetate from HMG-CoA; and (e) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetate decarboxylase that catalyzes the production of acetone from acetoacetate.

In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase encodes an amino acid sequence comprising ERG10 (SEQ ID NO: 209), thlA (SEQ ID NO: 210), atoB (SEQ ID NO: 211), H16_B0759 (SEQ ID NO: 212), Msed_0656 (SEQ ID NO: 213), or AAT1 (SEQ ID NO: 214). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase encode an amino acid sequence comprising ERG10 (SEQ ID NO: 209). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a acetoacetyl-CoA synthase encodes an amino acid sequence comprising nphT7 (SEQ ID NO: 285).

In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a acetoacetyl-CoA transferase encodes an amino acid sequence comprising atoA/atoD (SEQ ID NO: 215 and 216), ctfA/ctfB (SEQ ID NO: 219 and 220), ctfA/ctfB (SEQ ID NO:221 and 222) or ctfA/ctfB (SEQ ID NO:223 and 224). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA transferase encodes an amino acid sequence comprising atoA/atoD (SEQ ID NO: 215 and 216)

In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a hydroxymethylglutaryl-CoA synthase and a hydroxymethylglutaryl-CoA lyase encode an amino acid sequence comprising ERG13 (SEQ ID NO: 283) and yngG (SEQ ID NO: 284). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a acetoacetate decarboxylase encodes an amino acid sequence comprising Adc.Ca (SEQ ID NO: 225), Adc.Cbe (SEQ ID NO: 226), Adc (SEQ ID NO: 227), Adc (SEQ ID NO: 228), Adc (SEQ ID NO: 229) or Adc.Pp (SEQ ID NO: 230). In some embodiments, the endogenous and/or exogenous nucleic acid molecule encoding a acetoacetate decarboxylase encodes an amino acid sequence comprising Adc.Pp (SEQ ID NO: 230).

The disclosure provides a method of co-producing 3-HP, and/or derivatives thereof and Acetyl-CoA and/or derivatives thereof by contacting the recombinant microorganism of any of the claim 1 or 2 with a fermentable carbon source under conditions and for a sufficient period of time to produce 3-HP, or derivatives and Acetyl-CoA or derivatives. In some embodiments, the carbon source is selected from sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, lignocellulose, proteins, carbon dioxide, and carbon monoxide. In some embodiments, the recombinant microorganism produces 3-hydroxypropionic acid (3-HP), acetyl-CoA, 1-propanol and/or 2-propanol, in an aerobic or anaerobic production process, preferably an anaerobic process.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a novel combined pathway for 3-HP and acetyl-CoA production with malonate semialdehyde as a common single intermediate. The shaded offshoot identifies the usual pathway for malonate semialdehyde production with malonyl-CoA as an intermediate, which is replaced by a malonate semialdehyde in the present pathway.

FIG. 2 depicts the two routes for oxaloacetate generation in yeast cells, through pyruvate carboxylase (pyc gene) or malate dehydrogenase (mdh gene) activity.

FIG. 3 depicts the pathways from oxaloacetate to malonate semialdehyde. The typical pathway utilizes aspartate and beta-alanine as intermediates, and the new route utilizes a decarboxylase (oxdc) to directly produce malonate semialdehyde from oxaloacetate.

FIG. 4 depicts the two pathways for the production of acetyl-CoA, through malonyl-CoA as an intermediate (standard route) and directly with malonate semialdehyde (new route).

FIG. 5 depicts the pathways for production of 1-propanol from 3-HP by a four-step process and by a 2-step process.

FIG. 6 the pathways to acetone production from glucose, and the excess of NAD(P)H produced in the pathways.

FIG. 7 depicts the combined pathways from oxaloacetate to malonate semialdehyde to 1-propanol and acetone, and the redox neutral status of the combined pathways.

FIG. 8 depicts a pathway from glucose to malonate semialdehyde, including catalytic enzymes and corresponding malonate semialdehyde intermediates, particularly identifying the oxaloacetate decarboxylase utilized to produce malonate semialdehyde from oxaloacetate.

FIG. 9 depicts a pathway from malonate semialdehyde to acetone, including catalytic enzymes and corresponding malonate semialdehyde intermediates.

FIG. 10 depicts a pathway from malonate semialdehyde to propanol, including malonate semialdehyde intermediates and corresponding cofactors.

FIG. 11 depicts a pathway from malonate semialdehyde to propanol, including catalytic enzymes and corresponding malonate semialdehyde intermediates.

FIG. 12 depicts a pathway from malonate semialdehyde to acrylic acid, including malonate semialdehyde intermediates and corresponding cofactors.

FIG. 13 depicts a pathway and yeast genotype relating to the mutants relating to Example 2.

FIG. 14 depicts a co-production pathway of 1-propanol and acetone that shows a neutral redox balance using selected enzymes.

FIG. 15 depicts the stoichiometry of co-production of 1-propanol and acetone in aerobic conditions.

FIG. 16 depicts the stoichiometry of co-production of 1-propanol and acetone in anaerobic conditions.

FIG. 17 depicts the stoichiometry of co-production of 1-propanol and 2-propanol in aerobic conditions.

FIG. 18 depicts the stoichiometry of co-production of 1-propanol and 2-propanol in anaerobic conditions.

FIG. 19 depicts the stoichiometry of production of 2-propanol in aerobic conditions.

FIG. 20 depicts the workflow for engineering an organism to produce 3-HP from glucose.

FIG. 21 is a plot and values demonstrating kinetic parameters for Enzyme No 1.

FIG. 22 is a plot and values demonstrating kinetic parameters for Enzyme No 6.

FIG. 23 is a plot and values demonstrating kinetic parameters for Enzyme No 7.

FIG. 24 is a plot and values demonstrating kinetic parameters for Enzyme No 8.

FIG. 25 is a plot and values demonstrating kinetic parameters for Enzyme No 9

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure is generally drawn to methods for utilizing genetically modified microbes to co-produce 3-hydroxypropionic acid (3-HP) and acetyl-CoA derivatives from malonate semialdehyde as a common single intermediate. The disclosure further provides modified microbe that co-produce the 3-HP and acetyl-CoA derivatives from malonate semialdehyde.

The co-production of 3-HP and acetyl-CoA, and derivatives thereof from malonate semialdehyde is novel, and the co-production results in a redox balanced set of pathways that co-produces the products at a high yield.

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an enzyme” includes a plurality of such enzymes and reference to “the microorganism” includes reference to one or more microorganisms, and so forth.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having, “contains,” “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or.”

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. Sequence identity, such as for the purpose of assessing percent complementarity, may be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see e.g. the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss needle/nucleotide.html, optionally with default settings), the BLAST algorithm (see e.g. the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see e.g. the EMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss water/nucleotide.html, optionally with default settings). Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.

As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

As used herein, the term “about” is used synonymously with the term “approximately.” Illustratively, the use of the term “about” with regard to an amount indicates that values slightly outside the cited values, e.g., plus or minus 0.1% to 10%.

The term “biologically pure culture” or “substantially pure culture” refers to a culture of a microbe species described herein containing no other microbe species in quantities sufficient to interfere with the replication of the culture or be detected by normal microbiological techniques.

As used herein, a “control sequence” refers to an operator, promoter, silencer, or terminator.

As used herein, “introduced” refers to the introduction by means of modern biotechnology, and not a naturally occurring introduction.

As used herein, a “constitutive promoter” is a promoter, which is active under most conditions and/or during most development stages. There are several advantages to using constitutive promoters in expression vectors used in biotechnology, such as: high level of production of proteins used to select transgenic cells or organisms; high level of expression of reporter proteins or scorable markers, allowing easy detection and quantification; high level of production of a transcription factor that is part of a regulatory transcription system; production of compounds that requires ubiquitous activity in the organism; and production of compounds that are required during all stages of development.

As used herein, a “non-constitutive promoter” is a promoter which is active under certain conditions, in certain types of cells, and/or during certain development stages. For example, inducible promoters, and promoters under development control are non-constitutive promoters. As used herein, “inducible” or “repressible” promoter is a promoter which is under chemical or environmental factors control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, certain chemicals, the presence of light, acidic or basic conditions, etc.

As used herein, the term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the disclosure can be operably linked, either directly or indirectly, 5′ to the target mRNA, or 3′ to the target mRNA, or within the target mRNA, or a first complementary region is 5′ and its complement is 3′ to the target mRNA.

The term “signal sequence” as used herein refers to an amino acid sequence that targets peptides and polypeptides to cellular locations or to the extracellular environment. Signal sequences are typically at the N-terminal portion of a polypeptide and are typically removed enzymatically. Polypeptides that have their signal sequences are referred to as being full-length and/or unprocessed. Polypeptides that have had their signal sequences removed are referred to as being mature and/or processed.

The term “exogenous” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature. On the other hand, the term “endogenous” or “native” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

The term “heterologous” as used herein in the context of a modified host cell refers to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true: (a) the molecule(s) is/are foreign (“exogenous”) to (i.e., not naturally found in) the host cell; (b) the molecule(s) is/are naturally found in (e.g., is “endogenous to”) a given host microorganism or host cell but is either produced in an unnatural location or in an unnatural amount in the cell; and/or (c) the molecule(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid as found endogenously is produced in an unnatural (e.g., greater than naturally found) amount in the cell.

The term “homolog,” as used herein with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural, or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Homologs most often have functional, structural, or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homologs can be confirmed using functional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. Thus, the term “homologous proteins” is intended to mean that the two proteins have similar amino acid sequences. In certain instances, the homology between two proteins is indicative of its shared ancestry, related by evolution. The terms “homologous sequences” or “homologs” are thought, believed, or known to be functionally related. A functional relationship may be indicated in any one of a number of ways, including, but not limited to: (a) degree of sequence identity and/or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated. The degree of sequence identity may vary, but in one aspect, is at least 50% (when using standard sequence alignment programs known in the art), at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus (Scientific and Educational Software, Pennsylvania). Other non-limiting alignment programs include Sequencher (Gene Codes, Ann Arbor, Mich.), AlignX, and Vector NTI (Invitrogen, Carlsbad, Calif.). A similar biological function may include, but is not limited to: catalyzing the same or similar enzymatic reaction; having the same or similar selectivity for a substrate or co-factor; having the same or similar stability; having the same or similar tolerance to various fermentation conditions (temperature, pH, etc.); and/or having the same or similar tolerance to various metabolic substrates, products, by-products, intermediates, etc. The degree of similarity in biological function may vary, but in one aspect, is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%, according to one or more assays known to one skilled in the art to determine a given biological function.

Genes of the present disclosure may be referenced with their common names, their nucleic acid sequences, and the amino acid sequences that are translated from the nucleic acid sequences. Using the references given in accession numbers for known genes, a practitioner is able to determine equivalent genes in other organisms, bacterial strains, yeast, fungi, mammals, plants, etc. This work is performed utilizing consensus sequences that can be determined by carrying out sequence alignments with genes derived from other microorganisms and designing degenerated probes to clone the corresponding gene in another organism.

As used herein, “overexpression” means that the expression of a gene or of an enzyme is increased as compared to the non-modified microorganism. Increasing the expression of an enzyme is obtained by increasing the expression of a gene encoding said enzyme. When a non-modified microorganism did not express a given gene, modifying said microorganism in order to express this gene is thus also considered as being an increase expression of said gene, and thus as an overexpression. Increasing the expression of a gene may be carried out by all techniques known by the one skilled in the art. In this regard, it may be notably cited the implementation of a strong promoter upstream the nucleic acid intended to be overexpressed or the introduction of a plurality of copies of the said nucleic acid between a promoter, especially a strong promoter, and a terminator.

As used herein, “inducible” promoter” means a promoter whose activity is induced, i.e. increased (1) in the presence of one or more particular metabolite(s)—the higher the metabolite concentration in the medium, the stronger the promoter activity; or (2) in the presence of a low concentration, or in the absence, of one or more metabolite(s). These metabolites are different from those whose increasing presence induces the activity of the promoter. The lower the metabolite concentration in the medium, the stronger the promoter activity.

As used herein, enzyme/protein “activity” and “function” are used interchangeably and designates, in the context of the disclosure, the capacity of (1) an enzyme to catalyze a desired reaction or (2) a protein to act in a certain manner.

As used herein, “aerobic conditions” refer to concentrations of oxygen in the culture medium that are sufficient for an aerobic or facultative anaerobic microorganism to use oxygen as a terminal electron acceptor.

As used herein, “anaerobic conditions” refer to culture or growth conditions with regard to the concentration of oxygen, which is intended to mean that the amount of oxygen is less than about 0% saturation of dissolved oxygen in liquid media. The term is also intended to include sealed chambers of liquid or solid media maintained with an atmosphere of less than about 0% oxygen.

As used herein, “microaerobic conditions” refer to concentrations of oxygen in the culture medium in which the concentration of oxygen is less than that in air under standard temperature and pressure, i.e., an oxygen concentration of up to ˜6% of the total gas present.

The term “variant” refers to any polypeptide or enzyme described herein. A variant also encompasses one or more components of a multimer, multimers comprising an individual component, multimers comprising multiples of an individual component (e.g., multimers of a reference molecule), a chemical breakdown product, and a biological breakdown product. In particular, non-limiting aspect, an enzyme may be a “variant” relative to a reference enzyme by virtue of alteration(s) in any part of the polypeptide sequence encoding the reference enzyme. A variant of a reference enzyme can have enzyme activity of at least 10%, at least 30%, at least 50%, at least 80%, at least 90%, at least 100%, at least 105%, at least 110%, at least 120%, at least 130% or more in a standard assay used to measure enzyme activity of a preparation of the reference enzyme. In some aspects, a variant may also refer to polypeptides having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the full-length, or unprocessed enzymes of the present disclosure. In some aspects, a variant may also refer to polypeptides having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least

91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the mature, or processed enzymes of the present disclosure.

As used herein the terms “microorganism” or “microbe” should be taken broadly. These terms, used interchangeably, include but are not limited to, the two prokaryotic domains, Bacteria and Archaea.

As used herein, “isolate,” “isolated,” “isolated microbe,” and like terms, are intended to mean that the one or more microorganisms has been separated from at least one of the materials with which it is associated in a particular environment (for example media, water, reaction chamber, etc.). Thus, an “isolated microbe” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain or isolated microbe may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain). In aspects, the isolated microbe may be in association with an acceptable carrier, which may be a commercially or industrial acceptable carrier.

In certain aspects of the disclosure, the isolated microbes exist as “isolated and biologically pure cultures.” It will be appreciated by one of skill in the art that an isolated and biologically pure culture of a particular microbe, denotes that said culture is substantially free of other living organisms and contains only the individual microbe in question. The culture can contain varying concentrations of said microbe. The present disclosure notes that isolated and biologically pure microbes often “necessarily differ from less pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussing purified prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA 1979)(discussing purified microbes), see also, Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned Hand discussing purified adrenaline), aff'd in part, rev'd in part, 196 F. 496 (2d Cir. 1912), each of which are incorporated herein by reference. Furthermore, in some aspects, the disclosure provides for certain quantitative measures of the concentration, or purity limitations, that must be found within an isolated and biologically pure microbial culture. The presence of these purity values, in certain aspects, is a further attribute that distinguishes the presently disclosed microbes from those microbes existing in a natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limitations for vitamin B12 produced by microbes), incorporated herein by reference.

Microbes of the present disclosure may include spores and/or vegetative cells. In some aspects, microbes of the present disclosure include microbes in a viable but non-culturable (VBNC) state. As used herein, “spore” or “spores” refer to structures produced by bacteria and fungi that are adapted for survival and dispersal. Spores are generally characterized as dormant structures; however, spores are capable of differentiation through the process of germination. Germination is the differentiation of spores into vegetative cells that are capable of metabolic activity, growth, and reproduction. The germination of a single spore results in a single fungal or bacterial vegetative cell. Fungal spores are units of asexual reproduction, and in some cases are necessary structures in fungal life cycles. Bacterial spores are structures for surviving conditions that may ordinarily be nonconductive to the survival or growth of vegetative cells.

As used herein, “microbial composition” refers to a composition comprising one or more microbes of the present disclosure.

As used herein, “carrier,” “acceptable carrier,” “commercially acceptable carrier,” or “industrial acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the microbe can be administered, stored, or transferred, which does not detrimentally effect the microbe.

The term “yield potential” as used herein refers to a yield of a product from a biosynthetic pathway. In one aspect, the yield potential may be expressed as a percent by weight of end product per weight of starting compound.

The term “thermodynamic maximum yield” as used herein refers to the maximum yield of a product obtained from fermentation of a given feedstock, such as glucose, based on the energetic value of the product compared to the feedstock. In a normal fermentation, without use of additional energy sources such as light, hydrogen gas or methane or electricity, for instance, the product cannot contain more energy than the feedstock. The thermodynamic maximum yield signifies a product yield at which all energy and mass from the feedstock is converted to the product. This yield can be calculated and is independent of a specific pathway. If a specific pathway towards a product has a lower yield than the thermodynamic maximum yield, then it loses mass and can most likely be improved upon or substituted with a more efficient pathway towards the product.

The term “redox balanced” refers to a set of reactions, which taken together produce as much redox cofactors as they consume. Designing metabolic pathways and engineering an organism such that the redox cofactors are balanced or close to being balanced usually results in a more efficient, higher yield production of the desired compounds. Redox reactions always occur together as two half-reactions happening simultaneously, one being an oxidation reaction and the other a reduction reaction. In redox processes, the reductant transfers electrons to the oxidant. Thus, in the reaction, the reductant or reducing agent loses electrons and is oxidized, and the oxidant or oxidizing agent gains electrons and is reduced. In one aspect, the redox reactions take place in a biological system. Biological energy is frequently stored and released by means of redox reactions. Photosynthesis involves the reduction of carbon dioxide into sugars and the oxidation of water into molecular oxygen. The reverse reaction, respiration, oxidizes sugars to produce carbon dioxide and water. As intermediate steps, the reduced carbon compounds are used to reduce nicotinamide adenine dinucleotide (NAD+), which then contributes to the creation of a proton gradient, which drives the synthesis of adenosine triphosphate (ATP) and is maintained by the reduction of oxygen. The term redox state is often used to describe the balance of GSH/GSSG, NAD+/NADH and NADP+/NADPH in a biological system such as a cell or organ. The redox state is reflected in the balance of several sets of metabolites (e.g., lactate and pyruvate, beta-hydroxybutyrate, and acetoacetate), whose interconversion is dependent on these ratios. An abnormal redox state can develop in a variety of deleterious situations, such as hypoxia, shock, and sepsis.

As used herein, the term “productivity” refers to the total amount of bioproduct (the products described herein) produced per hour.

As used herein “malonate semialdehyde,” “malonic semialdehyde,” and “3-oxopropanoic acid are used interchangeably to describe the C₃H₄O₃molecule of the present disclosure.

As used herein, “enhanced activity” of an enzyme designates either an increased specific catalytic activity of the enzyme, and/or an increased quantity/availability of the enzyme in the cell, obtained, for example, by overexpression of the gene encoding the enzyme.

The present application generally relates to methods of utilizing modified microbes for co-producing 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof; and further related to the modified microbes per se.

In some aspects, the present disclosure is drawn to a recombinant microorganism capable of co-producing 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof, from a feedstock comprising one or more carbon sources, wherein the recombinant microorganism comprises one or more of the following: (a) at least one endogenous and/or exogenous nucleic acid molecule encoding an 3-hydroxypropionic acid dehydrogenase that catalyzes the production of 3-HP from malonate semialdehyde; (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a (i) malonate semialdehyde dehydrogenase that catalyzes the production of acetyl-CoA from malonate semialdehyde, and/or (ii) malonyl-CoA reductase and/or 2-keto acid decarboxylase that catalyzes the conversion of malonate semialdehyde into malonyl-CoA, and malonyl-CoA decarboxylase that catalyzes the production of acetyl-CoA from malonyl-CoA, and/or (iii) malonyl-CoA synthetase that catalyzes the conversion of oxaloacetate to malonyl-CoA, and malonyl-CoA decarboxylase that catalyzes the production of acetyl-CoA from malonyl-CoA; (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an enzyme that is able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde.

In some aspects, the recombinant microorganism is capable of producing 1-propanol. In some aspects, the recombinant microorganism comprises one or more of the following: (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a propionyl-CoA synthase that catalyzes the production of propionyl-CoA from 3-HP; (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionyl-CoA synthetase/transferase, 3-hydroxypropionyl-CoA dehydratase, and acrylyl-CoA reductase that catalyzes the production of propionyl-CoA from 3-HP; (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an alcohol/aldehyde dehydrogenase that catalyzes the production of 1-propanol from propionyl-CoA; (d) at least one endogenous and/or exogenous nucleic acid molecule encoding an aldehyde dehydrogenase (acetylating) that catalyzes the production of propionaldehyde from propionyl-CoA and alcohol dehydrogenase that catalyzes the production of 1-propanol from propionaldehyde.

In some aspects, the recombinant microorganism is capable of producing acetone. In some aspects, the recombinant microorganism comprises one or more of the following: (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase that catalyzes the production of acetoacetyl-CoA from acetyl-CoA; (b) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA synthase that catalyzes the production of acetoacetyl-CoA from malonyl-CoA; (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA transferase that catalyzes the production of acetoacetate from acetoacetyl-CoA; (d) at least one endogenous and/or exogenous nucleic acid molecule encoding a (i) hydroxymethylglutaryl-CoA synthase that catalyzes the production of HMG-CoA from acetoacetyl-CoA, and (ii) hydroxymethylglutaryl-CoA lyase that catalyzes the production of acetoacetate from HMG-CoA; and (e) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetate decarboxylase that catalyzes the production of acetone from acetoacetate.

In some aspects, the recombinant microorganism catalyzes malonate semialdehyde for the production of both 3-HP and acetyl-CoA, and/or derivatives thereof. In some aspects, the recombinant microorganism comprises a decarboxylase capable of acting on oxaloacetate to produce malonate semialdehyde. In some aspects, the derivatives of 3-HP are selected from the group consisting of: acrylic acid, 1-propanol, propene, and polypropylene. In some aspects, the derivatives of acetyl-CoA are selected from the group consisting of: acetone, 2-propanol, propene, and polypropylene.

In some aspects, at least a portion of excess NAD(P)H produced by the recombinant microorganism in the production of acetyl-CoA is utilized as a source of reducing equivalents in the production of 3-HP.

In some aspects, the microorganism is selected from a bacterium, a fungus, or a yeast. In some aspects, the recombinant microorganism is a yeast. In some aspects, the yeast is Saccharomyces cerevisiae. In some aspects, the yeast is capable of aerobic and anaerobic growth.

In some aspects, the recombinant microorganism is derived from a parental microorganism selected from the group consisting of: Clostridium sp., Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillus sp, Candida sp., Candida krusei, Corynebacterium sp., Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacter glycolicus.

In some aspects, the disclosure is drawn to a method of producing 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof, the method comprising culturing the recombinant microorganism in a culture medium containing a feedstock comprising a carbon source until the 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof, are produced.

In some aspects, the disclosure is drawn to a method of producing a recombinant microorganism capable of co-producing 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof, from a feedstock comprising one or more carbon sources, the method comprising introducing into and/or overexpressing in the recombinant microorganism one or more of the following: (a) at least one endogenous and/or exogenous nucleic acid molecule encoding an 3-hydroxypropionic acid dehydrogenase that catalyzes the production of 3-HP from malonate semialdehyde; (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a (i) malonate semialdehyde dehydrogenase that catalyzes the production of acetyl-CoA from malonate semialdehyde, and/or or (ii) malonyl-CoA reductase and/or 2-keto acid decarboxylase that catalyzes the conversion of malonate semialdehyde into malonyl-CoA, and malonyl-CoA decarboxylase that catalyzes the production of acetyl-CoA from malonyl-CoA, and/or (iii) malonyl-CoA synthetase that catalyzes the conversion of oxaloacetate to malonyl-CoA, and malonyl-CoA decarboxylase that catalyzes the production of acetyl-CoA from malonyl-CoA; (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an enzyme that is able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde.

In some aspects, the recombinant microorganism is capable of producing 1-propanol.

In some aspects, the method further comprises introducing into and/or overexpressing in the recombinant microorganism one or more of the following: (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a propionyl-CoA synthase that catalyzes the production of propionyl-CoA from 3-HP; (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionyl-CoA synthetase/transferase, 3-hydroxypropionyl-CoA dehydratase, and acrylyl-CoA reductase that catalyzes the production of propionyl-CoA from 3-HP; (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an alcohol/aldehyde dehydrogenase that catalyzes the production of 1-propanol from propionyl-CoA; (d) at least one endogenous and/or exogenous nucleic acid molecule encoding an aldehyde dehydrogenase (acetylating) that catalyzes the production of propionaldehyde from propionyl-CoA and alcohol dehydrogenase that catalyzes the production of 1-propanol from propionaldehyde. In some aspects, the recombinant microorganism is capable of producing acetone. In some aspects, the method further comprises introducing into and/or overexpressing in the recombinant microorganism one or more of the following: (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase that catalyzes the production of acetoacetyl-CoA from acetyl-CoA; (b) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA synthase that catalyzes the production of acetoacetyl-CoA from malonyl-CoA; (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA transferase that catalyzes the production of acetoacetate from acetoacetyl-CoA; (d) at least one endogenous and/or exogenous nucleic acid molecule encoding a (i) hydroxymethylglutaryl-CoA synthase that catalyzes the production of HMG-CoA from acetoacetyl-CoA, and (ii) hydroxymethylglutaryl-CoA lyase that catalyzes the production of acetoacetate from HMG-CoA; and (e) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetate decarboxylase that catalyzes the production of acetone from acetoacetate.

In some aspects, the recombinant microorganism catalyzes malonate semialdehyde for the production of both 3-HP and acetyl-CoA, and/or derivatives thereof. In some aspects, the derivatives of 3-HP are selected from the group consisting of: acrylic acid, 1-propanol, propene, and polypropylene. In some aspects, the derivatives of acetyl-CoA are selected from the group consisting of: acetone, 2-propanol, propene, and polypropylene. In some aspects, at least a portion of excess NAD(P)H produced by the recombinant microorganism in the production of acetyl-CoA is utilized as a source of reducing equivalents in the production of 3-HP. In some aspects, the recombinant microorganism comprises a decarboxylase capable of acting on oxaloacetate to produce malonate semialdehyde.

In some aspects, the microorganism is selected from a bacterium, a fungus, or a yeast. In some aspects, the recombinant microorganism is a yeast. In some aspects, the yeast is capable of aerobic and anaerobic growth. In some aspects, the yeast is Saccharomyces cerevisiae.

In some aspects, the recombinant microorganism is derived from a parental microorganism selected from the group consisting of: Clostridium sp., Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillus sp, Candida sp Candida krusei, Corynebacterium sp., Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacter glycolicus.

Generation of Microbial Populations
Genetic Modification

The genetic modification introduced into one or more microbes of the present disclosure may alter or abolish a regulatory sequence of a target gene. In some aspects, the genetic modification introduced into one or more microbes of the present disclosure may introduce a new trait or phenotype into the one or more microbes. One or more regulatory sequences may also be inserted, including heterologous regulatory sequences and regulatory sequences found within a genome of an animal, plant, fungus, yeast, bacteria, or virus corresponding to the microbe into which the genetic variation is introduced. Moreover, regulatory sequences may be selected based on the expression level of a gene in a microbial culture. The genetic variation may be a pre-determined genetic variation that is specifically introduced to a target site. In some aspects the genetic variation is a nucleic acid sequence that is introduced into one or more microbial chromosomes. In some aspects, the genetic variation is a nucleic acid sequence that is introduced into one or more extrachromosomal nucleic acid sequence. The genetic variation may be a random mutation within the target site. The genetic variation may be an insertion or deletion of one or more nucleotides. In some cases, a plurality of different genetic variations (e.g. 2, 3, 4, 5, 10, or more) are introduced into one or more of the isolated bacteria. The plurality of genetic variations can be any of the above types, the same or different types, and in any combination. In some cases, a plurality of different genetic variations are introduced serially, introducing a first genetic variation after a first isolation step, a second genetic variation after a second isolation step, and so forth so as to accumulate a plurality of desired modifications in the microbes.

In general, the term “genetic variation” refers to any change introduced into a polynucleotide sequence relative to a reference polynucleotide, such as a reference genome or portion thereof, or reference gene or portion thereof. A genetic variation may be referred to as a “mutation,” and a sequence or organism comprising a genetic variation may be referred to as a “genetic variant” or “mutant”. Genetic variations can have any number of effects, such as the increase or decrease of some biological activity, including gene expression, metabolism, and cell signaling. Genetic variations can be specifically introduced to a target site, or introduced randomly. A variety of molecular tools and methods are available for introducing genetic variation. For example, genetic variation can be introduced via polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, recombineering, lambda red mediated recombination, CRISPR/Cas9 systems, chemical mutagenesis, and combinations thereof. Chemical methods of introducing genetic variation include exposure of DNA to a chemical mutagen, e.g., ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-nitrosourea (EN U), N-methyl-N-nitro-N′-nitrosoguanidine, 4-nitroquinoline N-oxide, diethyl sulfate, benzopyrene, cyclophosphamide bleomycin, triethylmelamine, acrylamide monomer, nitrogen mustard, vincristine, diepoxyalkanes (for example, diepoxybutane), ICR-170, formaldehyde, procarbazine hydrochloride, ethylene oxide, dimethylnitrosamine, 7,12 dimethylbenz(a)anthracene, chlorambucil, hexamethylphosphoramide, bisulfan, and the like. Radiation mutation-inducing agents include ultraviolet radiation, γ-irradiation, X-rays, and fast neutron bombardment. Genetic variation can also be introduced into a nucleic acid using, e.g., trimethylpsoralen with ultraviolet light. Random or targeted insertion of a mobile DNA element, e.g., a transposable element, is another suitable method for generating genetic variation. Genetic variations can be introduced into a nucleic acid during amplification in a cell-free in vitro system, e.g., using a polymerase chain reaction (PCR) technique such as error-prone PCR. Genetic variations can be introduced into a nucleic acid in vitro using DNA shuffling techniques (e.g., exon shuffling, domain swapping, and the like). Genetic variations can also be introduced into a nucleic acid as a result of a deficiency in a DNA repair enzyme in a cell, e.g., the presence in a cell of a mutant gene encoding a mutant DNA repair enzyme is expected to generate a high frequency of mutations (i.e., about 1 mutation/100 genes-1 mutation/10,000 genes) in the genome of the cell. Examples of genes encoding DNA repair enzymes include but are not limited to Mut H, Mut S, Mut L, and Mut U, and the homologs thereof in other species (e.g., MSH 1 6, PMS 1 2, MLH 1, GTBP, ERCC-1, and the like). Example descriptions of various methods for introducing genetic variations are provided in e.g., Stemple (2004) Nature 5:1-7; Chiang et al. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; and U.S. Pat. Nos. 6,033,861, and 6,773,900.

In some aspects, recombinant microbes of the present disclosure may comprise any one or more of the disclosed nucleic acid sequence listings. In some aspects, recombinant microbes of the present disclosure may comprise any one or more nucleic acid sequences that share at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one or more of the disclosed nucleic acid sequence listings. In some aspects, recombinant microbes of the present disclosure may comprise any one or more nucleic acid sequence that shares at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with any one or more of the disclosed nucleic acid sequence listings.

In some aspects, recombinant microbes of the present disclosure may comprise one or more nucleic acid sequences that encode one or more amino acid sequences of the present disclosure. In some aspects, recombinant microbes of the present disclosure may comprise one or more nucleic acid sequences that encode one or more amino acid sequences that share at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one or more of the disclosed amino acid sequence listings. In some aspects, recombinant microbes of the present disclosure may comprise one or more nucleic acid sequences that encode one or more amino acid sequences that share at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with any one or more of the disclosed amino acid sequence listings.

Genetic variations introduced into microbes may be classified as transgenic, cisgenic, intragenomic, intrageneric, intergeneric, synthetic, evolved, rearranged, or SNPs. CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats)/CRISPR-associated (Cas) systems can be used to introduce desired mutations. CRISPR/Cas9 provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. The Cas9 protein (or functional equivalent and/or variant thereof, i.e., Cas9-like protein) naturally contains DNA endonuclease activity that depends on the association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently link to form a single molecule (also called a single guide RNA (“sgRNA”). Thus, the Cas9 or Cas9-like protein associates with a DNA-targeting RNA (which term encompasses both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and guides the protein to a target nucleic acid sequence. If the Cas9 or Cas9-like protein retains its natural enzymatic function, it will cleave target DNA to create a double-stranded break, which can lead to genome alteration (i.e., editing: deletion, insertion (when a donor polynucleotide is present), replacement, etc.), thereby altering gene expression. Some variants of Cas9 (which variants are encompassed by the term Cas9-like) have been altered such that they have a decreased DNA cleaving activity (in some cases, they cleave a single strand instead of both strands of the target DNA, while in other cases, they have severely reduced to no DNA cleavage activity). Further exemplary descriptions of CRISPR systems for introducing genetic variation can be found in, e.g. US879596 and Di Carlo et al. (2013. Nucl. Acids Res., 7(41):4336-4343).

Oligonucleotide-directed mutagenesis, also called site-directed mutagenesis, typically utilizes a synthetic DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so that it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion, or insertion, or a combination of these. The single-strand primer is then extended using a DNA polymerase, which copies the rest of the gene. The gene thus copied contains the mutated site, and may then be introduced into a host cell as a vector and cloned. Finally, mutants can be selected by DNA sequencing to check that they contain the desired mutation.

Genetic variations can be introduced using error-prone PCR. In this technique, the gene of interest is amplified using a DNA polymerase under conditions that are deficient in the fidelity of replication of sequence. The result is that the amplification products contain at least one error in the sequence. When a gene is amplified and the resulting product(s) of the reaction contain one or more alterations in sequence when compared to the template molecule, the resulting products are mutagenized as compared to the template. Another means of introducing random mutations is exposing cells to a chemical mutagen, such as nitrosoguanidine or ethyl methanesulfonate (Nestmann, Mutat Res 1975 June; 28(3):323-30), and the vector containing the gene is then isolated from the host.

Homologous recombination mutagenesis involves recombination between an exogenous DNA fragment and the targeted polynucleotide sequence. After a double-stranded break occurs, sections of DNA around the 5′ ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3′ end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. The method can be used to delete a gene, remove exons, add a gene, and introduce point mutations. Homologous recombination mutagenesis can be permanent or conditional. Typically, a recombination template is also provided. A recombination template may be a component of another vector, contained in a separate vector, or provided as a separate polynucleotide. In some aspects, a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a site-specific nuclease. A template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some aspects, the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence. When optimally aligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In some aspects, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence. Non-limiting examples of site-directed nucleases useful in methods of homologous recombination include zinc finger nucleases, CRISPR nucleases, TALE nucleases, and meganuclease. For a further description of the use of such nucleases, see e.g. U.S. Pat. No. 8,795,965 and US20140301990.

Introducing genetic variation may be an incomplete process, such that some bacteria in a treated population of bacteria carry a desired mutation while others do not. In some cases, it is desirable to apply a selection pressure so as to enrich for bacteria carrying a desired genetic variation. Traditionally, selection for successful genetic variants involved selection for or against some functionality imparted or abolished by the genetic variation, such as in the case of inserting antibiotic resistance gene or abolishing a metabolic activity capable of converting a non-lethal compound into a lethal metabolite. It is also possible to apply a selection pressure based on a polynucleotide sequence itself, such that only a desired genetic variation need be introduced (e.g. without also requiring a selectable marker). In this case, the selection pressure can comprise cleaving genomes lacking the genetic variation introduced to a target site, such that selection is effectively directed against the reference sequence into which the genetic variation is sought to be introduced. Typically, cleavage occurs within 100 nucleotides of the target site (e.g. within 75, 50, 25, 10, or fewer nucleotides from the target site, including cleavage at or within the target site). Cleaving may be directed by a site-specific nuclease selected from the group consisting of a Zinc Finger nuclease, a CRISPR nuclease, a TALE nuclease (TALEN), or a meganuclease. Such a process is similar to processes for enhancing homologous recombination at a target site, except that no template for homologous recombination is provided. As a result, bacteria lacking the desired genetic variation are more likely to undergo cleavage that, left unrepaired, results in cell death. Bacteria surviving selection may then be isolated for assessing conferral of an improved trait.

A CRISPR nuclease may be used as the site-specific nuclease to direct cleavage to a target site. An improved selection of mutated microbes can be obtained by using Cas9 to kill non-mutated cells. Microbes can then be re-isolated from tissues. CRISPR nuclease systems employed for selection against non-variants can employ similar elements to those described above with respect to introducing genetic variation, except that no template for homologous recombination is provided. Cleavage directed to the target site thus enhances death of affected cells.

Other options for specifically inducing cleavage at a target site are available, such as zinc finger nucleases, TALE nuclease (TALEN) systems, and meganuclease. Zinc-finger nucleases (ZFNs) are artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double stranded breaks. Transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENS can be quickly engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. Meganucleases (homing endonuclease) are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs. Meganucleases can be used to replace, eliminate or modify sequences in a highly targeted way. By modifying their recognition sequence through protein engineering, the targeted sequence can be changed. Meganucleases can be used to modify all genome types, whether bacterial, plant or animal and are commonly grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TeeI, I-TevII and I-TevIII.

In some aspects, methods of enhancing expression of endogenous or exogenous genes is to introduce one or more supplementary copies of the gene into a chromosome or plasmid. In some aspects, another way of enhancing expression of endogenous or exogenous genes is to replace the endogenous promoter of a gene with, or to use an exogenous promoter of a gene, a stronger promoter. In some aspects, the promoters are homologous or heterologous.

In some aspects, the microbes of the present disclosure are modified such that they comprise one or more selectable markers useful for the selection of transformed microbial cells. In some aspects, the selectable markers are introduced via DNA constructs comprising the genes, polynucleotides, oligonucleotides, and/or pathways of the present disclosure.

In some aspects, the selectable marker is an antibiotic resistance marker. Illustrative examples of antibiotic resistance markers include, but are not limited to the, NAT 1, AUR1-C, HPH, DSDA, KAN<R>, and SH BLE gene products. The NAT 1 gene product from S. noursei confers resistance to nourseothricin; the AUR1-C gene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA); the HPH gene product of Klebsiella pneumonia confers resistance to Hygromycin B; the DSDA gene product of E. coli allows cells to grow on plates with D-serine as the sole nitrogen source; the KAN<R> gene of the Tn903 transposon confers resistance to G418; and the SH BLE gene product from Streptoalloteichus hindustanus confers resistance to Zeocin (bleomycin). In some aspects, the antibiotic resistance marker is deleted after the genetically modified microbial cell of the present disclosure is isolated.

In some aspects, the selectable marker rescues an auxotrophy (e.g., a nutritional auxotrophy) in the genetically modified microbial cell. In such aspects, a parent microbial cell comprises a functional disruption in one or more gene products that function in an amino acid or nucleotide biosynthetic pathway, such as, for example, the HIS3, LEU2, LYS1, LYS2, MET 15, TRP1, ADE2, and URA3 gene products in yeast, which renders the parent microbial cell incapable of growing in media without supplementation with one or more nutrients (auxotrophic phenotype). The auxotrophic phenotype can then be rescued by transforming the parent microbial cell with a chromosomal integration encoding a functional copy of the disrupted gene product (NB: the functional copy of the gene can originate from close species, such as Kluveromyces, Candida etc.), and the genetically modified microbial cell generated can be selected for based on the loss of the auxotrophic phenotype of the parent microbial cell.

When a gene from a different type of microorganism is introduced into a cell (yeast gene to bacteria, bacterial gene to yeast, viral gene to yeast, etc), the gene may be transcoded (codon-optimized) such that the genes are synthesized with an optimal codon usage for expression in the host to which the gene is being introduced. In some aspects, the nucleotide sequence (and not the amino acid sequence) of some genes may be transcoded to minimize recombination with an endogenous copy of the same gene or homolog. In some aspects, the gene may be rendered inducible by deleting the endogenous copy of the gene, if necessary, and placing a new copy of the gene under the control of an inducible promoter.

In some aspects, the disclosure is drawn to a nucleic acid sequence encoding an enzyme capable of catalyzing the decarboxylation of oxaloacetate into malonate semialdehyde, or one of its salts, and can be expressed in a microbe using to types of non-mutually exclusive manners: (1) overexpression, i.e., one or a plurality of copies is/are introduced into the microorganism; and/or (2) the at least one nucleic acid is placed under the control of a strong or inducible promoter.

Promoters

In some aspects, various promoters may be used for the desired expression of the coding sequences of interest, which include (i) constitutive strong promoters (sometimes referred to as strong promoters), and (ii) inducible promoters.

In some aspects, the promoters are yeast promoters. A list of yeast promoters with their relative activities in different media can be found in Keren et al. (2013. Molecular Systems Biology, 9:701). Promoters allowing the constitutive over-expression of a given gene, may be found in Velculescu et al. (1997. Cell, 88:243-251).

In some aspects, strong promoters may be selected from the following: pTDH3 (SEQ ID NO: 13), pENO2 (SEQ ID NO: 14), pTEF KI (SEQ ID NO: 15), pTEF3 (SEQ ID NO: 16), pTEF1 (SEQ ID NO: 17), pADH1 (SEQ ID NO: 18), pGMP1 (SEQ ID NO: 19), pFBA1 (SEQ ID NO: 20), pPDC1 (SEQ ID NO: 21), pCCW12 (SEQ ID NO: 22), and pGK1 (SEQ ID NO: 23).

In some aspects, the strong promoter according to the disclosure is, independently, selected from the group consisting of pTDH3, pENO2, pTEF-KI, pTEF3, pTEF1, pADH1, pGMP1, pFBA1, pPDC1, pCCW12 and pGK1.

As described herein, inducible promoters are promoters whose activity is controlled by the presence or absence of biotic or abiotic factors, and also by the quantity of said factor. Accordingly, their activity will be induced and thus increased when the quantity of a given factor increases or is increased.

In some aspects, increasing the quantity of methionine in a culture medium of a recombinant yeast comprising a pSAM4 promoter will induce and thus increase transcription of the gene under the control of this promoter. In some aspects, reducing the quantity of copper in a culture medium of a recombinant yeast comprising a pCTR1 promoter will lead to an induced, and thus an increased, transcription of the gene under the control of this promoter.

For this reason, the following promoters are referred to in the present text as being “inducible promoters.”

In some aspects, inducible promoters may be selected from the group comprising promoters inducible with copper, promoters inducible with methionine and promoters inducible with threonine, and may be selected from the group consisting of: pSAM4—methionine inducible (SEQ ID NO: 24), pCUP1-1—copper inducible (SEQ ID NO: 25), pCUP1.cgla—copper inducible (SEQ ID NO: 26), pCUP1.sba—copper inducible (SEQ ID NO: 27), pACU1—copper inducible (SEQ ID NO: 28), pACU2—copper inducible (SEQ ID NO: 29), pACU3p—copper inducible (SEQ ID NO: 30), pACU4p—copper inducible (SEQ ID NO: 31), pACU5—copper inducible (SEQ ID NO: 32), pACU6—copper inducible (SEQ ID NO: 33), pACU7—copper inducible (SEQ ID NO: 34), pACU8—copper inducible (SEQ ID NO: 35), pACU9—copper inducible (SEQ ID NO: 36), pACU10p—copper inducible (SEQ ID NO: 37), pACU11—copper inducible (SEQ ID NO: 38), pACU12—copper inducible (SEQ ID NO: 39), pACU13—copper inducible (SEQ ID NO: 40), pACU14—copper inducible (SEQ ID NO: 41), pACU15—copper inducible (SEQ ID NO: 42), pGAL/CUP1p—copper inducible (SEQ ID NO: 43), pCRS5—copper inducible (SEQ ID NO: 44), and pCHA1—threonine inducible (SEQ ID NO: 45).

In some aspects, the inducible promoter(s) may be selected from the group consisting of pSAM4, pCUP1-1, pCUP1.Cgla, pCUP1.Sba, pACU1, pACU2, pACU3p, pACU4p, pACU5, pACU6, pACU7, pACU8, pACU9, pACU10p, pACU11, pACU12, pACU13, pACU14, pACU15, pGAL/CUP1p, pCRS5, and pCHA1. The activity of these promoters is thus induced by the increasing presence of methionine, copper or threonine as indicated above.

In some aspects, inducible promoters may be selected from: (1) promoters inducible due to the absence of copper (i.e. promoter's activity is increased by the absence of copper—the fewer copper there is in the medium, the higher the activity of these promoters); (2) promoters inducible due to the absence of lysine (i.e. promoter's activity is increased by the absence of lysine—the fewer lysine there is in the medium, the higher the activity of these promoters); and (3) promoters inducible due to the absence of methionine (i.e. promoter's activity is increased by the absence of methionine—the fewer methionine there is in the medium, the higher the activity of these promoters). In some aspects, the inducible promoters are selected from the group consisting of: pCTR1—copper inducible (SEQ ID NO: 46), pCTR3—copper inducible (SEQ ID NO: 47), pCUR1—copper inducible (SEQ ID NO: 48), pCUR2—copper inducible (SEQ ID NO: 49), pCUR3—copper inducible (SEQ ID NO: 50), pCUR4—copper inducible (SEQ ID NO: 51), pCUR5p—copper inducible (SEQ ID NO: 52), pCUR6—copper inducible (SEQ ID NO: 53), pCUR7—copper inducible (SEQ ID NO: 54), pCUR8—copper inducible (SEQ ID NO: 55), pCUR9—copper inducible (SEQ ID NO: 56), pCUR10—copper inducible (SEQ ID NO: 57), pCUR11—copper inducible (SEQ ID NO: 58), pCUR12—copper inducible (SEQ ID NO: 59), pCUR13—copper inducible (SEQ ID NO: 60), pCUR14—copper inducible (SEQ ID NO: 61), pCUR15—copper inducible (SEQ ID NO: 62), pCUR16—copper inducible (SEQ ID NO: 63), pCUR17—copper inducible (SEQ ID NO: 64), pLYS1—lysine inducible (SEQ ID NO: 65), pLYS4—lysine inducible (SEQ ID NO: 66), pLYS9—lysine inducible (SEQ ID NO: 67), pLYR1p—lysine inducible (SEQ ID NO: 68), pLYR2p—lysine inducible (SEQ ID NO: 69), pLYR3p—lysine inducible (SEQ ID NO: 70), pLYR4p—lysine inducible (SEQ ID NO: 71), pLYR5p—lysine inducible (SEQ ID NO: 72), pLYR6p—lysine inducible (SEQ ID NO: 73), pLYR7p—lysine inducible (SEQ ID NO: 74), pLYR8—lysine inducible (SEQ ID NO: 75), pLYR9—lysine inducible (SEQ ID NO: 76), pLYR10—lysine inducible (SEQ ID NO: 77), pLYR11—lysine inducible (SEQ ID NO: 78), pMET17—methionine inducible (SEQ ID NO: 79), pMET6—methionine inducible (SEQ ID NO: 80), pMET14—methionine inducible (SEQ ID NO: 81), pMET3—methionine inducible (SEQ ID NO: 82), pSAM1—methionine inducible (SEQ ID NO: 83), pSAM2—methionine inducible (SEQ ID NO: 84), pMDH2—glucose inducible (SEQ ID NO: 85), pJEN1—glucose inducible (SEQ ID NO: 86), pICL1—glucose inducible (SEQ ID NO: 87), pADH2—glucose inducible (SEQ ID NO: 88), and pMLS1—glucose inducible (SEQ ID NO: 89).

In some aspects, the inducible promoter(s) may be selected from the group consisting of pCTR1, pCTR3, pCUR1, pCUR2, pCUR3, pCUR4, pCUR5p, pCUR6, pCUR7, pCUR8, pCUR9, pCUR10, pCUR11, pCUR12, pCUR13, pCUR14, pCUR15, pCUR16, pCUR17, pLYS1, pLYS4, pLYS9, pLYR1p, pLYR2p, pLYR3p, pLYR4p, pLYR5p, pLYR6p, pLYR7p, pLYR8, pLYR9, pLYR10, pLYR11, pMET17, pMET6, pMET14, pMET3, pSAM1, pSAM2, pMDH2, pJEN1, pICL1, pADH2, and pMLS1.

In some aspects, inducible promoters may be selected from the group comprising promoters inducible with copper, promoters inducible due to the absence of copper, promoters inducible due to the absence of glucose, promoters inducible due to the absence of lysine, promoters inducible with methionine, promoters inducible due to the absence of methionine, and promoters inducible with threonine.

In some aspects, inducible promoter may be selected from the group consisting of pSAM4, pCUP1-1, pCUP1.Cgla, pCUP1.Sba, pACU1, pACU2, pACU3p, pACU4p, pACU5, pACU6, pACU7, pACU8, pACU9, pACU10p, pACU11, pACU12, pACU13, pACU14, pACU15, pGAL/CUP1p, pCRS5, pCHA1, pCTR1, pCTR3, pCUR1, pCUR2, pCUR3, pCUR4, pCUR5p, pCUR6, pCUR7, pCUR8, pCUR9, pCUR10, pCUR11, pCUR12, pCUR13, pCUR14, pCUR15, pCUR16, pCUR17, pLYS1, pLYS4, pLYS9, pLYR1p, pLYR2p, pLYR3p, pLYR4p, pLYR5p, pLYR6p, pLYR7p, pLYR8, pLYR9, pLYR10, pLYR11, pMET17, pMET6, pMET14, pMET3, pSAM1, pSAM2, pMDH2, pJEN1, pICL1, pADH2 and pMLS1.

In some aspects, the inducible promoters, identical or different, may be preferably characterized by a sequence of nucleic acids selected from the group consisting of sequences having at least 80/85/90/95/96/97/98/99/100% identity with sequences SEQ ID NOs: 13-89.

In some aspects, synthetic promoters, as described in Blazeck & Alper (2013) Biotechnol. J. 8 46-58, can also be used.

The strong and inducible or repressible promoters of the disclosure can originate from any organism from Saccharomycetes class and can in particular originate, independently, from an organism selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces castelii, Saccharomyces bayanus, Saccharomyces arboricola, Saccharomyces kudriavzevii, Ashbya gossypii, Kluveromyces lactis, Pichia pastoris, Candida glabrata, Candida tropicalis, Debaryomyces castelii, Yarrowia hpolitica, and Cyberlindnera jadinii.

In some aspects, strong, weak, and inducible promoters may originate from an organism selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces castelii, Saccharomyces bayanus, Saccharomyces arboricola, Saccharomyces kudriavzevii, and Kluveromyces lactis.

Terminators

In some aspects, the recombinant microbes comprise appropriate transcription terminator sequences that are functional in yeast cells, including in Saccharomyces cerevisiae. In some aspects, the transcription terminators, identical or different, may be found in Yamanishi et al., (2013) ACS synthetic biology 2, 337-347.

In some aspects, terminators may be selected from the group comprising:

- tTDH2 from the gene coding for Glyceraldehyde-3-phosphate dehydrogenase, isozyme 2 (TDH2 gene=Sequence SEQ ID NO: 90),
- tCYC1 (=Sequence SEQ ID NO: 91),
- tTDH3 (=Sequence SEQ ID NO: 92),
- tADH1 from gene coding for the alcohol dehydrogenase (ADH1 gene=Sequence SEQ ID NO: 93),
- tADH2 from gene coding for the alcohol dehydrogenase (ADH2 gene=Sequence SEQ ID NO: 94),
- tTPI1 from the gene encoding for the Triose Phosphate Isomerase (TPI1 gene=Sequence SEQ ID NO: 95),
- tMET17 from the gene encoding for the O-acetyl homoserine-O-acetyl serine sulfhydrylase (Met17 gene=Sequence SEQ ID NO: 96),
- tENO2 from the gene coding for Enolase II (ENO2 gene=Sequence SEQ ID NO: 97),
- tMET3 (=Sequence SEQ ID NO: 98),
- tPGK1 from the gene encoding for the 3-phosphoglycerate kinase (PGK1 gene=Sequence SEQ ID NO: 99),
- tDIT1 (=Sequence SEQ ID NO: 100)
- tRPL3 (=Sequence SEQ ID NO: 101)
- tRPL41B (=Sequence SEQ ID NO: 102)
- tRPL15A (=Sequence SEQ ID NO: 103)
- tIDP1 (=Sequence SEQ ID NO: 104)

In some aspects, the terminator, identical or different, may be preferably characterized by a sequence of nucleic acid selected from the group consisting of sequences having at least 80/85/90/95/96/97/98/99/100% identity with SEQ ID NOs: 90-104.

Malonate Semialdehyde, Salts Thereof and Derivatives Thereof

Malonate semialdehyde and its salts thereof are a key intermediate for the production of valuable compounds. These compounds of economic interest include those produced directly from malonate semialdehyde or its salts, such as acrylate, 1-propanol, isopropanol, 3-hydroxypropionate, and propionate, but also those derived from malonyl-CoA, mostly produced by polyketides synthases such as phloroglucinol and flavonoids, and the fatty acids synthase, or those derived from the mevalonate such as farnesyl-PP, squalene and derivatives or the 3-hydroxy-3-methyl-butyrate pathways.

Malonate semialdehyde and its salts are naturally produced in yeast from malonyl-CoA and beta-alanine. However, production of malonyl-CoA and its salts is in competition with the ethanol biosynthesis pathway, thus rendering difficult the flux derivation to malonate semialdehyde.

Moreover, production from beta-alanine requires the amination of oxaloacetate followed by the deamination of beta-alanine, involving a great number of enzymes.

In order to facilitate the production of malonate semialdehyde and its salts in yeasts, it has been proposed to obtain malonate semialdehyde in one step by decarboxylation of oxaloacetate (US2010/0021978). However, no natural enzyme is known as being able to perform this transformation in a natural pathway efficiently. The enzymatic activity of the decarboxylation of oxaloacetate into malonate semialdehyde is herein referred to as oxaloacetate 1-decarboxylase (MSA forming), and is not to be confused with oxaloacetate decarboxylase (EC 4.1.1.3) which yields pyruvate.

US2010/0021978 proposes the use of a promiscuous decarboxylase such as the benzoylformate decarboxylase, the alpha-ketoglutarate decarboxylase, the alpha-ketoisovalerate decarboxylase or the pyruvate decarboxylase to perform this decarboxylation of oxaloacetate in malonate semialdehyde or one of its salts. This document exemplifies the use of benzoylformate decarboxylase in Escherichia coli to produce 3 hydroxypropionate through malonate semialdehyde.

US2018/032830 proposes and exemplifies the use of a decarboxylase such as pyruvate decarboxylase to perform this decarboxylation of oxaloacetate in 3-oxopropanoate (malonate semialdehyde) to produce 3-hydroxipropionate.

U.S. Pat. No. 8,809,027 proposes and exemplifies the use of pyruvate decarboxylase, 2-oxoglutarate decarboxylase and alpha-ketoglutarate decarboxylase to perform the decarboxylation of oxaloacetate in malonate semialdehyde to produce 3-hydroxipropionate.

The inventors in the aforementioned pre-grant publication could detect carboxylase activity both in cellulo and in vitro on their cognate substrate, the inventors were unable to detect any activity of all these enzymes on oxaloacetate.

Accordingly, there is still a need in the art for enzymes able to efficiently catalyze the transformation of oxaloacetate into malonate semialdehyde, or one of its salts, when expressed in a yeast, and in particular in the yeast Saccharomyces cerevisiae.

Malonate semialdehyde is a compound having the following structure:

[Image Omitted]

In some aspects, the compound may exist in the form of a base or of a salt. In some aspects, the salt can be malonate semialdehyde having the following structure:

[Image Omitted]

In some aspects, organic cation salts such as ammonium, sodium, potassium, phosphonium, or sulfonium salts may also be concerned.

In some aspects, malonate semialdehyde derivatives are compounds that may be obtained from malonate semialdehyde, or from one of its salts, after modification by at least one enzyme naturally or artificially present in the microorganism producing the malonate semialdehyde, or one of its salts.

In some aspects, derivatives of malonate semialdehyde include propanol, propanal, acrylic acid, acrylyl-CoA, acetyl-CoA, 3-HP, acrylate, acetone, isopropanol, propionate, propionyl-CoA, 3-hydroxypropionate, 3-hydroxypropionyl-CoA, 3-hydroxy-3-methyl-butyrate, phloroglucinol, flavonoids, cannabinoids, farnesyl-PP, and squalene.

In some aspects, 3-hydroxypropionate, Acrylyl-CoA, Propionyl-CoA, propanal and propanol can be obtained from malonate semialdehyde or from one of its salts through the steps detailed in FIG. 11 PCS represents a propionyl-CoA synthase, such as the PCS of Chloroflexus aggregans, Roseiflexus castenholzii, or Chloroflexus aurantiacus. This reaction can be catalyzed by enzymes with, but not restricted to, EC number 6.2.1.17/6.2.1.36 such as listed in Table 3. ADHE represents an alcohol dehydrogenase E, such as the ADHE from Clostridium beijerinckii or Clostridium arbusti. This reaction can be catalyzed by enzymes with, but not restricted to, EC number 1.1.1.1/1.2.1.4/1.2.1.5 such as those listed in Table 7.

In some aspects, acrylic acid and acrylate can be obtained from malonate semialdehyde or from one of its salts by multi-step enzymatic reactions involving the CoA attachment to 3-HP, dehydration of 3-HP-CoA to acrylyl-CoA as depicted in FIG. 12 and detachment of CoA from acrylyl-CoA as a synthetic pathway already demonstrated in the literature (Chu et al. Direct fermentation route for the production of acrylic acid. Metabolic Engineering, 32 (2015), 23-29). The steps from malonate semialdehyde to acrylyl-CoA can be catalyzed by 3-hydroxypropionic acid dehydrogenase with, but not restricted to, EC number 1.1.1.381 such as those listed in Table 1; 3-hydroxypropionyl-CoA synthetase/CoA transferases that can be used have, but are not restricted to, EC numbers 2.8.3.1/6.2.1.17/6.2.1.36 such as those listed in Table 4; 3-hydroxypropionyl-CoA dehydratase/enoyl-CoA hydratases that can be used have, but are not restricted to, EC numbers 4.2.1.116/4.2.1.55/4.2.1.150/4.2.1.17 such as those listed in Table 5 and acyl-CoA hydrolase or thioesterase (Table 14). Acrylic acid can be derived chemically via a dehydration reaction. Dehydration methods of 3-hydroxypropionic acid are well known in the art. For example, incorporated herein for its teachings of conversion of 3-HP, U.S. Pat. No. 8,846,353 B2 describes a method where 3-HP present in a fermentation broth can be dehydrated in a vapor phase reaction in the presence of an acid catalyst like but not limited to NaH2PO4-silica gel, H3PO4-silica gel, CuSO4-silica gel and zeolite H-β-H3PO4. The U.S. Pat. No. 2,469,701 describes other example for the reaction by adding 3-hydroxypropionic acid gradually to a concentrated dehydration catalyst, such as phosphoric acid or sulfuric acid maintained at a temperature of about 150-190° C. under reduced pressure, in the presence of powdered metallic copper and separating the aqueous acrylic acid formed from the catalyst by distillation. An aqueous phase containing 3-HP can also be dehydrated via reactive distillation to give a fluid acrylic acid solution that may proceed to an optional purification step by a suspension crystallization or a layer crystallization as described in U.S. Pat. No. 8,198,481 B2.

In some aspects, malonate semialdehyde can be transformed into acetyl-CoA by a malonate semiadehyde dehydrogenase (acetylating (E.C. 1.2.1.18), such as KES23460 from Pseudonymous putida described in Wilding et al. (2016. Appl. Microbiol Biotechnol, 82:3846-3856). See FIG. 9 Acetyl-CoA is then a starting point to produce isopropanol as described in Tamakawa et al. Appl Microbiol Biotechnol (2013) 97:6231-6239.

In some aspects, propionyl-CoA is obtained from malonate semialdehyde. Propionyl-CoA can then be transformed into propionate through the successive catalysis of phosphotransacetylase (E.C. 2.3.1.) and an acetate kinase (E.C. 2.7.2.1), described in Erbilgin et al. (2016) PLoS Biol 14(3): e1002399.doi:10.1371/journal.pbio.1002399 and Reinscheid et al. (1999) Microbiology 145, 503-513

In some aspects, malonate semialdehyde can be transformed into malonyl-CoA by a malonyl-CoA reductase (E.C 1.2.1.75) as described in Alber et al. (2006) Journal of bacteriology 188, 8551-8559. See FIG. 4 Malonyl-CoA is then the starting point to synthetize phloroglucinol and derivatives using a phloroglucinol synthase (E.C.2.3.1.253) as described in Yang and Cao (2012) Appl Microbiol Biotechnol 93:487-495.

Malonyl-CoA is a major building block and often a bottleneck required for flavonoids biosynthesis (Johnson et al. (2017) Metabolic Engineering 44: 253-264). Malonate semialdehyde can be transformed into malonyl-CoA by a malonyl-CoA reductase (E.C 1.2.1.75) as described in Alber et al. (2006) Journal of bacteriology 188, 8551-8559. Malonyl-CoA can then be used to fuel flavonoids synthesis as described in Batra, Priya, and Anil K. Sharma. (2013) 3 Biotech 6: 439-59; and in Mou, et al. (2015) PLoS ONE 10(6).

Acetyl-CoA is also a major building block and often a bottleneck required for farnesyl-PP and derivatives biosynthesis, as for example squalene. As mentioned above, malonate semialdehyde can be transformed into acetyl-CoA by a malonate semialdehyde dehydrogenase (acetylating) (E.C 1.2.1.18) as for example KES23460 from Pseudomonas putida described in Wilding et al. (2016) Appl. Env. Microbiology, 82, 3846-3856. Acetyl-CoA is then a starting point to produce Farnesyl PP and derivatives as described in Wang, J.; Li, Y.; Liu, D. Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos. Int. J. Mol. Sci. 2014, 15, 22188-22202.

In some aspects, cannabinoids can be obtained from malonate semialdehyde or from one of its salts from acetyl-CoA which is the precursor of all cannabinoids. Malonate semialdehyde can be transformed into acetyl-CoA by a malonate semialdehyde dehydrogenase (acetylating) (E.C 1.2.1.18) as for example KES23460 from Pseudomonas putida described in Wilding et al. Acetyl-CoA is then a starting point to produce cannabinoids as described in Carvalho et al. (2017) FEMS Yeast Research, 17, fox037. doi: 10.1093/femsyr/fox037.

In some aspects, 3-hydroxy-3-methyl-butyrate can be obtained from malonic semialdehyde or from one of its salts as follows. Malonate semialdehyde can, as mentioned above, be transformed into acetyl-CoA by a malonate semialdehyde dehydrogenase (acetylating) (E.C 1.2.1.18) as for example KES23460 from Pseudomonas putida described in Wilding et al. Acetyl-CoA is then the starting point for 3-hydroxy-3-methyl-butyrate biosynthesis as described in Gogerty and Bobic (2010) Appl Microbiol Biotechnol 76: 8004-8010.

In some aspects, a recombinant microbe of the present disclosure may comprise one or more nucleic acid sequences encoding the enzymes mentioned above in order to obtain the malonate semialdehyde and/or derivatives of interest. The one or more nucleic acid sequences encoding the enzymes performing the necessary transformations of malonate semialdehyde, or one of its salts, to the malonate semialdehyde derivative of interest can be naturally present in the microbe (endogenous) and/or can be incorporated into the microbe as transgenes according to methods well known to the man skilled in the art.

In some aspects, a recombinant microbe of the present disclosure may comprise, in addition to a nucleic acid sequence encoding an enzyme able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde as defined above, at least one nucleic acid encoding a 3-hydroxy acid dehydrogenase and/or a malonyl-CoA reductase.

In some aspects, the 3-hydroxy acid dehydrogenase and/or malonyl-CoA reductase according to this embodiment can be, but not limited to, the 3-hydroxy acid dehydrogenase with EC number 1.1.1.381 such as those listed in Table 1.

In some aspects, a recombinant microbe of the present disclosure may comprise, in addition to a nucleic acid gene encoding an enzyme able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde as defined above, (i) at least one nucleic acid encoding a 3-hydroxy acid dehydrogenase or a malonyl-CoA reductase and (ii) at least one nucleic acid encoding a propionyl-CoA synthase.

In some aspects, the 3-hydroxy acid dehydrogenase and/or malonyl-CoA reductase according to this embodiment can be, but not limited to, the 3-hydroxy acid dehydrogenase with EC number 1.1.1.381 such as those listed in Table 1. The propionyl-coA synthase (PCS) that can be used has, but is not restricted to, EC numbers 6.2.1.17/6.2.1.36 as those listed in Table 3.

In some aspects, a recombinant microbe of the disclosure may comprise, in addition to a nucleic acid gene encoding an enzyme able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde as defined above, (i) at least one nucleic acid encoding a 3-hydroxy acid dehydrogenase or a malonyl-CoA reductase, (ii) at least one nucleic acid encoding a propionyl-CoA synthase or 3-hydroxypropionyl-CoA synthetase/transferase, 3-hydroxypropionyl-CoA dehydratase, and acrylyl-CoA reductase and (iii) at least one alcohol dehydrogenase E or aldehyde dehydrogenase and alcohol dehydrogenase.

In some aspects, the 3-hydroxy acid dehydrogenase and/or malonyl-CoA reductase of the disclosure may be the 3-hydroxy acid dehydrogenase with, but not restricted to, EC number 1.1.1.381 such as those listed in Table 1. In some aspects, the propionyl-CoA synthase (PCS) of the disclosure may be the PCS with, but not limited to, EC numbers 6.2.1.17/6.2.10.36 such as those listed in Table 3.

In some aspects, the ADHE of the disclosure that can be used, but is not restricted to, is a enzyme with EC numbers 1.1.1.1/1.2.1.4/1.2.1.5 such as those listed in Table 7. In some aspects, it is possible to use two enzymes to convert propanol from propionyl-CoA. Can be used, but are not restricted to, enzymes with EC numbers 1.2.1.10/1.2.1.87 such as those listed in Table 8 or enzymes with EC numbers 2.3.1.8/2.7.2.1 such as those listed in Table 10 and enzymes with EC numbers 1.1.1.1/1.1.1.2 such as those listed in Table 9.

In some aspects, the recombinant microbe may comprise, in addition to a nucleic acid sequence encoding an enzyme able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde as defined above, (i) at least one nucleic acid encoding a 3-hydroxy acid dehydrogenase or a malonyl-CoA reductase and (ii) at least one nucleic acid encoding a propionate-CoA transferase.

In some aspects, a propionate-CoA transferase may be, but not restricted to, the enzyme with the reference E.C.2.8.3.1.

In some aspects, the 3-hydroxy acid dehydrogenase and/or malonyl-CoA reductase may be, but not limited to, the 3-hydroxy acid dehydrogenase or the malonyl-CoA reductase with EC number 1.1.1.381 such as those listed in Table 1. In some aspects, the propionate-CoA transferase may be, but not limited to, the propionate-CoA transferases or 3-hydroxypropionyl-CoA synthetases with EC number 2.8.3.1/6.2.1.17/6.2.1.36 such as those listed in Table 4.

In some aspects, a recombinant microbe may comprise, in addition to a nucleic acid gene encoding an enzyme able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde as defined above, (i) at least one nucleic acid encoding a 3-hydroxy acid dehydrogenase or a malonyl-CoA reductase, (ii) at least one nucleic acid encoding a propionate-CoA transferase and (iii) at least one 3-hydroxypropionyl coenzyme A dehydratase.

In some aspects, the 3-hydroxy acid dehydrogenase and/or malonyl-CoA reductase may be, but are not restricted to enzymes with EC number 1.1.1.381 such as those listed in Table 1. In some aspects, the propionate-CoA transferase or 3-hydroxypropionyl-CoA synthetase that can be used has, but is not restricted to, EC number 2.8.3.1/6.2.1.17/6.2.1.36 such as those listed in Table 4. In some aspects, the 3-hydroxypropionyl coenzyme A dehydratase may be, but is not restricted to enzymes with EC numbers 4.2.1.116/4.2.1.55/4.2.1.150/4.2.1.17 such as those listed in Table 5.

In some aspects, the disclosure is drawn to a recombinant microbial organism, a microbe in some aspects, able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde. In some aspects, these enzymes are characterized by SEQ ID NO: 1, as follows.

(SEQ ID NO: 1)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACV

VGIADGYAQASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQT

RAMIGVEAX₁X₂TNVDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMASM

APQGPVYLSVPYDDWDKDADPQSHHLFDRHVSSSVRLNDQDLDILVKALN

SASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSAPRCPFPTRHPC

FRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHQYDPGQYLKPGTRLISV

TCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQ

DAGRLHPETVFDTLNDMAPENAIYLNESX₃STTAQMWQRLNMRNPGSYY

X₄X₅AAGGX₆GFALPAAIGVQLAEPERQVIAVIGDGSANYSISALWTAAQY

NIPTIFVIMNNGTYGX₇LRWFAGVLEAENVPGLDVPGIDFRALAKGYGVQ

ALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK;

wherein:

X₁represents an amino acid selected from the group consisting of leucine, lysine, arginine and valine;

X₂represents an amino acid selected from the group consisting of leucine and lysine;

X₃represents an amino acid selected from the group consisting of threonine and serine;

X₄represents an amino acid selected from the group consisting of phenylalanine, asparagine, alanine, isoleucine and valine;

X₅represents an amino acid selected from the group consisting of cysteine and arginine;

X₆represents an amino acid selected from the group consisting of leucine, asparagine and alanine; and

X₇represents an amino acid selected from the group consisting of alanine and leucine, with the proviso that the enzyme cannot have the sequence SEQ ID NO: 1 wherein X₁represents leucine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; and X₇represents alanine.

In some aspects, X₁represents an amino acid selected from the group consisting of lysine, arginine and valine. In some aspects, X₁represents an amino acid selected from the group consisting of arginine and valine. In some aspects, X₁is arginine.

In some aspects, an enzyme of the disclosure is that of SEQ ID NO: 1 as defined above, with X₁being arginine.

In some aspects, X₂represents leucine.

In some aspects, X₁represents valine and X₂represents lysine.

In some aspects, X₃represents threonine.

In some aspects, X₁is arginine, X₂represents leucine and X₃represents threonine.

In some aspects, X₄represents phenylalanine or asparagine.

In some aspects, an enzyme of sequence of SEQ ID NO: 1 is such that:

X₁is arginine or lysine, X₂represents leucine, X₃represents threonine and X₄represents phenylalanine; or

X₁is arginine or lysine, X₂represents leucine, X₃represents threonine and X₄represents asparagine.

In some aspects, X₅represents cysteine.

In some aspects, an enzyme of sequence SEQ ID NO: 1 is such that:

X₁is arginine or lysine, X₂represents leucine, X₃represents threonine, X₄represents phenylalanine and X₅represents cysteine; or

X₁is arginine or lysine, X₂represents leucine, X₃represents threonine, X₄represents asparagine, and X₅represents cysteine.

In some aspects, X₆represents an amino acid selected from the group consisting of leucine and asparagine. In some aspects, X₆is leucine.

In some aspects, X₁represents valine, X₂represents lysine and X₆represents asparagine.

In some aspects, an enzyme of sequence SEQ ID NO: 1 is such that:

X₁is arginine or lysine, X₂represents leucine, X₃represents threonine, X₄represents phenylalanine, X₅represents cysteine and X₆is leucine; or

X₁is arginine or lysine, X₂represents leucine, X₃represents threonine, X₄represents asparagine, X₅represents cysteine and X₆is leucine.

In some aspects, X₇represents alanine.

In some aspects, an enzyme of sequence SEQ ID NO: 1 is such that:

X₁is arginine or lysine, X₂represents leucine, X₃represents threonine, X₄represents phenylalanine, X₅represents cysteine, X₆is leucine and X₇is alanine; or

X₁is arginine or lysine, X₂represents leucine, X₃represents threonine, X₄represents asparagine, X₅represents cysteine, X₆is leucine and X₇is alanine.

In some aspects, an enzyme of the disclosure is selected from the group consisting of:

(i) an enzyme of sequence SEQ ID NO: 1 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; and X₇represents alanine; (i.e. an enzyme of amino acid sequence SEQ ID NO: 2)

(ii) an enzyme of sequence SEQ ID NO: 1 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents asparagine; X₅represents cysteine; X₆represents leucine; and X₇represents alanine; (i.e. an enzyme of amino acid sequence SEQ ID NO: 3)

(iii) an enzyme of sequence SEQ ID NO: 1 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents asparagine; X₅represents cysteine; X₆represents leucine; and X₇represents alanine; (i.e. an enzyme of amino acid sequence SEQ ID NO: 4)

(iv) an enzyme of sequence SEQ ID NO: 1 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents asparagine; X₅represents cysteine; X₆represents leucine; and X₇represents leucine; (i.e. an enzyme of amino acid sequence SEQ ID NO: 5), and

(v) an enzyme of sequence SEQ ID NO: 1 wherein X₁represents valine; X₂represents lysine; X₃represents threonine; X₄represents asparagine; X₅represents cysteine; X₆represents asparagine; and X₇represents alanine. (i.e. an enzyme of amino acid sequence SEQ ID NO: 6)

In some aspects, the disclosure is drawn to a recombinant microbial organism, a microbe in some aspects, able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde. In some aspects, these enzymes are characterized by an amino acid sequence comprising:

(SEQ ID NO: 274)

MASVHGTTYELLRRQGIDX₈VFGNPGSNELPFLKDFPEDFRYILALQEAC

VVGIADGYAQASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQ

TRAMIGVEAX₁X2TNVDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMAS

MAPQGPVYLSVPYDDWDKDADPQSHHLFDRHVX₉SSVRLNDQDLDILVKA

LNSASNPX₁₀IVLGPDVDAANANADCVMLAERLKAPVWVAPSAPRCPFPT

RHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYX₁₁X₁₂YDPGQYLK

PGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAA

PEPAKVDQDAGRLHPETVFDTLNDMAPEX₁₃AIYLNESX₃STTAQMWQRL

X₁₄MRNPGSYYX₄X₅AAGGX₆GFALPAAIGVQLAEPX₁₅RQVIAVIGDGS

ANYSISALWTAAQYNX₁₆PTIFVIMNNGTYGX₇LRWX₁₇AGVLX₁₈AENV

PGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVST

VSPVK

wherein:

X₁represents an amino acid selected from the group consisting of leucine, lysine, arginine and valine;

X₂represents an amino acid selected from the group consisting of leucine and lysine;

X₃represents an amino acid selected from the group consisting of threonine and serine;

X₄represents an amino acid selected from the group consisting of phenylalanine, asparagine, alanine, isoleucine, valine, leucine, tryptophan and arginine;

X₅represents an amino acid selected from the group consisting of cysteine and arginine;

X₆represents an amino acid selected from the group consisting of leucine, asparagine, alanine, valine and serine;

X₇represents an amino acid selected from the group consisting of alanine, leucine, threonine, glycine and asparagine;

X₈represents an amino acid selected from the group consisting of threonine or isoleucine;

X₉represents an amino acid selected from the group consisting of serine or threonine;

X₁₀represents an amino acid selected from the group consisting of alanine or valine;

X₁₁represents an amino acid selected from the group consisting of histidine or arginine;

X₁₂represents an amino acid selected from the group consisting of glutamine or arginine;

X₁₃represents an amino acid selected from the group consisting of asparagine or aspartic acid;

X₁₄represents an amino acid selected from the group consisting of asparagine or aspartic acid;

X₁₅represents an amino acid selected from the group consisting of glutamic acid or glycine;

X₁₆represents an amino acid selected from the group consisting of isoleucine or valine;

X₁₇represents an amino acid selected from the group consisting of phenylalanine or serine; and

X₁₈represents an amino acid selected from the group consisting of glutamic acid or glycine;

with the proviso that the enzyme cannot have the sequence SEQ ID NO: 274 wherein X₁represents leucine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents alanine, X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine and X₁₈represents glutamic acid.

An enzyme according to the invention can in particular be selected from the group consisting of:

(i) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₈represents cysteine; X₆represents leucine; X₇represents alanine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 2);

(ii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents leucine; X₈represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents valine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 242);

(iii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₈represents cysteine; X₆represents leucine; X₇represents leucine; X₈represents isoleucine; X₉represents serine; X₁₀represents alanine; X₁₁represents arginine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents aspartic acid; X₁₅represents glycine; X₁₆represents isoleucine; X₁₇represents serine; and X₁₈represents glycine (i.e. an enzyme of amino acid sequence SEQ ID NO: 243);

(iv) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents glycine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents arginine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 244);

(v) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 245);

(vi) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents leucine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 247);

(vii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents alanine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents arginine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 248);

(viii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents arginine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 249);

(ix) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents alanine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 250);

(x) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents leucine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 251);

(xi) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents leucine; X₅represents cysteine; X₆represents leucine; X₇represents leucine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents valine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 252);

(xii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents leucine; X₅represents cysteine; X₆represents leucine; X₇represents glycine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents valine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 253);

(xiii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents leucine; X₅represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents valine; X₁₇represents serine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 254);

(xiv) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents leucine; X₅represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glycine; X₁₆represents valine; X₁₇represents serine; and X₁₈represents glycine (i.e. an enzyme of amino acid sequence SEQ ID NO: 255);

(xv) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents leucine; X₅represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glycine; X₁₆represents isoleucine; X₁₇represents serine; and X₁₈represents glycine (i.e. an enzyme of amino acid sequence SEQ ID NO: 256);

(xvi) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents leucine; X₈represents isoleucine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents aspartic acid; X₁₅represents glycine; X₁₆represents isoleucine; X₁₇represents serine; and X₁₈represents glycine (i.e. an enzyme of amino acid sequence SEQ ID NO: 257);

(xvii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents leucine; X₈represents isoleucine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents aspartic acid; X₁₅represents glycine; X₁₆represents isoleucine; X₁₇represents serine; and X₁₈represents glycine (i.e. an enzyme of amino acid sequence SEQ ID NO: 258);

(xviii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents tryptophan; X₅represents cysteine; X₆represents leucine; X₇represents alanine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents arginine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 259);

(xix) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents arginine; X₅represents cysteine; X₆represents valine; X₇represents alanine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents arginine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 260);

(xx) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents tryptophan; X₅represents cysteine; X₆represents leucine; X₇represents asparagine; X₈represents isoleucine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents aspartic acid; X₁₅represents glycine; X₁₆represents isoleucine; X₁₇represents serine; and X₁₈represents glycine (i.e. an enzyme of amino acid sequence SEQ ID NO: 261);

(xxi) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents phenylalanine; X₅represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents threonine; X₁₀represents valine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 262);

(xxii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents alanine; X₅represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 263);

(xxiii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents leucine; X₅represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 264);

(xxiv) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents arginine; X₅represents cysteine; X₆represents valine; X₇represents alanine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents valine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 265);

(xxv) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents arginine; X₅represents cysteine; X₆represents valine; X₇represents glycine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents valine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 266);

(xxvi) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents tryptophan; X₅represents cysteine; X₆represents serine; X₇represents asparagine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents valine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 267);

(xxvii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents tryptophan; X₅represents cysteine; X₆represents leucine; X₇represents glycine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents arginine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 268);

(xxviii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents arginine; X₅represents cysteine; X₆represents leucine; X₇represents glycine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents arginine; X₁₂represents glutamine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 269);

(xxix) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents tryptophan; X₅represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 270);

(xxx) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents arginine; X₅represents cysteine; X₆represents leucine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents asparagine; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents isoleucine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 271);

(xxxi) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents arginine; X₅represents cysteine; X₆represents valine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents valine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 272); and

(xxxii) an enzyme of sequence SEQ ID NO: 274 wherein X₁represents arginine; X₂represents leucine; X₃represents threonine; X₄represents tryptophan; X₅represents cysteine; X₆represents serine; X₇represents threonine; X₈represents threonine; X₉represents serine; X₁₀represents alanine; X₁₁represents histidine; X₁₂represents arginine; X₁₃represents aspartic acid; X₁₄represents asparagine; X₁₅represents glutamic acid; X₁₆represents valine; X₁₇represents phenylalanine; and X₁₈represents glutamic acid (i.e. an enzyme of amino acid sequence SEQ ID NO: 273).

3-HP and Derivatives Thereof

3-HP derivatives include, but are not limited to acrylic acid, 1-propanol, propene, acrylyl-CoA, and polypropylene.

Acetyl-CoA and Derivatives Thereof

Acetyl-CoA derivatives include, but are not limited to acetone, 2-propanol, propene, and polypropylene.

Co-Production of 3-HP and Acetyl-CoA, Salts Thereof, and Derivatives Thereof

The combination of the 3-HP pathway with the acetyl-CoA pathway is adopted here to create a redox balanced pathway resulting in high yield. Between 3-HP derivatives, is possible to cite acrylyc acid, 1-propanol, propene, and polypropylene. Between acetyl-CoA derivatives, is possible to cite acetone, 2-propanol, propene, and polypropylene.

Looking for a solution for the loss of yield for production of 3-HP and acetyl-CoA derivatives, we identify a novel pathway combination of 3-HP derivative production with acetyl-CoA derivative production in a recombinant yeast. 1-propanol biosynthesis from glucose is highly dependent on the availability of reducing power cofactors for the conversion of malonate semialdehyde into 1-propanol: 4 NAD(P)H cofactors are required to convert 1 molecule of malonate semialdehyde into 1 molecule of 1-propanol. Although the biosynthesis of malonate semialdehyde from glucose generates some NAD(P)H cofactors, there is not enough NAD(P)H being formed though to sustain such malonate semialdehyde conversion into 1-propanol. The remaining NAD(P)H cofactors required for the biosynthesis of 1-propanol itself should be provided burning some glucose under aerobic conditions, but reducing so its overall yield potential. The current invention overcomes such redox unbalance and yield potential restriction by combining the biosynthesis of 1-propanol with the biosynthesis of acetone. As acetone is a highly oxidized molecule, its biosynthesis from glucose is linked to the net production of reducing power NAD(P)H cofactors. So, the co-production of both 1-propanol and acetone is redox balanced under certain carbon flow ratio to the target products, since the NAD(P)H required for 1-propanol biosynthesis would come from both glycolysis and acetone biosynthesis.

As described on FIG. 15 and FIG. 16, the co-production of 1-propanol and acetone is redox balanced under both aerobic and anaerobic fermentation conditions, with a yield potential increase under anaerobic condition. The stoichiometry of 1-propanol and acetone co-production pathway under aerobic fermentation condition is: 1 Glucose+0.33 02→0.67 acetone+0.67 1-propanol+1.33 H2O+2 CO2 with a maximum theoretical yield of 0.437 g/g products per glucose. As shown on FIG. 15, 6 molecules of malonate semialdehyde is produced from 3 molecules of glucose, wherein 4 molecules of malonate semialdehyde goes to the acetone biosynthesis and 2 molecules goes to the 1-propanol biosynthesis adjusting the NAD(P)H cofactors net to a neutral redox balance.

In contrast, the stoichiometry of 1-propanol and acetone co-production pathway under anaerobic fermentation condition is: 1 Glucose→0.6 acetone+0.8 1-propanol+0.8 H2O+1.8 CO2 with a maximum theoretical yield of 0.46 g/g products per glucose. As shown on FIG. 16, 10 molecules of malonate semialdehyde is produced from 5 molecules of glucose, wherein 6 molecules of malonate semialdehyde goes to acetone biosynthesis and 4 goes to 1-propanol biosynthesis in order to have a neutral redox balance. An example of redox balanced 1-propanol and acetone co-production pathway is shown in the FIG. 14 wherein cofactors NADH and NADPH are perfectly equilibrated: the 6 NADH cofactors generated in the upper glycolysis and acetone biosynthesis are consumed in the 1-propanol biosynthesis, and the 2 NADPH cofactors generated in the upper glycolysis are used in the 1-propanol biosynthesis, considering 2.5 molecules of glucose consumed for the production of 2 molecules of 1-propanol and 1.5 molecules of acetone. This can be achieved by using specific enzymes described and listed in the current invention. More precisely, a glyceraldehyde 3-phosphate dehydrogenase enzyme candidate (GAPDH) from Kluyveromyces lactis can be used, which show high activity either with NAD+ and NADP+ (and so generating NADH and NADPH), instead of using the native genes from Saccharomyces cerevisiae (TDH1, TDH2 and TDH3), which display a strict requirement for NAD+(generating NADH only). Besides, malonate semialdehyde dehydrogenase enzyme candidates from Pseudomonas aeruginosa or Candida albicans can convert malonate semialdehyde into acetyl-CoA displaying a strict requirement for NAD+ as cofactor. A highly active NADH-dependent 3-hydroxypropionic acid dehydrogenase enzyme from Candida albicans (encoded by the gene HPD1) can be used to convert malonate semialdehyde into 3-HP. Finally, the conversion of 3-HP into propionyl-CoA shall be done by using NADPH-dependent enzymes, while 1-propanol production from propionyl-CoA can be performed by NADH-dependent dehydrogenase enzymes. This co-production pathway is redox neutral and with a small excess of ATP, resulting in a more efficient and higher yield production of the desired compounds. Furthermore, the balanced pathway has the potential to be performed under anaerobic conditions, which brings several process advantages when compared with an aerobic process with the same yield. Like for example no air needs to be supplied to the production fermenters, so there is a CAPEX reduction with air compressors and an OPEX reduction with the utilities consumed by these equipment. Another significant advantage is that anaerobic fermenters can have larger maximum sizes than aerobic fermenters, mainly because in aerobic fermenters the oxygen transfer from gas to liquid phase gets more difficult as the size of the fermenter increase, limiting its size to a maximum value, so for the same annual production anaerobic process can have a smaller number of larger fermenters than an aerobic process, which at the end represents a smaller CAPEX with fermenters and its accessories for the anaerobic process. Besides that, there is an improvement of yield in anaerobic conditions, where co-production of 1-propanol and acetone leads to a higher total yield of 0.46 g of solvents/g of glucose, assuming these products are produced in a 3:4 ratio (acetone:1-propanol).

Under anaerobic conditions, the proposed pathway, which includes acetyl-CoA generation from malonate semialdehyde, avoids some of the biggest pathway engineering challenges for acetyl-CoA derivative production, which is acetyl-CoA availability in anaerobic conditions.

Furthermore, the new route for acetyl-CoA production is independent of pyruvate, which is distinct from the typical acetyl-CoA production pathways. In addition, the pathway for 3-HP derivatives is also distinct from the typical 3-HP production pathways, being independent of malonyl-CoA.

In some aspects, the recombinant microorganism is capable of producing acetone from malonate semialdehyde via acetyl-CoA. The proposed acetone pathway relies on the conversion of malonate semialdehyde to acetyl-CoA that is independent of pyruvate, and differs from the typical acetyl-CoA production pathways, mitigating the yield loss by pyruvate deviation to the TCA cycle. Also, malonate semialdehyde is produced by the decarboxylation of oxaloacetate or by the β-alanine pathway, which differs from the naturally malonate semialdehyde produced from malonyl-CoA in yeast. The natural production of malonyl-CoA and its salts is in competition with the ethanol biosynthesis, thus rendering difficult the flux derivation to malonate semialdehyde.

In some aspects, the native glyceraldehyde-3-phosphate dehydrogenase of the microbe is replaced by an exogenous version. The native yeast glyceraldehyde-3-phosphate dehydrogenase catalyzes glyceraldehyde-3-phosphate conversion to 1,3-bisphosphoglycerate resulting in the oxidation of an aldehyde with conversion of NAD⁺ to NADH. Other versions of this enzyme are described in literature where the enzyme activity results in conversion of NADP⁺ to NADPH. See Martinez et al. (2008. Metabolic Engineering, 10(6):352-359). Replacement of the native NAD-dependent enzyme with a NADP-dependent enzyme provides a better balanced pathway favoring production of desired compounds. In some aspects, one or more non-native glyceraldehyde-3-phosphate dehydrogenases replace the native enzyme. In some aspects, the glyceraldehyde-3-phosphate dehydrogenase is selected from a fungi, yeast, bacterium, insect, animal, plant, or flagellate. In some aspects, the glyceraldehyde-3-phosphate dehydrogenase that can be used has, but is not restricted to, EC number 1.2.1.12, such as from Clostridium acetobutylicum (gapC) or Kluyveromyces lactis (gdp1).

The novel combined pathway for 3-HP and acetyl-CoA production has malonate semialdehyde as a common single intermediate. The usual pathway for malonate semialdehyde production has malonyl-CoA as intermediate (Suyama et al., 2017). Otherwise, in the instant pathway, malonate semialdehyde has oxaloacetate as intermediate. See FIG. 1.

The aforementioned issues relating to the traditional pathways of the producing the products, as described above, depress the availability of intermediates and result in enzymes with low activity. As indicated in FIG. 1, the traditional route to malonyl-CoA is from acetyl-CoA, but a considerable portion of the acetyl-CoA is directed towards the TCA cycle. Thus a limited amount of malonyl-CoA is formed (0.01-0.023 nmolmg-1 cell dry weight, which is only 0.5% of the total CoA pool) (Rathnasingh et al., 2012). In addition, malonyl-CoA is an important intermediate in fatty acid metabolism, and it is difficult to redirect this compound into the pathway for the products described herein. Furthermore, the conversion of malonyl-CoA into malonate semialdehyde is performed by a reductase with low activity at 37° C. (maximum activity at 50° C., decrease of 65% at 37° C.) and high Km value for malonyl-CoA (30 μM) (Rathnasingh et al., 2012). The instant pathway is able to clear these issues by utilizing oxaloacetate as an intermediate for malonate semialdehyde production.

As depicted in FIG. 2, yeast cells have two routes for oxaloacetate generation; through pyruvate carboxylase (pyc gene) or malate dehydrogenase (mdh gene) activity. As the pyruvate carboxylase is a biotin-dependent enzyme with low catalytic activity, its catalytic turnover is not sufficient to provide high levels of oxaloacetate. Furthermore, oxaloacetate generated by the TCA cycle was found not to be useful to fuel high carbon fluxes. The instant pathway is able to clear these issues by producing oxaloacetate from phosphoenol pyruvate (PEP) instead of from pyruvate. To achieve a high concentration of oxaloacetate, the bacterial enzyme phosphoenol pyruvate carboxylase and/or phosphoenol pyruvate carboxykinase is utilized in the recombinant microbe. In some aspects, the microbe of interest is modified by introduction of one or more phosphoenol pyruvate carboxylase (ppc) and/or one or more phosphoenol pyruvate carboxykinase (pepck). In some aspects, the one or more phosphoenol pyruvate carboxylase (ppc) and/or one or more phosphoenol pyruvate carboxykinase (pepck) are selected from a fungi, yeast, bacterium, insect, animal, plant, or flagellate. In some aspects, the one or more phosphoenol pyruvate carboxylase (ppc) and/or one or more phosphoenol pyruvate carboxykinase (pepck) are from E. coli.

Once oxaloacetate is formed, the molecule can be converted to malonate semialdehyde directly from oxaloacetate or through the intermediary formation of β-alanine, as depicted in FIG. 3.

In some aspects, the malonate semialdehyde directed production from oxaloacetate is performed by a decarboxylase, such as an alpha-ketoisovalerate decarboxylase, benzoylformate decarboxylase, or a 2-oxoglutarate decarboxylase. In some aspects, the recombinant microbe comprises one or more alpha-ketoisovalerate decarboxylase (kdca), benzoylformate decarboxylase (mdlc), and/or 2-oxoglutarate decarboxylase (oxdc). In some aspects, the one or more alpha-ketoisovalerate decarboxylase, benzoylformate decarboxylase, and/or 2-oxoglutarate decarboxylase are selected from a fungi, yeast, bacterium, insect, animal, plant, or flagellate. In some aspects, the alpha-ketoisovalerate decarboxylase is from Lactococcus lactis. In some aspects, the benzoylformate decarboxylase is from Pseudomonas putida. In some aspects, the 2-oxoglutarate decarboxylase is from Oenococcus oeni or Euglena gracilis.

The malonate semialdehyde production through β-alanine involved oxaloacetate transamination to aspartate by an aspartate amino transferase (aat2), aspartate conversion to β-alanine by an aspartate decarboxylase (pand) and β-alanine conversion to malonate semialdehyde by a β-alanine pyruvate amino transferase (baat) and/or β-alanine transaminase (pyd4). In some aspects, the recombinant microbe comprises one or more aspartate amino transferase, aspartate decarboxylase, β-alanine pyruvate amino transferase, and/or β-alanine transaminase. In some aspects, the aspartate amino transferase, aspartate decarboxylase, β-alanine pyruvate amino transferase, and/or β-alanine transaminase are selected from a fungi, yeast, bacterium, insect, animal, plant, or flagellate. In some aspects, the aspartate amino transferase is from S. cerevisiae. In some aspects, the aspartate decarboxylase is from Tribolium castaneum. In some aspects, the aspartate decarboxylase is from Corynebacterium glutamicum. In some aspects, the beta-alanine pyruvate amino transferase is from Bacillus cereus. In some aspects, the beta-alanine transaminase is from Lachancea kluyveri (Table 16).

Malonate semialdehyde can be converted to 3-HP by a 3-hydroxypropionic acid dehydrogenase. In some aspects, the recombinant microbe comprises one or more 3-hydroxypropionic acid dehydrogenases that can be, but are not restricted to, enzymes with EC number 1.1.1.381 such as those listed in Table 1. In some aspects, the 3-hydroxypropionic acid dehydrogenase is from a fungi, yeast, bacterium, insect, animal, plant, or flagellate. In some aspects, the 3-hydroxypropionic acid dehydrogenase (mcr-1) is from Chloroflexus aurantiacus. In some aspects, the 3-hydroxypropionic acid dehydrogenase (adh) is from Arthrobacter enclensis. In some aspects, the 3-hydroxypropionic acid dehydrogenase (mmsb) is from Bacillus cereus. In some aspects, the 3-hydroxypropionic acid dehydrogenase (ydfg-0) is from E. coli. In some aspects, the 3-hydroxypropionic acid dehydrogenase (YDF1) is from Saccharomyces cerevisiae. In some aspects, the 3-hydroxypropionic acid dehydrogenase is preferentially (HPD1) from Candida albicans,

TABLE 1

Enzymes candidates for conversion

to 3-HP from malonate semialdehyde.

EC
SEQ

Enzymes
Origin
UniProt number
Number
ID NO

MCR-
Chloroflexus
YP_001636209.1
—
105

Nterm.Cau
aurantiacus
(NCBI ref seq)

ADH.Ae
Arthrobacter
WP_058267460.1
—
106

enclensis
(NCBI ref seq)

MMSB.Bce
Bacillus
NP_833760.1
—
107

cereus
(NCBI ref seq)

YDFG-0.Ec
Escherichia
BAA15241.1
—
108

coli
(seq nucleotídeo)

YMR226C
Saccharomyces
Q05016
1.1.1.381
109

(YDF1)
cerevisiae

HPD1
Candida
A0A1D8PQ07

110

albicans

Malonate semialdehyde can be converted to acetyl-CoA by a one-step reaction using a malonate semialdehyde dehydrogenase or by a two-step reaction through malonyl-CoA including a malonyl-CoA synthetase or a malonyl-CoA reductase and a malonyl-CoA decarboxylase. In some aspects, a plurality or majority of the malonate semialdehyde is produced from oxaloacetate. In some aspects, a plurality or majority of the malonate semialdehyde is not produced from malonyl-CoA. The enzymes candidates to this step can be, but are not restricted to, enzymes with EC number 1.2.1.18/1.2.1.27 such as those listed in Table 2.

TABLE 2

Enzymes candidates for conversion to

acetyl-CoA from malonate semialdehyde

UniProt
EC
SEQ

Genes
Origin
number
Number
ID NO

MSD.Pa
Pseudomonas
Q9I702
1.2.1.18
111

aeruginosa

MSD.Cal
Candida
A0A1D8PM94
1.2.1.18
112

albicans

iolA
Listeria
Q8Y9Y4
1.2.1.27
113

monocytogenes

iolA
Bacillus
A7BJC4
1.2.1.27
114

subtilis

iolA
Cupriavidus
F8GGV48
1.2.1.18
115

necator

mmsA
Bacillus
A0A2C1ZL92
1.2.1.27
116

cereus

dddC
Halomonas
C8YX90
—
117

sp. HTNK1

iolA
Lactobacillus
A5YBJ3
1.2.1.27
118

casei

Alternatively, another option to produce acetyl-CoA is a two-step pathway from oxaloacetate and through malonyl-CoA including a 2-keto acid decarboxylase and a malonyl-CoA decarboxylase. See FIG. 4 None of the routes presented utilizes pyruvate as an intermediate, being different from usual pathways and an interesting solution for acetyl-CoA generation in anaerobic conditions, where the availability of cytosolic acetyl-CoA is compromised.

In some aspects, the recombinant microbe comprises one or more malonate semialdehyde dehydrogenase, malonyl-CoA synthetase, malonyl-CoA reductase, malonyl-CoA decarboxylase, and/or 2-keto acid decarboxylase. In some aspects, one or more malonate semialdehyde dehydrogenase, malonyl-CoA synthetase, malonyl-CoA reductase, malonyl-CoA decarboxylase, and/or 2-keto acid decarboxylase are from a fungi, yeast, bacterium, insect, animal, plant, or flagellate. In some aspects, the malonate semialdehyde dehydrogenase that can be used has, but is not restricted to, EC number 1.2.1.18/1.2.1.27 such as those listed in Table 2, preferentially MSD from Candida albicans or Pseudomonas aeruginosa.

In some aspects, the malonyl-CoA synthetase (MatB) is from Rhizobium trifohi. In some aspects, the malonyl-CoA synthetase (AAE13) is from Arabidopsis thaliana. In some aspects, the malonyl-CoA synthetase (ACSF3B) is from Homo sapiens. In some aspects, the malonyl-CoA reductase (mcr) is from Chloroflexus aurantiacus.

In some aspects, the malonyl-CoA decarboxylase (MatA) is from Rhizobium trifohi. In some aspects, the malonyl-CoA decarboxylase (MLYCD) is from Homo sapiens. In some aspects, the 2-keto acid decarboxylase (kivd) is from Lactococcus lactis. In some aspects, the 2-keto acid decarboxylase (KdcA) is from Lactococcus lactis. In some aspects, the 2-keto acid decarboxylase (ARO10) is from Saccharomyces cerevisiae.

From 3-HP, several compound can be produced, including 1-propanol as an example. For 1-propanol production, 3-HP needs to be converted to propionyl-CoA by a single-step reaction performed by a propionyl-CoA synthase or by multi-step reactions involving a 3-hydroxypropionyl-CoA synthetase, 3-hydroxypropionyl-CoA dehydratase, and acrylyl-CoA reductase. Then propionyl-CoA needs to be converted to 1-propanol by a bifunctional alcohol/aldehyde dehydrogenase (EC number 1.1.1.1/1.2.1.4/1.2.1.5 such as listed in Table 7) or by an aldehyde dehydrogenase (acetylating) (table 8) and an alcohol dehydrogenase (EC number 1.2.1.10/1.2.1.87 such as listed in Table 9). As depicted in FIG. 5, 1-propanol production has a high demand for NAD(P)H, with there being no balanced pathway.

In some aspects, the recombinant microbe comprises one or more propionyl-CoA synthase, 3-hydroxypropionyl-CoA synthetase, 3-hydroxypropionyl-CoA dehydratase, acrylyl-CoA reductase, alcohol/aldehyde dehydrogenase, alcohol dehydrogenase and/or aldehyde dehydrogenase from a fungi, yeast, bacterium, insect, animal, plant, or flagellate. In some aspects, the one or more propionyl-CoA synthase that can be used are enzymes with, but not limited to, EC number 6.2.1.17/6.2.1.36 such as those listed in Table 3. In some aspects, the 3-hydroxypropionyl-CoA synthetase/transferase that can be used, but is not restricted to, is an enzyme with EC number 2.8.3.1/6.2.1.17/6.2.1.36 such as those listed in Table 4. In some aspects, the 3-hydroxypropionyl-CoA dehydratase that can be used, but is not restricted to, is an enzyme with EC number 4.2.1.116/4.2.1.55/4.2.1.150/4.2.1.17 such as those listed in Table 5. In some aspects, the acrylyl-CoA reductase is from, but not limited to, the organisms and/or correspond to the genes indicated in Table 6. In some aspects, the alcohol/aldehyde dehydrogenase, that can be used, but is not restricted to, is an enzyme with EC number 1.1.1.1/1.2.1.4/1.2.1.5 such as those listed in Table 7. In some aspects, other candidates to convert propionyl-CoA to propanol are used (EC number: 1.2.1.10/1.2.1.87 such as listed in Table 8, EC number 2.3.1.8/2.7.2.1 such as listed in Table 9 and EC number 2.3.1.8/2.7.2.1 such as listed in Table 10).

TABLE 47

Enzymes candidates for the conversion of oxaloacetate

to acetyl-CoA trough malonyl-CoA

Uniprot
EC
SEQ

Genes
Origin
number
number
ID NO

AAE13
Arabidopsis
Q8H151
6.2.1.—
276

thaliana

matB
Rhizobium
—
—

trifolii

ACSF3
Homo sapiens
Q4G176
6.2.1.—
277

mcr
Chloroflexus
Q6QQP7
1.1.1.298
278

aurantiacus

mata
Rhizobium
—
—
—

trifolii

MLYCD
Homo sapiens
O95822
4.1.1.9
279

kivD
Lactococcus
D2BR82
—
280

lactis

kdcA
Lactococcus
Q6QBS4
—
281

lactis

ARO10
Saccharomyces
Q06408
—
282

cerevisiae

TABLE 3

Enzymes candidates for trifunctional propionyl-CoA synthase.

Uniprot
EC
SEQ

Genes
Origin
number
number
ID NO

PCS.Cau
Chloroflexus
Q8VRG6
6.2.1.17
119

aurantiacus

PCS.Cag
Chloroflexus
B8G4K4
6.2.1.17
120

aggregans

PCS.Rca
Roseiflexus
WP_012121416.1
6.2.1.17
121

castenholzii
(genebank)

PCS.No
Natronococcus
WP_015322855.1
6.2.1.17
122

occultus
(NCBI ref Seq)

PCS.Hj
Halioglobus
—
6.2.1.17
—

japonicus

NAP1_02725
Erythrobacter
A3WE14
6.2.1.36
123

sp NAP1

4.2.1.116

1.3.1.84

AP017312.1
Aneurinibacillus

—

soli

CP0022600.1
Porphyrobacter

—

HT-58-2

CP017057.1
Erythrobacter

—

litoralis

CP020083.1
Blasromonas

—

fulva

CP003929.1
Natronococcus

—

occultus

CP007793.1
Azospirillum

—

brasilense

CP019450.1
Halioglobus

—

japonicus

TABLE 4

Enzymes candidates for conversion to 3-hydroxypropionyl-CoA from

3-HP (3-hydroxypropionyl-CoA synthetase/CoA transferase).

Uniprot
EC
SEQ

Genes
Organism
number
number
ID NO

PCT-0.Cne (pct)
Cupriavidus
Q0K874
2.8.3.1
124

necator

PCT-0.Cp (pct)
Clostridium
Q9L3F7
2.8.3.1
125

propionicum

PCT or YdiF
Megasphaera
G0VND6
2.8.3.1
126

elsdenii

PrpE
Salmonella
P55912
6.2.1.17
127

enterica

Nmar_1309
Nitrosopumilus
A9A2G6
—
128

maritimus

Msed_1456
Metallosphaera
A4YGR1
6.2.1.36
129

sedula

PrpE
Escherichia
P77495
6.2.1.17
130

coli

YdiF
Cupriavidus
Q0K874
2.8.3.1
131

necator

STK_07830
Sulfolobus
Q973W5
6.2.1.36
132

tokodaii

TABLE 5

Enzymes candidates for conversion to acrylyl-CoA from 3-Hydroxypropionyl-

CoA (3-hydroxypropionyl-coA dehydratase/enoyl-CoA hydratase)

Uniprot
EC
SEQ

Genes
Organism
number
number
ID NO

HPCD.Mse
Metallosphaera
A4YI89
4.2.1.116
133

sedula

HPDC.Bsp
Bacillus sp.
A0A1B9ACR9
—
134

HPCD.Sac
Sporanaerobacter
A0A1M5Y529
—
135

acetigenes

ENCD.Rp
Ruegeria
Q5LMB7
—
136

pomeroyi

3HPCD
Sulfolobus
F9VNG3
4.2.1.116
137

tokodaii

Nmar_1308
Nitrosopumilus
A9A2G5
4.2.1.116
138

maritimus

Hpcd
Chloroflexus
—
4.2.1.116
—

aurantiacus

crt
Clostridium
P52046
4.2.1.55/
139

acetobutylicum

4.2.1.150

3-hydrobutyryl-
Clostridium
L7EP14
4.2.1.55
140

coa dehydratase
pasteuranum

crt
Clostridium
P81357
4.2.1.150
141

pasteuranum

MELS_1449
Megasphaera
G0VQE2
4.2.1.55
142

elsdenii

Aflv_0566
Anoxybacillus
B7GGZ2
4.2.1.17
143

flavithermus

TABLE 6

Enzymes candidates for conversion to Propionyl-

CoA from Acrylyl-CoA (acrylyl-CoA reductase).

Uniprot
EC
SEQ

Genes
Organism
number
number
ID NO

ACR-0.Rp
Ruegeria
Q5LS56
1.3.1.84
144

pomeroyi

ACR-0.Ec
Escherichia
P26646
1.3.1.84
145

coli

ACR-0.Rs
Rhodobacter
Q3J6K9
1.3.1.84
146

sphaeroides

acuI
Ruegeria
Q5LS56
1.3.1.84
147

pomeroyi

acuI
Rhodobacter
Q3J6K9
1.3.1.84
148

sphaeroids

β-ETF,
Clostridium
G3KIM6,
1.3.1.95
149

α-ETF and
propionicum
G3KIM7 and

Propionyl-

G3KIM8

coA dehydro-

genase

acuI
Alcaligenes
A0A3G6HCN9
1.3.1.95
150

faecalis

ACR
Sulfurisphaera
Q975C8
1.3.1.84
151

tokodaii

AcuI
Escherichia
P26646
1.3.1.84
152

coli

Acr
Metallosphaera
A4YGN2
1.3.1.84
153

sedula

Nmar_1565
Nitrosopumilus
A9A5Y3
—
154

maritimus

TABLE 7

Enzymes candidates for conversion to Propanol from

Propionyl-CoA (alcohol/aldehyde dehydrogenase)

UniProt
EC
SEQ

Genes
Origin
number
number
ID NO

ADHE.Ca
Clostridium
Q9ANR5
—
155

acetobutylicum

ADHE.Cbe
Clostridium
A0A1S8PSK3
—
156

beijerinckii

ADHE.St
Salmonella
P74880
—
157

typhimurium

ADHE.Car
Clostridium
WP_010241373.1
—
158

arbusti
(NCBI Ref Seq)

adhE
Escherichia
P0A9Q7
1.1.1.1
159

coli

adhP
Escherichia
P39451
1.1.1.1
160

coli

bdhB
Clostridium
Q04945
1.1.1.1
161

acetobutylicum

ADH2
Saccharomyces
P00331
1.1.1.1
162

cerevisiae

adhE
Clostridium
—

—

roseum

adhA
Thermoanaerobacterium
I3VX46
—
163

saccharolyticum

ald6
Saccharomyces
P54115
1.2.1.4
164

cerevisiae

aldh3A1
Homo sapiens
P30838
1.2.1.5
165

TABLE 8

Enzymes candidates for conversion of propionyl-CoA to

propionaldehyde (aldehyde dehydrogenase (acetylating))

EC
SEQ

Genes
Origin
UniProt number
number
ID NO

MHPF.Ec
Escherichia
WP_097337838.1
1.2.1.10
166

coli
(NCBI ref seq)

MHPF.Ppu
Pseudomonas
YP_003617188.1
1.2.1.10
167

putida
(NCBI ref seq)

MHPF.Pse
Pseudomonas sp.
CRM99844.1
1.2.1.10
168

(NCBI ref Seq)

MHPF.Pf
Pseudomonas
BAA09693.1
1.2.1.10
169

fluorescens
(gene Bank)

MHPF.Px
Paraburkholderia
Q79AF6
1.2.1.10
170

xenovorans

PDUP.Sen
Salmonella
AAD39015.1
—
171

enterica
(H9L4I6)

PDUP1.Lmo
listeria
NP 464690.1
—
172

monocytogenes
(Q8Y7V4)

PDUP2.Lmo
listeria
A0A0E0UUX4
—
173

monocytogenes

PDUP.Kp
Klebsiella
YP_001336844.1
—
174

pneumoniae

aldH
Acinetobacter sp.
A5JT11
1.2.1.10
175

Ald
Clostridium
Q9X681
1.2.1.10
176

beijerinckii

Cphy1178
Clostridium
A9KN57
—
177

phytofermentans

tesF
Comamonas
Q83VZ4
1.2.1.10
178

testosteroni

BN476_01309
Eubacterium
R6G856
—
179

hallii

PduP
Lactobacillus
Q845A7
—
180

collinoides

EutE
Lactobacillus
A0A1B7LQN5
—
181

reuteri

HsaG
Mycobacterium
A0A2I7WCV7
1.2.1.10
182

tuberculosis

MhpF
Mycobacterium
P9WQH3
1.2.1.10
183

tuberculosis

bphJ
Paraburkholderia
Q79AF6
1.2.1.10
184

xenovorans

1.2.1.87

SAMN04244550_00489
Rhodobacter
A0A1G7D6K3
—
185

capsulatus

hsaG
Rhodococcus
Q0S816
1.2.1.10
186

jostii

RPC_1174
Rhodopseudomonas
Q21A49
—
187

palustris

TTHB247
Thermus
Q53WH9
1.2.1.10
188

thermophilus

1.2.1.87

TABLE 9

Enzymes candidates for conversion

to propanol from propionaldehyde

UniProt
EC
SEQ

Genes
Origin
number
number
ID NO

alrA
Acinetobacter sp
Q9F1R1
1.1.1.2
189

Aldehyde
Acinetobacter sp.
Q9F1Q8
1.1.1.2
190

reductase

bdhI
Clostridium
Q04944
1.1.1.—
191

acetobutylicum

bdhII
Clostridium
Q04945
1.1.1.—
192

acetobutylicum

adhA
Corynebacterium
A0A169S5A7
1.1.1.1
193

glutamicum
Q8NLX9

194

yqhD
Escherichia
Q46856
1.1.1.2
195

coli

yjgB
Escherichia
P27250
1.1.1.2
196

coli

adhP
Escherichia
P39451
1.1.1.1
197

coli

PduQ
Propionibacterium
A0A2C8A2U1
—
198

freudenreichii

ADH1
Saccharomyces
P00330
1.1.1.1
199

cerevisiae

ADH2
Saccharomyces
P00331
1.1.1.1
200

cerevisiae

ADH4
Saccharomyces
P10127
1.1.1.1
201

cerevisiae

ADH6
Saccharomyces
Q04894
1.1.1.2
202

cerevisiae

PduQ
Salmonella
H9L4H8
—
203

(adhE_2)
enterica

Adh
Sulfolobus
Q96XE0
1.1.1.1
204

tokodaii

adhA
Synechocystis
P74721
—
205

sp

adhA
Zymommonas
P20368
1.1.1.1
206

mobilis

TABLE 10

Enzymes candidates for conversion to

propionaldehyde from propionyl-CoA

UniProt
EC
SEQ

Genes
Origin
number
number
ID NO

PTA.Cg
Corynebacterium
WP_011015350
2.3.1.8
207

glutamicum
(NCBI ref seq)

ACKA.Cg
Corynebacterium
P77845
2.7.2.1
208

glutamicum

From acetyl-CoA, several compound can be produced, being possible to cite acetone as an example. For acetone production, acetyl-CoA needs to be converted to acetoacetyl-CoA by a thiolase or an acetyl-CoA acetyltransferase. Alternatively, acetoacetyl-CoA can be formed through malonyl-CoA by acetoacetyl-CoA synthase. Once acetoacetyl-CoA is formed, its conversion to acetoacetate can be done by an acetoacetyl-CoA transferase or through HMG-CoA by hydroxymethylglutaryl-CoA synthase and hydroxymethylglutaryl-CoA lyase. Acetoacetate conversion to acetone is done by an acetoacetate decarboxylase. As depicted in FIG. 6, acetone production has an excess of NAD(P)H, being also a no balanced pathway.

In some aspects, the recombinant microbe comprises one or more thiolase, acetyl-CoA acetyltransferase, acetoacetyl CoA synthase, acetoacetyl-CoA transferase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA lyase, and/or acetoacetate decarboxylase from a fungi, yeast, bacterium, insect, animal, plant, or flagellate. In some aspects, the one or more thiolase or acetyl-CoA acetyltransferase that can be used has, but is not restricted to, EC number 2.3.1.9 such as those listed in Table 11 and acetoacetyl-CoA transferase that can be used has, but is not restricted to, EC number 2.8.3.8/2.8.3.9 such as those listed in Table 12, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA lyase, and/or acetoacetate decarboxylase that can be used has, but is not restricted to, an enzyme with EC number 4.1.1.4 such as those listed in Table 13.

TABLE 11

Enzymes candidates for conversion to acetoacetyl-

CoA from acetyl-CoA (acetyltransferase)

UniProt
EC
SEQ

Genes
Origin
number
number
ID NO

ERG10
Saccharomyces
P41338
2.3.1.9
209

cerevisiae

thlA
Clostridium
P45359
2.3.1.9
210

acetobutylicum

atoB
Escherichia
P76461
2.3.1.9
211

coli

H16_B0759
Cupriviadus
Q0K368
2.3.1.9
212

necator

Msed_0656
Metallosphaera
A4YEH9
2.3.1.9
213

sedula

AAT1
Arabidopsis
Q8S4Y1
2.3.1.9
214

thaliana

TABLE 12

Enzymes candidates for conversion to acetoacetate from

acetoacetyl-CoA (acetoacetyl-CoA transferase/synthase)

UniProt
EC
SEQ

Genes
Origin
number
number
ID NO

ATOA.Ec/
Escherichia
P76459
2.8.3.8
215

ATOD.Ec
coli
P76458

216

C7401_123119
Paraburkholderia
A0A328X8N5
2.8.3.8
217

unamae

YdiF
Escherichia
Q8X5X6
2.8.3.8
218

coli

ctfA.Ca/ctfB
Clostridium
P33752
2.8.3.9
219

acetobutylicum
P23673

220

ctfA./ctfB
Clostridium
U5MXH7
2.8.3.9
221

saccharobutylicum
U5MUQ0

222

ctfA/ctfB
Escherichia
A0A2X1MWL8
2.8.3.9
223

coli
A0A0K5TTP4

224

ERG13
Saccharomyces
P54839
2.3.3.10
283

cerevisiae

yngG
Bacillus
O34873
4.1.3.4
284

subtilis

nphT7
Streptomyces sp.
D7URV0
2.3.1.194
285

TABLE 13

Enzymes candidates for conversion to acetone from

acetoacetate (acetoacetate decarboxylase)

UniProt
EC
SEQ

Genes
Origin
number
number
ID NO

Adc.Ca
Clostridium
P23670
4.1.1.4
225

acetobutylicum

Adc.Cbe
Clostridium
A6M020
4.1.1.4
226

beijerinckii

Adc
Clostridium
P23650
4.1.1.4
227

pasteurianum

Adc
Pseudomonas
Q8XR10
4.1.1.4
228

solanacearum

Adc
Paraburkholderia
Q141C9
4.1.1.4
229

xenovorans

Adc.Pp
Paenibacillus
A0A378XWA9
—
230

polymyxa

To solve high demand of NAD(P)H related to 3-HP derivatives production and excess of NADH related to acetyl-CoA derivatives production, we are suggesting the combination of both pathways to result in a balanced pathway. As depicted in FIG. 7, combination of these pathways for 1-propanol and acetone, for example, leads to a high total yield of 0.437 g of solvents/g of glucose, assuming these products are produced in a 1:1 ratio (acetone:1-propanol) in aerobic conditions (FIG. 15). The proposed co-production pathway is redox neutral and with a small excess of ATP, results in a more efficient and high yield production of the desired compounds. Furthermore, balanced pathway has potential to be performed in an anaerobic condition, which may represent a potential for a lower production cost process, due to advantages already mentioned in paragraph [0264], like CAPEX and OPEX reduction with air compressors, and CAPEX decrease with production fermenters. Besides that, there is an improvement of yield in anaerobic conditions, where co-production of 1-propanol and acetone leads to a higher total yield of 0.46 g of solvents/g of glucose, assuming these products are produced in a ratio 3:4 (acetone:1-propanol) (FIG. 16).

In some aspects, 2-propanol can be obtained by dehydrogenation of acetone and can be coproduced with 1-propanol. The NADPH/NADH requirements in the 3-HPA derivative pathway complements the NADPH/NADH excess from the acetyl-CoA derivative pathway. The combination of these pathways for the production of 1-propanol and 2-propanol, leads to a high total yield of 0.39 g of solvents/g of glucose, assuming these products are produced in a 2:5 ratio (1-propanol:2-propanol), in aerobic conditions, according to FIG. 17.

In some aspects, 2-propanol can be obtained by enzymatic dehydrogenation of acetone and can be coproduced with 1-propanol in anaerobic conditions, that leads to a higher total yield of 0.44 g of solvents/g of glucose, assuming these products are produced in a 1:1 ratio (1-propanol:2-propanol), in anaerobic conditions, according to FIG. 18. This process may represent a potential for a lower cost process, in relation to an aerobic process.

TABLE 14

Enzymes to convert acrylyl-CoA to acrylic

acid (acyl-CoA hydrolase or thioesterase)

UniProt
EC
SEQ

Genes
Origin
number
number
ID NO

yciA
Escherichia
P0A8Z0
—
231

coli

ACIAD3139
Escherichia
Q6F7Y5
—
232

coli

TABLE 15

Enzymes candidates to 2-propanol dehydrogenase

UniProt
EC

Genes
Origin
number
number

PRDH.Dr
Devosia
—
—

riboflavina

PRDH.Sp
Sporotrichum
—
—

pulverulentum

TABLE 16

Genes involved on malonate semialdehyde

production by p-alanine pathway

UniProt
EC
SEQ

Genes
Origin
number
number
ID NO

aat2
Saccharomyces
P23542
2.6.1.1
233

cerevisiae

PAND
Corynebacterium
A4QAD0
4.1.1.11
234

glutamicum

PAND.Tca
Tribolium
A7U8C7
4.1.1.11
275

castaneum

Baat
Saccharomyces
P47176
2.6.1.42
235

cerevisiae

PYD4.Lk
Lachancea
A5H0J5
2.6.1.19
236

kluyveri

TABLE 17

Genes of glycolysis

UniProt
EC

Genes
Origin
Number
number
SEQ ID

PEPCK
Escherichia
P22259
4.1.1.49
237

coli

GAPDH
Saccharomyces
P00359
1.2.1.13
238

cerevisiae

GDP1.K1
Kluyveromyces
Q8J0C9
1.2.1.13
239

lactis

PYK1
Saccharomyces
P00549
2.7.1.40
240

cerevisiae

gapC
Clostridium
Q97D25
1.2.1.13
241

acetobutylicum

Methods of Detecting Genetic Modification

The present disclosure teaches primers, probes, and assays that are useful for detecting the microbes taught herein. In some aspects, the disclosure provides for methods of detecting the WT parental strains. In other aspects, the disclosure provides for methods of detecting the engineered or modified microbes derived from parent strains or WT strains. In some aspects, the present disclosure provides methods of identifying genetic alterations in a microbe.

In some aspects, the genomic engineering methods of the present disclosure lead to the creation of non-natural nucleotide “junction” sequences in the modified microbes. These non-naturally occurring nucleotide junctions can be used as a type of diagnostic that is indicative of the presence of a particular genetic alteration in a microbe taught herein.

The present techniques are able to detect these non-naturally occurring nucleotide junctions via the utilization of specialized quantitative PCR methods, including uniquely designed primers and probes. In some aspects, the probes of the disclosure bind to the non-naturally occurring nucleotide junction sequences. In some aspects, traditional PCR is utilized. In other aspects, real-time PCR is utilized. In some aspects, quantitative PCR (qPCR) is utilized. In some aspects, the PCR methods are used to identify heterologous sequences that have been inserted into the genomic DNA or extra-genomic DNA of the microbes.

Thus, the disclosure can cover the utilization of two common methods for the detection of PCR products in real-time: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence. In some aspects, only the non-naturally occurring nucleotide junction will be amplified via the taught primers, and consequently can be detected via either a non-specific dye, or via the utilization of a specific hybridization probe. In other aspects, the primers of the disclosure are chosen such that the primers flank either side of a junction sequence, such that if an amplification reaction occurs, then said junction sequence is present.

Aspects of the disclosure involve non-naturally occurring nucleotide junction sequence molecules per se, along with other nucleotide molecules that are capable of binding to said non-naturally occurring nucleotide junction sequences under mild to stringent hybridization conditions. In some aspects, the nucleotide molecules that are capable of binding to said non-naturally occurring nucleotide junction sequences under mild to stringent hybridization conditions are termed “nucleotide probes.”

In some aspects, genomic DNA can be extracted from samples and used to quantify the presence of microbes of the disclosure by using qPCR. The primers utilized in the qPCR reaction can be primers designed by Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify unique regions of the wild-type genome or unique regions of the engineered non-intergeneric mutant strains. The qPCR reaction can be carried out using the SYBR GreenER qPCR SuperMix Universal (Thermo Fisher P/N 11762100) kit, using only forward and reverse amplification primers; alternatively, the Kapa Probe Force kit (Kapa Biosystems P/N KK4301) can be used with amplification primers and a TaqMan probe containing a FAM dye label at the 5′ end, an internal ZEN quencher, and a minor groove binder and fluorescent quencher at the 3′ end (Integrated DNA Technologies).

Quantitative polymerase chain reaction (qPCR) is a method of quantifying, in real time, the amplification of one or more nucleic acid sequences. The real time quantification of the PCR assay permits determination of the quantity of nucleic acids being generated by the PCR amplification steps by comparing the amplifying nucleic acids of interest and an appropriate control nucleic acid sequence, which may act as a calibration standard.

TaqMan probes are often utilized in qPCR assays that require an increased specificity for quantifying target nucleic acid sequences. TaqMan probes comprise an oligonucleotide probe with a fluorophore attached to the 5′ end and a quencher attached to the 3′ end of the probe. When the TaqMan probes remain as is with the 5′ and 3′ ends of the probe in close contact with each other, the quencher prevents fluorescent signal transmission from the fluorophore. TaqMan probes are designed to anneal within a nucleic acid region amplified by a specific set of primers. As the Taq polymerase extends the primer and synthesizes the nascent strand, the 5′ to 3′ exonuclease activity of the Taq polymerase degrades the probe that annealed to the template. This probe degradation releases the fluorophore, thus breaking the close proximity to the quencher and allowing fluorescence of the fluorophore. Fluorescence detected in the qPCR assay is directly proportional to the fluorophore released and the amount of DNA template present in the reaction.

The features of qPCR allow the practitioner to eliminate the labor-intensive post-amplification step of gel electrophoresis preparation, which is generally required for observation of the amplified products of traditional PCR assays. The benefits of qPCR over conventional PCR are considerable, and include increased speed, ease of use, reproducibility, and quantitative ability.

Microbes

As described herein, in some aspects, the recombinant microorganisms are prokaryotic microorganism. In some aspects, the prokaryotic microorganisms are bacteria. “Bacteria”, or “eubacteria”, refers to a domain of prokaryotic organisms. Bacteria include at least eleven distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic+non-photosynthetic Gram-negative bacteria (includes most “common” Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, and sporulating rods. The genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

In some aspects, the microorganisms of the present disclosure are fungi or yeasts.

In some aspects, the recombinant microorganism is a eukaryotic microorganism. In some aspects, the eukaryotic microorganism is a yeast. In exemplary aspects, the yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, and Myxozyma.

In some aspects, the recombinant microorganism is a prokaryotic microorganism. In exemplary aspects, the prokaryotic microorganism is a member of a genus selected from the group consisting of Escherichia, Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium.

In some aspects, microorganism for use in the methods of the present disclosure can be selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, Myxozyma, Escherichia, Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium.

In some aspects, a microbe resulting from the methods described herein may be a species selected from any of the following genera: Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, Fusobacterium, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, Streptomyces, Saccharomyces, Pichia, and Aspergillus.

In some aspects, microorganisms for use in the methods of the present disclosure include Clostridium sp., Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Candida krusei, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillus sp, Corynebacterium sp., Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacter glycolicus. In some aspects, the recombinant microorganisms are derived from a parental microorganism selected from any one of the microorganisms disclosed herein.

In some aspects, yeast can be cultivated more rapidly and at a higher density than bacteria, and does not require an aseptic environment in the commercial/industrial setting. In some aspects, yeast cells can be more easily separated from the culture medium than can bacteria, simplifying the process for product extraction and purification.

In some aspects, the microorganism(s) used in the methods of the present disclosure include yeasts selected from genus Saccharomyces, Candida, Ashbya, Dekkera, Pichia (Hansenula), Debaryomyces, Clavispora, Lodderomyces, Yarrowia, Zigosaccharomyces, Schizosaccharomyces, Torulaspora, Kluyveromyces, Brettanomycces, Cryptococcus, or Malassezia.

In some aspects, the yeast may be a Crabtree-positive yeast selected from genus Saccharomyces, Dekkera, Schizosaccharomyces, Kluyveromyces, Torulaspora Zigosaccharomyces, or Brettanomycces.

In some aspects, the yeast may be selected from Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces douglasii, Saccharomyces bayanus, Zigosaccharomyces bailii, Schizosaccharomyces pombe, Dekkera brucelensis, Dekkera intermedia, Brettanomycces custersii, Brettanomycces intermedius, Kluyveromyces themotolerens, Torulaspora globosa, and Torulaspora glabrata.

In some aspects, the yeast may be from genus Saccharomyces. In some aspects, the yeast may be Saccharomyces cerevisiae.

In some aspects, a recombinant yeast of the present disclosure is able to decarboxylate oxaloacetate into malonate semialdehyde due to the insertion of at least one nucleic acid sequence or gene described herein. In some aspects, a recombinant yeast of the present disclosure is further able to co-produce one or more 3-HP and acetyl-CoA derivatives. In some aspects the malonate semialdehyde is the substrate for the co-production of the one or more 3-HP and acetyl-CoA derivatives.

The term “recombinant microorganism” and “recombinant host cell” are used interchangeably herein and refer to microorganisms that have been genetically modified to express or to overexpress endogenous enzymes, to express heterologous enzymes, such as those included in a vector, in an integration construct, or which have an alteration in expression of an endogenous gene. By “alteration” it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression level, or activity is greater than or less than that observed in the absence of the alteration. For example, the term “alter” can mean “inhibit,” but the use of the word “alter” is not limited to this definition. It is understood that the terms “recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but also to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

Culture Conditions

Culturing of the microorganisms used in the methods of the disclosure may be conducted using any number of processes known in the art for culturing and fermenting substrates using the microorganisms of the present disclosure.

The fermentation may be carried out in any suitable bioreactor, such as Continuous Stirred Tank Bioreactor, Bubble Column Bioreactor, Airlift Bioreactor, Fluidized Bed Bioreactor, Packed Bed Bioreactor, Photo-Bioreactor, Immobilized Cell Reactor, Trickle Bed Reactor, Moving Bed Biofilm Reactor, Bubble Column, Gas Lift Fermenter, Membrane Reactors such as Hollow Fiber Membrane Bioreactor. In some aspects, the bioreactor comprises a first, growth reactor in which the microorganisms are cultured, and a second, fermentation reactor, to which fermentation broth from the growth reactor is fed and in which most of the fermentation product is produced. In some aspects, the bioreactor simultaneously accomplishes the culturing of microorganism and the producing the fermentation product from carbon sources such substrates and/or feedstocks provided.

In some aspects, the present disclosure relates to the use of a recombinant microbe, for the production of (1) malonate semialdehyde, one of its salts, or a malonate semialdehyde derivative; (2) 3-HP, one of its salts, or a 3-HP derivative; and (3) acetyl-CoA, one of its salts, or a acetyl-CoA derivative. In some aspects, the recombinant microorganism is a yeast.

In some aspects, the present disclosure further relates to a method for producing (1) malonate semialdehyde, one of its salts, or a malonate semialdehyde derivative; (2) 3-HP, one of its salts, or a 3-HP derivative; and (3) acetyl-CoA, one of its salts, or a acetyl-CoA derivative; the method comprising the steps of:

(a) culturing a recombinant microorganism described herein in a culture medium; and

(b) recovering the (2) 3-HP, one of its salts, or a 3-HP derivative; and (3) acetyl-CoA, one of its salts, or an acetyl-CoA derivative from the culture medium. In some aspects, the recombinant microorganism is a yeast.

In some aspects, microorganisms described herein are grown at a temperature in the range of about 20° C. to about 37° C., preferably at a temperature ranging from 27 to 34° C., in an appropriate culture medium.

In some aspects, the recombinant yeast of the present disclosure belongs to the S. cerevisiae species, the temperature may range from 27 to 34° C., in an appropriate culture medium.

In some aspects, suitable growth media for yeast are common commercially prepared media such as broth that includes yeast nitrogen base, ammonium sulfate, and dextrose as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most. In some aspects, other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science.

Examples of culture media for a recombinant yeast of the present disclosure are described by D. Burke et al., Methods in yeast Genetics—A cold spring harbor laboratory course Manual (2000).

In some aspects, suitable pH ranges for the fermentation may be between pH 3.0 to pH 7.5, where pH 4.5 to pH 6.5 is preferred as the initial condition.

In some aspects, fermentations may be performed under aerobic conditions, microaerobic conditions, or anaerobic conditions.

In some aspects, the amount of product(s) in the fermentation medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC).

In some aspects, the present process may employ a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation, the medium is inoculated with the desired organism or organisms, and fermentation is permitted to occur without adding anything to the system. Typically, however, a “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as temperature, pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly up to the time when the fermentation is stopped. Within batch cultures cells progress through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.

In some aspects, a fed-batch system may also be used. A fed-batch system is similar to a typical batch system with the exception that the carbon source substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression (e.g. glucose repression) is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2.

A variety of fermentations are described in Sunderland et al., (1992), herein incorporated by reference. In some aspects, the fermentation is performed in batch mode it is contemplated that the method would be adaptable to continuous fermentation.

Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to vary. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

In some aspects, the methods may be practiced using either batch, fed-batch, or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for production.

In order to still improve the production of (1) malonate semialdehyde, (2) 3-HP, and acetyl-CoA; one of their salts, or derivatives thereof, a particular embodiment may consist of culturing the recombinant yeast cells in an appropriate culture medium, such as above-mentioned, wherein the said culture medium comprises an optimal amount of carbon source, especially glucose.

In some aspects, the cells are cultured in such an optimal culture medium during only a part of the whole culture duration. In some aspects, the yeast cells are incubated in the said optimal culture medium 10 hours or more after initiation of the culture, which encompasses 11, 12, 13, 14, 15 or 16 hours or more after initiation of the culture.

In some aspects, the cells are cultured in such an optimal culture medium during a time period ranging from 5 hours to 15 hours, which includes from 6 hours to 10 hours, e.g. 8 hours after initiation of the culture.

In some aspects, the carbon source comprised in said optimal culture medium consists of glucose. In preferred embodiments, the said optimal culture medium comprises 12% w/w or more glucose, including 15% w/w or more glucose. In preferred embodiments, the said optimal culture medium comprises at most 40% w/w glucose, which includes at most 35% w/w glucose.

In some aspects, a method for producing (1) malonate semialdehyde, (2) 3-HP, and (3) acetyl-CoA; one of their salts, or derivatives thereof may further comprise, between steps (a) and (c), an intermediate step (b) consisting of cultivating the yeast cells in the said optimal culture medium.

Product Isolation/Purification

In some aspects, the method of producing (1) malonate semialdehyde, one of its salts, or a malonate semialdehyde derivative; (2) 3-HP, one of its salts, or a 3-HP derivative; and (3) acetyl-CoA, one of its salts, or a acetyl-CoA derivative comprises one or more steps of isolation of at least the (2) 3-HP, one of its salts, or a 3-HP derivative; and (3) acetyl-CoA, one of its salts, or a acetyl-CoA derivative. In some aspects, the method comprises, in a first reaction, producing and isolating/purifying malonate semialdehyde, one of its salts, or a malonate semialdehyde derivative in one fermentation to add the purified/isolated product to a second reaction (fermentation/bioreactor) to drive the production of (2) 3-HP, one of its salts, or a 3-HP derivative; and (3) acetyl-CoA, one of its salts, or a acetyl-CoA derivative.

In some aspects, recovering one or more of (1) malonate semialdehyde, one of its salts, or a malonate semialdehyde derivative; (2) 3-HP, one of its salts, or a 3-HP derivative; and (3) acetyl-CoA, one of its salts, or a acetyl-CoA derivative from a culture medium may be achieved by a number of techniques, including, but not limited to, distillation, gas-stripping, pervaporation, selective precipitation, or liquid extraction.

In some aspects, the recombinant microorganism exports (1) malonate semialdehyde, one of its salts, or a malonate semialdehyde derivative; (2) 3-HP, one of its salts, or a 3-HP derivative; and (3) acetyl-CoA, one of its salts, or a acetyl-CoA derivative into the culture medium, thus simplifying the culture process.

In some aspects, the products are referred to as one or more of (1) malonate semialdehyde, one of its salts, or a malonate semialdehyde derivative; (2) 3-HP, one of its salts, or a 3-HP derivative; and (3) acetyl-CoA, one of its salts, or a acetyl-CoA derivative.

In some aspects, the products are collected from the medium by distillation. In some aspects, distillation may involve a component different from the culture medium in order to facilitate the isolation of the products by forming azeotrope and notably with water. This component is an organic solvent such as cyclohexane, pentane, butanol, benzene, toluene, tricholoethylene, octane, diethylether, or a mixture thereof.

In some aspects, gas stripping is achieved with a stripping gas chosen among helium, argon, carbon dioxide, hydrogen, nitrogen, or mixtures thereof. In some aspects, liquid extraction is achieved with an organic solvent as the hydrophobic phase such as pentane, hexane, or dodecane.

In some aspects, once a desired number of microbes have been achieved, the spent media is subjected to a process for isolating the products. In some aspects, once a desired density of microbes has been achieved, the spent media is subject to a process for isolating the products. In some aspects, the microbes are lysed and the cellular debris is pelleted out of solution in a centrifuge. In some aspects, the products collected from the cell pellet fraction or the liquid fraction with the aid of a solvent extraction process or a gradient ultra-centrifugation process.

Microbial Compositions

In some aspects, the microbes of the disclosure are combined into microbial compositions.

In some aspects, the microbial compositions of the present disclosure are solid. Where solid compositions are used, it may be desired to include one or more carrier materials including, but not limited to: mineral earths such as silicas, talc, kaolin, limestone, chalk, clay, dolomite, diatomaceous earth; calcium sulfate; magnesium sulfate; magnesium oxide; zeolites, calcium carbonate; magnesium carbonate; trehalose; chitosan; shellac; albumins; starch; skim milk powder; sweet whey powder; maltodextrin; lactose; inulin; dextrose; and products of vegetable origin such as cereal meals, tree bark meal, wood meal, and nutshell meal.

In some aspects, the microbial compositions of the present disclosure are liquid. In further aspects, the liquid comprises a solvent that may include water or an alcohol or a saline or carbohydrate solution. In some aspects, the microbial compositions of the present disclosure include binders such as polymers, carboxymethylcellulose, starch, polyvinyl alcohol, and the like.

In some aspects, microbial compositions of the present disclosure comprise saccharides (e.g., monosaccharides, disaccharides, trisaccharides, polysaccharides, oligosaccharides, and the like), polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides, proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acid polymers, silica, inorganic salts and combinations thereof. In further aspect, microbial compositions comprise polymers of agar, agarose, gelrite, gellan gum, and the like. In some aspects, microbial compositions comprise plastic capsules, emulsions (e.g., water and oil), membranes, and artificial membranes. In some aspects, emulsions or linked polymer solutions may comprise microbial compositions of the present disclosure. See Harel and Bennett (U.S. Pat. No. 8,460,726 B2).

In some aspects, microbial compositions of the present disclosure occur in a solid form (e.g., dispersed lyophilized spores) or a liquid form (microbes interspersed in a storage medium). In some aspects, microbial compositions of the present disclosure are added in dry form to a liquid to form a suspension immediately prior to use.

In some aspects, the microbial composition of the present disclosure possesses a water activity (aw) of less than 0.750, 0.700, 0.650, 0.600, 0.550, 0.500, 0.475, 0.450, 0.425, 0.400, 0.375, 0.350, 0.325, 0.300, 0.275, 0.250, 0.225, 0.200, 0.190, 0.180, 0.170, 0.160, 0.150, 0.140, 0.130, 0.120, 0.110, 0.100, 0.095, 0.090, 0.085, 0.080, 0.075, 0.070, 0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030, 0.025, 0.020, 0.015, 0.010, or 0.005.

In some aspects, the microbial composition of the present disclosure possesses a water activity (aw) of less than about 0.750, about 0.700, about 0.650, about 0.600, about 0.550, about 0.500, about 0.475, about 0.450, about 0.425, about 0.400, about 0.375, about 0.350, about 0.325, about 0.300, about 0.275, about 0.250, about 0.225, about 0.200, about 0.190, about 0.180, about 0.170, about 0.160, about 0.150, about 0.140, about 0.130, about 0.120, about 0.110, about 0.100, about 0.095, about 0.090, about 0.085, about 0.080, about 0.075, about 0.070, about 0.065, about 0.060, about 0.055, about 0.050, about 0.045, about 0.040, about 0.035, about 0.030, about 0.025, about 0.020, about 0.015, about 0.010, or about 0.005.

The water activity values are determined by the method of Saturated Aqueous Solutions (Multon, “Techniques d′Analyse E De Controle Dans Les Industries Agroalimentaires” APRIA (1981)) or by direct measurement using a viable Robotronic BT hygrometer or other hygrometer or hygroscope.

Feedstock

In some aspects, the disclosure is drawn to a method of producing and/or recovering/isolating a 3-HP and acetyl-CoA derivative. The recovery/collection/isolation can be by methods known in the art, such as distillation, membrane-based separation gas stripping, solvent extraction, and expanded bed adsorption.

In some aspects, the feedstock comprises a carbon source. In some aspects, the carbon source may be selected from sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, lignocellulose, proteins, carbon dioxide, and carbon monoxide. In one aspect, the carbon source is a sugar. In one aspect, the sugar is glucose or oligomers of glucose thereof. In one aspect, the oligomers of glucose are selected from fructose, sucrose, starch, cellobiose, maltose, lactose and cellulose. In one aspect, the sugar is a five carbon sugar. In one aspect, the sugar is a six carbon sugar. In some aspects, the feedstock comprises one or more five carbon sugars and/or one or more six carbon sugars. In some aspects, the feedstock comprises one or more of xylose, glucose, arabinose, galactose, maltose, fructose, mannose, sucrose, and/or combinations thereof. In some aspects, the feedstock comprises one or more of xylose and/or glucose. In some aspects, the feedstock comprises one or more of arabinose, galactose, maltose, fructose, mannose, sucrose, and/or combinations thereof.

In some aspects, the microbes utilize one or more five carbon sugars (pentoses) and/or one or more six carbon sugars (hexoses). In some aspects, the microbes utilize one or more of xylose and/or glucose. In some aspects, the microbes utilize one or more of arabinose, galactose, maltose, fructose, mannose, sucrose, and/or combinations thereof. In some aspects, the microbes utilize one or more of xylose, glucose, arabinose, galactose, maltose, fructose, mannose, sucrose, and/or combinations thereof. The carbon source(s) utilized may encompass a wide variety of carbon-containing substrates and will generally only be limited by the choice of microorganism.

In some aspects, hexoses may be selected from D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, D-tagtose, D-sorbose, D-fructose, D-psicose, and other hexoses known in the art. In some aspects, pentoses may be selected from D-xylose, D-ribose, D-arabinose, D-lyxose, D-xylulose, D-ribulose, and other pentoses known in the art. In some aspects, the hexoses and pentoses may be selected from the levorotary or dextrorotary enantiomer of any of the hexoses and pentoses disclosed herein.

In some aspects, a fermentation is generally conducted in fermenters/bioreactors with an appropriate culture medium adapted to the microorganism(s) being cultivated. In some aspects, the medium contains at least one simple carbon source. In some aspects, the medium contains additional substrates.

In some aspects, additional substrates may include any one or more of the carbon sources described herein; polysaccharides such as start or cellulose, or mixtures thereof unpurified mixtures from renewable feedstocks such as cheese whey permeate, corn steep liquor, sugar beet molasses, and barley malt.

In some aspects, the media may further contain suitable minerals, salts, cofactors, buffers, and other components suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for the production of the desired product.

In some aspects, total amount of C5 and/or C6 carbohydrates fed to a bioreactor/growth medium during the growth phase is at least 5 kg carbohydrate/m3, at least 10 kg carbohydrate/m3, at least 20 kg carbohydrate/m3, at least 30 kg carbohydrate/m3, at least 40 kg carbohydrate/m3, at least 50 kg carbohydrate/m3, at least 60 kg carbohydrate/m3, at least 70 kg carbohydrate/m3, at least 80 kg carbohydrate/m3, at least 90 kg carbohydrate/m3, at least 100 kg carbohydrate/m3, at least 150 kg carbohydrate/m3, at least 200 kg carbohydrate/m3, at least 250 kg carbohydrate/m3, at least 300 kg carbohydrate/m3, at least 400 kg carbohydrate/m3 at least 500 kg carbohydrate/m3, at least 600 kg carbohydrate/m3, at least 700 kg carbohydrate/m3, up to 800 kg carbohydrate/m3. In some aspects, total amount of C5 and/or C6 carbohydrates fed to the bioreactor/growth medium during the growth phase ranges from about 10 kg carbohydrate/m3 up to 500 kg carbohydrate/m3.

In some aspects, time required for the growth phase varies between 1 to 200 hours. In further aspects, the time of the growth phase is between 5 to 50 hours. The time is dependent on carbohydrate feeds and/or feedstocks.

As used herein, an “appropriate culture medium” means a medium, such as a sterile liquid medium, comprising nutrients essential or beneficial to the maintenance and/or growth of the microbial cells such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium phosphate; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for example magnesium salts, cobalt salts, and/or manganese salts; as well as growth factors such as amino acids, vitamins, growth promoters, and the like. The term “carbon source” or “carbon substrate” or “source of carbon” according to the present disclosure denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a microorganism, including hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, cellulose, hemicelluloses and combinations thereof.

In some aspects, the total amount of C5 and/or C6 carbohydrates fed to the bioreactor/growth medium during the production phase is at least 50 kg carbohydrate/m3, at least 60 kg carbohydrate/m3, at least 70 kg carbohydrate/m3, at least 80 kg carbohydrate/m3, at least 90 kg carbohydrate/m3, at least 100 kg carbohydrate/m3, at least 150 kg carbohydrate/m3, at least 200 kg carbohydrate/m3, at least 250 kg carbohydrate/m3, at least 300 kg carbohydrate/m3, at least 400 kg carbohydrate/m3, at least 500 kg carbohydrate/m3, at least 600 kg carbohydrate/m3, at least 700 kg carbohydrate/m3, at least 800 kg carbohydrate/m3, at least 900 kg carbohydrate/m3 up to 1000 kg carbohydrate/m3. In some aspects, total amount of C5 and/or C6 carbohydrates fed to the bioreactor/growth medium during the production phase ranges from about 100 kg carbohydrate/m3 up to 800 kg carbohydrate/m3.

In some aspects, time required for the production phase varies between 5 to 500 hours. In further aspects, the time for the production phase varies from 10 to 300 hours for batch and fed-batch operations. In other aspects, the time of the production phase is up to 300 hours with continuous fermentation.

In some aspects, the total amount of C5 and/or C6 carbohydrates fed to the bioreactor/growth medium for one-phase process is at least 50 kg carbohydrate/m3, at least 60 kg carbohydrate/m3, at least 70 kg carbohydrate/m3, at least 80 kg carbohydrate/m3, at least 90 kg carbohydrate/m3, at least 100 kg carbohydrate/m3, at least 150 kg carbohydrate/m3, at least 200 kg carbohydrate/m3, at least 250 kg carbohydrate/m3, at least 300 kg carbohydrate/m3, at least 400 kg carbohydrate/m3, at least 500 kg carbohydrate/m3, at least 600 kg carbohydrate/m3, at least 700 kg carbohydrate/m3, at least 800 kg carbohydrate/m3, at least 900 kg carbohydrate/m3 up to 1000 kg carbohydrate/m3. In some aspects, total amount of C5 and/or C6 carbohydrates fed to the bioreactor/growth medium during the production phase ranges from about 100 kg carbohydrate/m3 up to 800 kg carbohydrate/m3.

In some aspects, time required for the production phase in the one-phase process varies between 5 to 500 hours. In further aspects, the time required for production phase in the one-phase process varies between 5 to 300 hours.

In some aspects, the one-phase or multi-phase production processes take about 5, about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300 about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500 hours.

In some aspects, the one-phase or multi-phase production processes take 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300 325, 350, 375, 400, 425, 450, 475, or 500 hours.

EXAMPLES
Example 1: Creating Recombinant S. cerevisiae Strain to Produce Malonate Semialdehyde

Recombinant Saccharomyces cerevisiae strains were constructed from standard strains using standard yeast molecular genetics procedure (Methods in yeast Genetics—A cold spring harbor laboratory course Manual (2000) by D. Burke, D. Dawson, T. Stearns CSHL Press).

The following genes were integrated in recombinant yeast using the ability of yeast to efficiently recombine free DNA ends sharing sequence homology.

More particularly, the coding sequences to be cloned were artificially synthetized. For heterologous sequences (non-yeast), the nucleic sequences were modified in order to obtain a synonymous coding sequence using the yeast codon usage. Using restriction enzyme and classical cloning technology, each synthetic sequence was cloned in between a transcription promoter and a transcription terminator. Each promoter sequence is preceded by a 50 to 200 nucleotides sequence homologous to the sequence of the terminator of the upstream gene. Similarly, the terminator of each gene (a gene comprising the promoter-coding sequence-terminator) is followed by sequences homologous to the gene immediately following. So that each of the unit to be integrated have a 50-200 nucleotide overlap with both the unit upstream and the unit downstream. For the first unit, the promoter is preceded by 50-200 nucleotides homologous to the yeast chromosome nucleotide for the locus in which it will be integrated. Similarly, for the last unit, the terminator is followed by 50-200 nucleotides homologous to the yeast chromosome nucleotide for the locus in which it will be integrated.

Each unit are then PCR amplified from the plasmids constructs, yielding X unit of linear DNA having overlapping sequences. At least one of this gene is an auxotrophic marker, in order to select for recombination event. All the linear fragments are transformed in the yeast at once, and recombinant yeast are selected for the auxotrophy related to the marker used. The integrity of the sequence is then verified by PCR and sequencing. Aspects of synthetic biology utilized described in Tian J. et al., Mol. Biosyst. 2009; 5(7):714-22.

Example 2: Method of Modifying a Yeast

The recombinant yeasts are mutant yeasts (Δfms1) which are impaired for β-alanine synthesis. As a consequence, the yeast is auxotroph for panthotenate and cannot grow on a medium deprived of pantothenate. In the same yeasts is expressed PYD4 from Lachancea kluyveri, a gene which encodes a β-alanine aminotransferase activity absent from Saccharomyces cerevisiae.

This enzyme is able to transform Malonic semialdehyde into β-alanine (Andersen et al. 2007 FEBS Journal 274, 1804-1817). This yeast then lacks an activity able to produce malonyl semialdehyde to be able to grow in absence of pantothenate in the medium. See FIG. 13.

This yeast is still unable to grow on a pantothenate free medium upon expression of the benzoylformate decarboxylase from P. putida. Indeed, benzoylformate decarboxylase is not able to catalyze the transformation of oxaloacetate into malonate semialdehyde.

The following enzymes have been tested in vivo, in separate yeasts.

Enzyme No 1:

(SEQ ID NO: 2)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIA

DGYAQASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTN

VDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADP

QSHHLFDRHVSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERL

KAPVWVAPSAPRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHQYDPG

QYLKPGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAK

VDQDAGRLHPETVEDTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGG

LGFALPAAIGVQLAEPERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGALR

WFAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIE

VSTVSPVK

Enzyme No 2:

(SEQ ID NO: 3)

MASVHGTTYELLRRQGIDTVEGNPGSNELPFLKDEPEDFRYILALQEACVVGIA

DGYAQASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTN

VDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADP

QSHHLFDRHVSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERL

KAPVWVAPSAPRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHQYDPG

QYLKPGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAK

VDQDAGRLHPETVFDTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYNCAAGG

LGFALPAAIGVQLAEPERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGALR

WFAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIE

VSTVSPVK

Enzyme No 3:

(SEQ ID NO: 4)

MASVHGTTYELLRRQGIDTVEGNPGSNELPFLKDEPEDFRYILALQEACVVGIA

DGYAQASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEAKLTN

VDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADP

QSHHLFDRHVSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERL

KAPVWVAPSAPRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHQYDPG

QYLKPGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAK

VDQDAGRLHPETVFDTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYNCAAGG

LGFALPAAIGVQLAEPERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGALR

WFAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIE

VSTVSPVK

Enzyme No 4:

(SEQ ID NO: 5)

MASVHGTTYELLRRQGIDTVEGNPGSNELPFLKDEPEDFRYILALQEACVVGIA

DGYAQASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTN

VDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADP

QSHHLFDRHVSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERL

KAPVWVAPSAPRCPEPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVERYHQYDPG

QYLKPGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAK

VDQDAGRLHPETVEDTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYNCAAGG

LGFALPAAIGVQLAEPERQVIAVIGDGSANYSISALWTAAQYNIPTIEVIMNNGTYGLLR

WFAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIE

VSTVSPVK

Enzyme No 5:

(SEQ ID NO: 6)

MASVHGTTYELLRRQGIDTVEGNPGSNELPFLKDEPEDFRYILALQEACVVGIA

DGYAQASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEAVKT

NVDAANLPRPLVKWSYEPASAAEVPHAMSRAIRMASMAPQGPVYLSVPYDDWDKDA

DPQSHHLFDRHVSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAE

RLKAPVWVAPSAPRCPEPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVERYHQYD

PGQYLKPGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEP

AKVDQDAGRLHPETVEDTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYNCAA

GGNGFALPAAIGVQLAEPERQVIAVIGDGSANYSISALWTAAQYNIPTIEVIMNNGTVGA

LRWFAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVL

IEVSTVSPVK

Enzyme No 6:

(SEQ ID NO: 242)

MASVHGTTYELLRRQGIDTVEGNPGSNELPFLKDEPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPEPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVERYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYLCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNVPTIEVIMNNGTYGTLRWFAGVLEAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 7:

(SEQ ID NO: 243)

MASVHGTTYELLRRQGIDIVEGNPGSNELPFLKDEPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYRQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLDMRNPGSYYFCAAGGLGFALPAAIGVQLAEP

GRQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGLLRWSAGVLGAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 8:

(SEQ ID NO: 244)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYRQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGGLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 9:

(SEQ ID NO: 245)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGTLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 10:

(SEQ ID NO: 246)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYACAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGALRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 11:

(SEQ ID NO: 247)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGLLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 12:

(SEQ ID NO: 248)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYRQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGALRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 13:

(SEQ ID NO: 249)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYRQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGTLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 14:

(SEQ ID NO: 250)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRC PFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGALRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 15:

(SEQ ID NO: 251)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGLLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 16:

(SEQ ID NO: 252)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYLCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNVPTIFVIMNNGTYGLLRWFAGVLEAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 17:

(SEQ ID NO: 253)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYLCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNVPTIFVIMNNGTYGGLRWFAGVLEAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 18:

(SEQ ID NO: 254)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYLCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNVPTIFVIMNNGTYGTLRWSAGVLEAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 19:

(SEQ ID NO: 255)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYLCAAGGLGFALPAAIGVQLAEP

GRQVIAVIGDGSANYSISALWTAAQYNVPTIFVIMNNGTYGTLRWSAGVLGAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 20:

(SEQ ID NO: 256)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYLCAAGGLGFALPAAIGVQLAEP

GRQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGTLRWSAGVLGAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 21:

(SEQ ID NO: 257)

MASVHGTTYELLRRQGIDIVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLDMRNPGSYYFCAAGGLGFALPAAIGVQLAEP

GRQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGLLRWSAGVLGAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 22:

(SEQ ID NO: 258)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYRQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYNCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGLLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 23:

(SEQ ID NO: 259)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYRQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYWCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTVGALRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 24:

(SEQ ID NO: 260)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYRQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYRCAAGGVGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTVGALRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 25:

(SEQ ID NO: 261)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYRQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYWCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGNLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 26:

(SEQ ID NO: 262)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VTSSVRLNDQDLDILVKALNSASNPVIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGTLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 27:

(SEQ ID NO: 263)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYACAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGTLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 28:

(SEQ ID NO: 264)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYLCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGTLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 29:

(SEQ ID NO: 265)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYRCAAGGVGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNVPTIFVIMNNGTYGALRWFAGVLEAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 30:

(SEQ ID NO: 266)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYRCAAGGVGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNVPTIFVIMNNGTYGGLRWFAGVLEAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 31:

(SEQ ID NO: 267)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYWCAAGGSGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNVPTIFVIMNNGTYGNLRWFAGVLEAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 32:

(SEQ ID NO: 268)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYRQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYWCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGLLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 33:

(SEQ ID NO: 269)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYRQYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYRCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGLLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 34:

(SEQ ID NO: 270)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYWCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGTLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 35:

(SEQ ID NO: 271)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYRCAAGGLGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGTLRWFAGVLEAENVPGLDVP

GIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 36:

(SEQ ID NO: 272)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYRCAAGGVGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNVPTIFVIMNNGTYGTLRWFAGVLEAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

Enzyme No 37:

(SEQ ID NO: 273)

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQAS

RKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEARLTNVDAANLPR

PLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRH

VSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSA

PRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHRYDPGQYLKPGTRLISVT

CDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVF

DTLNDMAPEDAIYLNESTSTTAQMWQRLNMRNPGSYYWCAAGGSGFALPAAIGVQLAEP

ERQVIAVIGDGSANYSISALWTAAQYNVPTIFVIMNNGTYGTLRWFAGVLEAENVPGLDV

PGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

When one of the enzymes of the invention of sequence SEQ ID NO:274 as described herein was expressed in the Δfms1-Pyd41k strain, this yeast was able to grow on a pantothenate free medium.

These in vivo results show that the enzymes of the disclosure catalyze the transformation of oxaloacetate into malonic semialdehyde or one of its derivatives, such as for example malonate semialdehyde.

Example 3: 3-HP Production from MSA
3-HP Dehydrogenase

Most 3-HP dehydrogenase enzymes (Table 1) accept NADPH as a cofactor to convert malonate semialdehyde to 3-hydroxypropionic acid, but the use of NADH is desirable to get a redox balanced pathway. To contemplate this issue, the 3-HP dehydrogenase encoded by the HPD1 gene from the yeast Candida albicans, also active in Saccharomyces cells was identified and characterized as a 3-HP dehydrogenase working in the Propionyl-CoA degradation pathway and it was demonstrated to sustain efficient 3-HP synthesis from malonate semialdehyde, while using preferentially NADH as cofactor. The full-length HPD1.Cal gene was therefore cloned and expressed on a plasmid under the control of a strong promoter in Saccharomyces cerevisiae cells and compared with other enzymes. On the assay, it was used 20 mM of malonate semialdehyde and 2 mM of cofactor (NADPH or NADH). 3-HP was measured by GC-MS/MS after derivatization with BSTFA (data not shown).

TABLE 18

Constructed strain to test the 3-HP dehydrogenase.

Strain
Genotype

YA3542-3
MAT-α, ade2, adh1::[ADH1-4-URA3.Kl-loxP],

adh3::RS, adh4::RS, adh5::RS, can1-100,

his3, leu2, pdc1::HIS5.Sp-loxP,

pdc6::LEU2.Kl-loxP, trp1, ura3

TABLE 19

Plasmids constructed with different 3-HP dehydrogenases.

Plasmid
Description

pAD4003
pRS314-pCCW12-YDFG.Ec-tRPL15A

pAD4042
pRS314-pCCW12-YDF1-tRPL15A

pAD4287
pRS314-pCCW12-YDF1-11-tRPL15A

pAD4346
pRS314-pCCW12-HPD1.Cal-tRPL15A

TABLE 20

Activity of 3-HP dehydrogenases based

on cofactor consuming (in-vitro).

Activity
Activity

(nmol ·
(nmol ·

min⁻¹· mg⁻¹)
min⁻¹· mg⁻¹)

Strain
Relevant genotype
NADPH
NADH

YA3542-3
WT
Not detected

YA3542-3 +
pRS314-pSTRONG-
50
Not detected

pAD4003
YDFG-0.Ec

YA3 542-3 +
pRS316-pSTRONG-
2750 (+/− 250)
2 (+/−1)

pAD4042
YDF1

YA3542-3 +
pRS316-pSTRONG-
50
48 (+/−2)

pAD4287
YDF1-11

(S22N +

A47D + R48F)

YA3542-3 +
pRS316-pSTRONG-
200
2500

pAD4346
HPDl.Cal

It was demonstrated that HPD1.Cal is highly active in the catalysis of 3-HP formation from MSA and further displays a preferential use of NADH as cofactor.

Example 4: 3-HP Production from Glucose—Carbon Flux Rewiring from PEP to Oxaloacetate

This example describes enzymatic conversion of malonate semialdehyde to 3-HP. The MSA was produced by the β-alanine pathway (FIG. 3).

The redirection of the carbon flow towards oxaloacetate can be achieved through the implementation of the Oxaloacetate shunt, based on the strong expression of phosphoenolpyruvate carboxykinase from E. coli (PEPCK.Ec), while the yeast pyruvate kinase activity is strongly attenuated by (i) expressing the PYK1 gene from a weak promoter (pNUP57 or pMET25ΔF) and (ii) decreasing the half-life of the protein itself by its fusion to a specific degron (PYK1-7). Such strategy is described in WO2019011945A1 and WO2019011948A1.

The engineering workflow (FIG. 20) starts with diploid strains that comprise an inactive oxaloacetate shunt, as the pLow-PYK1-7 is present in only one chromosome, and it ends by a sporulation step allowing the oxaloacetate shunt activation. The final haploid strains are YA4963 and YA4968 (Table 21).

Strains were thus assayed for 3-HP production in anaerobic growth conditions: 10 mL of rich medium in the presence of 8% glucose in 50 mL closed Falcon tubes. Stirring at 135 rpm (50 mm shaking diameter). The 3-HP analysis was done after 48 h of growth using GC/FID (after 3-HP extraction in presence of 1-butanol).

TABLE 21

Strains constructed to improve carbon flux rewiring from PEP.

Strain
Genotype

DA2124-14
MAT-a/MAT-α, can1-100/can1-100, his3/his3,

JLP1/jlp1::[TRP1.K1-loxP-PYK1-7], leu2/leu2,

MET14/met14::[HIS3.Sba-RS-PAND.Tca-

PYD4.Lk-YDFG-0.Ec], PYK1/pyk1::[LEU2.Kl-loxP-

PEPCK-1.Ec-AAT2-PEPCK-1.Ec], trp1/trp1,

ura3/ura3::[PAND.Tca-YDFG-0.Ec-URA3]x9

YA4963-25A
jlp1::[TRP1.Kl-loxP-pNUP57-PYK1-7],

met14::[HIS3.Sba-RS-PAND.Tca-PYD4.Lk-YDFG-

0.Ec], pyk1::[LEU2.Kl-loxP-PEPCK-1.Ec-AAT2-

PEPCK-1.Ec], ura3::[PAND.Tca-YDFG-0.Ec-

URA3]x9

YA4968-12C
jlp1::[TRP1.Kl-loxP-pMET25DF-PYK1],

met14::[HIS3.Sba-RS-PAND.Tca-PYD4.Lk-

YDFG-0.Ec], pyk1::[LEU2.Kl-loxP-

PEPCK-1.Ec-AAT2-PEPCK-1.Ec],

ura3::[PAND.Tca-YDFG-0.Ec-URA3]x10

In fermentative conditions, PYK1 attenuated strains (YA4963-21C and YA4963-25A) produced more 3-HP (7.8 g/L of 3-HP) than their parental strain (0.9 g/L of 3-HP) without PYK1 attenuation (DA2124-12) (Table 22)

TABLE 22

3-HP production (in-vivo) after PYK1-7 attenuation.

OD
3-HP
Glucose
Ethanol
Glycerol
PYK1 Activity

Strain
600 nm
(g · L⁻¹)
(g · L⁻¹)
(g · L⁻¹)
(g · L⁻¹)
(nmol · min⁻¹· mg⁻¹)

DA2124-12
59
0.9
0
39
2
6200

YA4963-21C
37
5.1
42
15
5
130

YA4963-25A
22
7.8
44
11
8
150

Pyruvate Kinase activity has been determined as described in Aust, A.; Yun, S. L.; Suelter, C. H. (1975) Methods Enzymol. 42C, 176-182

Example 5: 3-HP Production from Glucose Through Oxaloacetate Decarboxylase

Oxaloacetate decarboxylase enzyme candidates have been prospected and successfully engineered to deliver an active enzyme to convert oxaloacetate into malonate semialdehyde and in a second step to leverage a novel fermentative metabolic pathway to produce MSA, 3-HP and derivatives from glucose.

To illustrate the oxaloacetate decarboxylase enzyme engineering R&D efforts and the magnitude of the MDLC enzyme (Pseudomonas putida) engineering results, the resulting activity can be compared to the value obtained in a similar approach with the wild type aspartate decarboxylase (PAND.Tca of Tribolium castaneum), that catalyzes the aspartate conversion to β-alanine. As described previously, 5-8 g/L of 3-HP was successfully produced from glucose through the β-alanine route by the use of the enzyme aspartate decarboxylase (PAND.Tca). As described in the table below, the variant MDLC-54 has been shown to be as active as the aspartate decarboxylase PAND.Tca (4× lower activity only), indicating such variant would be able to sustain the in-vivo production of 3-HP from glucose (Table 23).

TABLE 23

Activities of MDLC-54 and PAND.Tca

Enzyme
Activity (nmol/min/mg)

Engineered variant MDLC-54
10-15

Wild type Pand.Tca
2-2.5

An Oxaloacetate decarboxylase assay was carried out with yeast cell extracts containing 50, 100, 150 and 200 μg of total protein in 100 mM phosphate buffer pH 6 for 40 min at 30° C. in the presence of oxaloacetate (20 mM), MgSO₄.7H₂O (2 mM), TPP (2 mM), NADPH (2 mM) and a purified YdfG enzyme (4 μg/100 μL) The formation of 3-HP was measured by GC/MS/MS after derivatization with BSTFA.

Aspartate decarboxylase (PAND.Tca) was carried out with yeast cell extracts containing 20, 40, 60 and 80 μg of total protein in 100 mM phosphate buffer pH 7 for 20 min at 30° C. in the presence of aspartate (20 mM). The formation of Beta-alanine was measured by UPLC/UV after derivatization with AQC (6-Aminoquinolyl-n-hydroxysuccimidyl carbamate).

Based on these results, assays are additionally performed to analyse the kinetic properties of enzymes of the invention through the measure of their K_mand V_max, using extracts of yeasts expressing the different MDLC variants.

The kinetic assays are carried out with 100 μg of yeast extracts for 30 minutes in the presence of increasing concentrations of oxaloacetate (2.5, 5, 10, 20, 40, 80 and 120 mM), of purified YdfG (NADP-dependent 3-hydroxy acid hydrogenase—EC 1.1.1.298) from E. coli (4 μg/100 μL) and 2 mM NADPH.

The efficiency of the enzymes of the invention is assayed through the formation of 3-HP that is measured by GC/MS/MS after derivatization with BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide).

As negative control, this assay is performed on a yeast extract not comprising an enzyme according to the invention. No significant activity is detected.

The results obtained with yeast strains comprising either enzyme No 1 (SEQ ID NO: 2), enzyme No 6 (SEQ ID NO: 242), enzyme No 7 (SEQ ID NO: 243), enzyme No 8 (SEQ ID NO: 244) or enzyme No 9 (SEQ ID NO: 245) are in particular represented in FIG. 21, FIG. 22, FIG. 23, FIG. 24, and FIG. 25, respectively.

It can be observed that very low K_mare obtained, demonstrating that the enzymes of the invention are very effective.

Example 6: Acetone Production from MSA

Acetyl-CoA Production from MSA

Genes of table 2 were cloned and expressed on a plasmid under the control of a strong promoter in Saccharomyces cerevisiae cells (Table 24).

No MSD activity was detected using NADP as a cofactor. Using NAD as the co-enzyme, both MSD.Cal and MSD.Pa were active with an activity of 40 nmol·min⁻¹·mg⁻¹(Table 25).

Yeast extract from the considered strains was incubated in the presence of 80 mM Beta-alanine, 20 mM Oxoglutarate, 100 μM Pyridoxal Phosphate, 1 mM NAD, 0.5 mM Coenzyme A and 1 mM DTT in phosphate buffer 100 mM pH 7.5. Beta-alanine and oxoglutarate were converted in MSD and β-alanine by the PYD-4 transaminase. The MSD activity was then monitored by following NADH appearance by UV absorbance at 340 nM. No increase of absorbance at 340 nM was observed if either oxoglutarate or beta-alanine was omitted, or in a strain in which no MSD activity was expressed. This assay was adapted from Andersen and Piskur FEBS journal, (2007) 274, 1804-1817 and Waters and Venables FEMS Microbiology Letters 34 (1986) 279-282.

TABLE 24

Strains constructed to acetone production.

Strain
Genotype

YA4666-1
MAT-a, ade2, can1-100, fms1::ADE2.Sba-

RS, his3, jlp1::[HIS3.Sba-

RS-PYD4.Lk], trp1, ura3

YA4750-2/-4
MAT-a, ade2, can1-100, fms1::ADE2.Sba-

RS, his3, jlp1::[HIS3.Sba-RS-PYD4.Lk],

met14::[TRP1.K1-RS-MSD.Cal], trp1, ura3

YA4751-2
MAT-a, ade2, can1-100, fms1::ADE2.Sba-

RS, his3, jlp1::[HIS3.Sba-RS-PYD4.Lk],

met14::[TRP1.Kl-RS-MSD.Pa], trp1, ura3

TABLE 25

Activity of malonate semialdehyde dehydrogenase

based on cofactor consumption.

Activity
Activity

(nmol ·
(nmol ·

Relevant
min⁻¹· mg⁻¹)
min⁻¹· mg⁻¹)

Strain
genotype
NADP
NAD

YA4666-1
WT
Not detected

YA4750-2
YA4666-1 +
Not detected
40

pSTRONG-MSD.Cal

YA4751-2
YA4666-1 +

40

YA4750-4
pSTRONG-MSD.Pa

40

Acetoacetyl-CoA Production from Acetyl-CoA

For the conversion of acetyl-CoA to acetoacetyl-CoA, the enzyme ERG10 (S. cerevisiae) that is able to catalyze this reaction was preferentially used (data not shown), according to Hiser L, et al. (1994) ERG10 from Saccharomyces cerevisiae encodes acetoacetyl-CoA thiolase. J Biol Chem 269(50):31383-9.

Acetone Production from Acetyl-CoA In Vivo

To identify the best combination of acetoacetyl-CoA transferase/acetoacetate decarboxylase, a combination of different gene candidates have been integrated as clusters in the YA4565-2 strain (Table 26), in which the carbon flux is directed through the β-Alanine shunt allowing malonate semialdehyde production (FIG. 3).

TABLE 26

Constructed strains to produce acetone.

Strain
Relevant Genotype

YA4565-2
his3, leu2, met14::[HIS3.Sba-RS-

PAND.Tca-PYD4.Lk], trp1, ura3

YA5060
YA4565-2 + jlp1::[LEU2.Sba-RS-

MSD.Pa-MSD.Cal-ERG10-ATOA-0.Ec-

ATOD-0.Ec-ADC-0.Ca-PTA.Cg-

ACKA.Ec]

YA5061
YA4565-2 + jlp1::[LEU2.Sba-RS-

MSD.Pa-MSD.Cal-ERG10-ATOA-0.Ec-

ATOD-0.Ec-ADC-0.Ca-PTA.Cg-

ACKA.Ec]

YA5062
YA4565-2 + jlp1::[LEU2.Sba-RS-

MSD.Pa-MSD.Cal-ERG10-ATOA-

0.Ec-ATOD-0.Ec-ADC-0.Pp-

PTA.Cg-ACKA.Ec]

YA5063
YA4565-2 + jlp1::[LEU2.Sba-

RS-MSD.Pa-MSD.Cal-ERG10-CTFA-

0.Ca-CTFB-0.Ca-ADC-0.Ca-

PTA.Cg-ACKA.Ec]

YA5064
YA4565-2 + jlp1::[LEU2.Sba-

RS-MSD.Pa-MSD.Cal-ERG10-CTFA-

0.Ca-CTFB-0.Ca-ADC-0.Cbe-

PTA.Cg-ACKA.Ec]

YA5065
YA4565-2 + jlp1::[LEU2.Sba-

RS-MSD.Pa-MSD.Cal-ERG10-

CTFA-0.Ca-CTFB-0.Ca-ADC-

0.Pp-PTA.Cg-ACKA.Ec]

Strains were cultured in 25 mL of rich medium in the presence of 2% glucose in Erlenmeyer flask closed with a silicon cap with two 1 mL pipette tip with filter and stirring was maintained at 135 rpm (50 mm shaking diameter). Samples are kept in ice until analysis or frozen at −20° C. The acetone was measured by GC/MS head space after 24 h of growth (Table 27).

TABLE 27

Acetone production (in vivo) using proposed genes.

Total culture
Supernatant

OD 600
Acetone
Acetone

Strain
nm
(g · L⁻¹)
(g · L⁻¹)

YA4565-2
36
0
0

YA5063-1
31
0.1
0.1

YA5063-2
35
0.1
0.1

YA5064-2
31
0.1
0.1

YA5064-3
33
0.1
0.1

YA5065-1
37
0.2
0.2

YA5065-2
36
0.2
0.2

YA5060-1
31
0.3
0.3

YA5060-2
33
0.3
0.3

YA5061-1
39
0.1
0.1

YA5061-2
31
0.1
0.1

YA5062-1
33
0.5
0.5

YA5062-2
33
0.5
0.5

In both series of strains, the most efficient acetoacetate decarboxylase was ADC-0 of Paenibacillus polymyxa. The best combination of enzymes corresponded to acetoacetyl-CoA transferase ATOA-0-ATOD-0 of Escherichia coli and acetoacetate decarboxylase ADC-0 of Paenibacillus polymyxa. To increase acetone production, additional copies of MSD.Pa and PanD.Tca were integrated within the genome of the YA5060 and YA5062 strains (Table 28).

TABLE 28

Strains constructed with additional

copies of genes to produce acetone.

Strain
Relevant Genotype

YA4565-2
his3, leu2, met14::[HIS3.Sba-RS-PAND.Tca-

PYD4.Lk], trp1, ura3

YA5060-1
jlp1::[LEU2.Sba-RS-MSD.Pa-MSD.Cal-ERG10-

ATOA-0.Ec-ATOD-0.Ec-ADC-0.Ca-PTA.Cg-

ACKA.Ec], leu2, met14::[HIS3.Sba-RS-

PAND.Tca-PYD4.Lk]

YA5182-4/-11
YA5060-1

ura3:: [PAND.Tca-MSD.Pa-URA3]x2/x4

YA5062-1
jlp1::[LEU2.Sba-RS-MSD.Pa-MSD.Cal-

ERG10-ATOA-0.Ec-ATOD-0.Ec-ADC-0Pp-

PTA.Cg-ACKA.Ec], leu2, met14::HIS3.Sba-

RS-PAND.Tca-PYD4.Lk

YA5183-1/-23
YA5062-1

ura3::[PAND.Tca-MSD.Pa-URA3]x1/x5

The resulting strains YA5182 and YA5183 were grown in 25 mL of rich medium in the presence of 8% glucose in Erlenmeyer flask with a silicon cap+two 1 mL pipette tips with filter. Stirring was maintained at 135 rpm (50 mm shaking diameter) and the production of acetone was measured after 48 h of growth by GC/MS-MS head space analysis (Table 29).

TABLE 29

Acetone production by strains having more

genes copies of MSD.Pa and PanD.Tca.

Acetone
Ethanol

Strain
OD 600 nm
(g · L⁻¹)
(g · L⁻¹)

YA4565-2
51
0
39

YA5060-1
69
0.7
36

YA5182-4
77
1.0 +/− 0.1
34

YA5182-11
75
1
35

YA5062-1
68
0.8
35

YA5183-1
70
1.1
34

YA5183-23
79
1.2 +/− 0.1
34

Example 7. Propanol Production from 3-HP

Propionyl-CoA Production from 3-HP Using Propionyl-CoA Synthase of Chloroflexus Aurantiacus In Vitro

The PCS of Chloroflexus aurantiacus activity was measured using two strategies: a) integration of multiple copies of the PCS.Cau gene under the control of a strong promoter and b) usage and evaluation of different synonymous sequences of the gene obtained by several re-encoding algorithms to avoid any deleterious recombination event. The best variant was PCS-A.Cau and the activity was higher according to the number of gene copies. It is noteworthy that at 30° C., the enzyme activity was 8-10-fold lower than at 50° C.

The results were measured by production of Propionyl-CoA by UPLC/UV as described in Alder and Fuchs (2002) Journal of biological chemistry, 277 (14), 12137-12143

TABLE 30

Strains constructed with different PCS genes to produce propanol.

Strain
Relevant Genotype

YA4788-2
MAT-a, can1-100, his3, jlp1::[LEU2.Sba-

RS-PCS.Cau], leu2, met14::[HIS3.Sba-

RSPAND.Tca-PYD4.Lk-YDFG-0.Ec], trp1,

ura3::[PAND.Tca-URA3]x14

YA4971
MAT-a, can1-100, his3, jlp1::[LEU2.Sba-

RS-PCS-A.Cau], leu2, met14::[HIS3.Sba-

RS-PAND.Tca-PYD4.Lk-YDFG-0.Ec], trp1,

ura3::[PAND.Tca-URA3]x14

YA5068
his3, jlp1::[LEU2.Sba-RS-PCS-A.Cau-

PCS-A.Cau-PCS-A.Cau-PCS-A.Cau], leu2,

met14::[HIS3.Sba-RS-PAND.Tca-PYD4.Lk-

YDFG-0.Ec], trp1, ura3::[PAND.Tca-

URA3]x14

YA5133
MAT-a, can1-100, his3, jlp1::[LEU2.Sba-

RS-PCS-D.Cau], leu2, met14::[HIS3.Sba-

RS-PAND.Tca-PYD4.Lk-YDFG-0.Ec], trp1,

ura3::[PAND.Tca-URA3]x14

TABLE 31

Activity of different variants of PCS in different

temperatures evaluated by cofactor consumption.

Activity
Activity

(nmol ·
(nmol ·

Relevant
min⁻¹· mg⁻¹)
min⁻¹· mg⁻¹)

Strain
genotype
50° C.
30° C.

YA4788-2
PCS.Cau
15
ND

YA4971-2/-3
PCS-A.Cau
42
5

YA5133-2/-6
PCS-D.Cau
41
5

YA5068-18
4x PCS-A.Cau
200
21

Propionyl-CoA Production from 3-HP Using Single Enzymes (In Vitro) Screening of 3-HP-CoA Transferase

The 3-hydroxypropionyl-coA transferase activities from Cupriavidus necator and Clostridium propionicum were assayed. The in vitro activity was measured essentially as described in Volodina E., Schürmann M., Lindenkamp N., Steinbüchel A. (2014) Appl Microbiol Biotechnol 98:3579-3589. Crude extracts of strains YA4951-4, YA4952-2 and YA5067 (Table 32) were diafiltrated (cut-off 3 Kda) and the protein content was evaluated. Extracts containing, 5, 10, 20, 40, 80 or 160 μg of protein were incubated in presence of 20 mM 3 HP, 2 mM acetyl-CoA, 1 mM NADPH and 2 mM MgCl₂for 30 minutes at 30° C. The reaction was then stopped by addition of HClO₄1% and the quantity of 3 hydroxypropionyl-CoA formed was determined by HPLC.The most active enzyme was PCT from Clostridium propionicum.

TABLE 32

Strains constructed with different PCT

genes to produce 3-HP-CoA from 3-HP.

Strain
Relevant Genotype

YA4951-4
jlp1::[LEU2.Sba-RS-PCT-0.Cne-PCT-0.Cne-

PCT-0.Cne-PCT-0.Cne]

YA4952-2
jlp1::[LEU2.Sba-RS-PCT-0.Cp-PCT-0.Cp-

PCT-0.Cp-PCT-0.Cp]

YA5067
MAT-a, ade2, can1-100, his3,

leu2::[HIS3.Sba-RS-PCT-0.Cp-HPCD-0.Sac-

HPCD-0.Sac-HPCD-0.Sac-HPCD-0.Sac],

trp1, ura3

TABLE 33

Activity of PCT measured by acetyl-CoA consumption.

Activity

Strain
Relevant genotype
(nmol · min⁻¹· mg⁻¹)

YA4951-4
PCT-0.Cne x4
140

YA4952-2
PCT-0.Cp x4
240

YA5067-1
PCT-0.Cp x1
77

YA5067-4

64

Screening of 3-HP-CoA Dehydratase

3-HP-CoA dehydratases from Metallosphaera sedula (HPCD.Mse), Bacillus sp. (HPCD.Bsp), Sporanaerobacter acetigenes (HPCD-O.Sac) and enoyl-CoA hydratase of Ruegeria pomeroyi (ENCD.Rp) were assayed. The strain YA4952-2 was transformed with the following plasmids.

TABLE 34

Plasmids constructed with 3-hydroxypropionyl-

CoA dehydratases/Enoyl-CoA hydratase.

Plasmid
Description

pAD3967
pFL45L-pCCW12.Sba-HPCD.Mse-tRPL15A

pAD3968
pFL45L-pCCW12.Sba-HPCD.Bsp-tRPL15A

pAD3969
pFL45L-pCCW12.Sba-HPCD-0.Sac-tRPL15A

pAD3970
pFL45L-pCCW12.Sba-ENCD.Rp-tRPL15A

The in vitro activity was measured essentially as described in Asao, M. & Alber, B. E (2013). Journal of Bacteriology 195, 4716-4725. Crude extracts of the strain YA4952-2 transformed with the plasmids described in table 34 were diafiltrated (cut-off 3 Kda) and the protein content was evaluated. Extracts containing, 5, 10, 20, 40 or 80 μg of protein were incubated in presence of 20 mM 3 HP, 2 mM acetyl-CoA, 1 mM NADPH and 2 mM MgCl₂for 30 minutes at 30° C. The reaction was then stopped by addition of HClO₄1% and the quantity of acrylyl-CoA formed was determined by HPLC—The 3-HP-CoA dehydratases of Metallosphaera sedula and Sporanaerobacter acetigenes were slightly more active than the other candidates.

TABLE 35

Activity of 3-hydroxypropionyl-CoA dehydratase

measured by acrylyl-CoA peak area.

μg of crude

Strain
extract
Peak area

YA4952-2
80
<LOQ*

YA4952-2 +
80
<LOQ

pAD3970

YA4952-2 +
10
4000

pAD3968
20
5000

40
7000

80
8000

YA4952-2 +
10
5000

pAD3969
20
8000

40
9000

80
10000

YA4952-2 +
10
5000

pAD3967
20
8000

40
11000

80
13000

*limit of quantification

Screening of Acrylyl-CoA Reductase

The acrylyl-CoA reductase of Ruegeria pomeroyi was assayed in vitro. essentially as described in Asao, M. & Alber, B. E (2013). Journal of Bacteriology 195, 4716-4725. Crude extracts of the strain YA4952-2 were diafiltrated (cut-off 3 Kda) and the protein content was evaluated. Extracts containing, 5, 10, 20 or 30 μg of protein were incubated in presence of 20 mM 3 HP, 2 mM acetyl-CoA, 1 mM NADPH and 2 mM MgCl₂for 30 minutes at 30° C. The reaction was then stopped by addition of HClO₄1% and the quantity of propionyl-CoA formed was determined by HPLC. It was possible to observe that ACR from Ruegeria pomeroyi was highly active in yeast cells.

TABLE 36

Strains constructed with acrylyl-CoA

reductase from Ruegeria pomeroyi.

Strain
Relevant Genotype

YA5057
MAT-a, ade2, can1-100, his3, jlp1::[LEU2.Sba-

RS-PCT-0.Cp-PCT-0.Cp-PCT-0.Cp-PCT-0.Cp],

leu2::[HIS3.Sba-RS-ACR-0.Rp-HPCD-0.Sac-

HPCD-0.Sac-HPCD-0.Sac-HPCD-0.Sac], trp1, ura3

YA5058
MAT-a, ade2, can1-100, his3, jlp1::[LEU2.Sba-

RS-PCT-0.Cp-PCT-0.Cp-PCT-0.Cp-PCT-0.Cp],

leu2::[HIS3.Sba-RS-HPCD-0.Sac-ACR-0.Rp-

ACR-0.Rp-ACR-0.Rp-ACR-0.Rp], trp1, ura3

TABLE 37

Activity of acrylyl-CoA reductase

measured by cofactor consumption.

Relevant
Activity

Strain
genotype
(nmol · min⁻¹· mg⁻¹)

YA4952-2
WT
ND

YA5057-2
ACR-0.Rp x1
700

YA5057-9

400

YA5058-5
ACR-0.Rp x4
1200

YA5058-15

1700

Propanol Production from Propionyl-CoA In Vitro

To this step, two different pathways can be used to convert propionyl-CoA into 1-propanol. The first one relies on the implementation of the multifunctional ADHE enzyme of Clostridium arbusti, while the second one proceeds through the intermediary formation of propionaldehyde by a propionyl-CoA reductase from Paraburkholderia xenovorans or Salmonella enterica with or without endogenous overexpression oh the alcohol dehydrogenase ADH1. The reaction was measured by monitoring NADH consumption at 340 nm using 1 mM propionyl-CoA as substrate. Results demonstrated that the PDUP enzyme from Salmonella enterica was the best candidate to catalyze the reaction.

TABLE 38

Strains constructed to test activity of propionyl-

CoA reductase and alcohol/aldehyde dehydrogenase.

Strain
Relevant Genotype

YA5051
jlp1::[LEU2.Sba-RS-PCT-0.Cne-PCT-0.Cne-PCT-

0.Cne-PCT-0.Cne]

YA5212
MAT-a, ade2::[TRP1.Sba-loxP-ADHE-0A.Car-

ADHE-0A.Car-ADHE-0A.Car], can1-100, his3,

jlp1::[LEU2.Sba-RS-PCT-0.Cp-PCT-0.Cp-PCT-

0.Cp-PCT-0.Cp], leu2::[HIS3.Sba-RS-ACR-

0.Rp-HPCD-0.Sac-HPCD-0.Sac-HPCD-0.Sac-HPCD-0.Sac

YA5214
MAT-a, ade2::[TRP1.Sba-loxP-PDUP.Sen-PDUP.Sen-

PDUP.Sen], can1-100, his3, jlp1::[LEU2.Sba-

RS-PCT-0.Cp-PCT-0.Cp-PCT-0.Cp-PCT-0.Cp], leu2::

[HIS3.Sba-RS-ACR-0.Rp-HPCD-0.Sac-HPCD-0.Sac-

HPCD-0.Sac-HPCD-0.Sac]

YA5215
MAT-a, ade2::[TRP1.Sba-loxP-PDUP.Sen-PDUP.Sen-

PDUP.Sen-ADH1], can1-100, his3, jlp1::[LEU2.Sba-

RS-PCT-0.Cp-PCT-0.Cp-PCT-0.Cp-PCT-0.Cp], leu2::

[HIS3.Sba-RS-ACR-0.Rp-HPCD-0.Sac-HPCD-0.Sac-

HPCD-0.Sac-HPCD-0.Sac]

TABLE 39

Activity of propionyl-CoA reductase and alcohol/aldehyde

dehydrogenase measured by cofactor consumption.

Activity

Strain
Relevant genotype
(nmol · min⁻¹· mg⁻¹)

YA5057-2
WT
ND

YA5051-1
+MHPF.Px x3
ND

YA5212-4
YA5057-2 + ADHE-0.Car x3
45

YA5214-1
YA5057-2 + PDUP. Sen x3
1500

YA5214-4

2200

YA5215-1
YA5057-2 + PDUP. Sen x3 + ADH1
2200

Propanol Production from 3-HP In Vitro

The production of propanol from 3-HP was assayed in vitro using cell extracts of the YA5214 and YA5215 strains. The assay was carried out in phosphate buffer 0.1M pH 6.5 in the presence of 2 mg/mL of cell extract at 30° C. for 60 minutes, using 20 mM of 3-HP+2 mM of acetyl-CoA+2.8 mM of NADPH+4 mM of NADH. 1-propanol production was monitored by GC/MS-MS head space analysis.

TABLE 40

In-vitro production of 1-propanol

Propanol
Activity
Propionyl-CoA

Strain
(mg · L⁻¹)
(nmol · min⁻¹· mg⁻¹)
(mg · L⁻¹)

YA5214-1
28
4
59

YA5214-4
29
4
60

YA5215-2
35
5
6

Propanol Production from 3-HP In Vivo

To evaluate 1-propanol synthesis, YA5212, YA5214 and YA5215 strains were growth in the presence of glucose and fed with 3-HP. The cells were grown in 25 mL of rich medium in the presence of 4% glucose in Erlenmeyer flasks with a silicon cap+two 1 mL pipette tip with filter. After 24 h of culture, another amount of 4% glucose was added. The stirring was maintained at 135 rpm (50 mm shaking diameter). The presence of 1-propanol into the growth medium was measured by GC/MS-MS head space analysis.

TABLE 41

Strains constructed to produce 1-propanol

Strain
Relevant Genotype

YA4613-7
MAT-a, can1-100, his3, leu2, met14::[HIS3.Sba-

RS-PAND.Tca-PYD4.Lk-YDFG-0.Ec], trp1,

ura3::[PAND.Tca-URA3]x8

TABLE 42

1-Propanol production after 48 h of culture.

OD
1-propanol

Strain
3-HP added
600 nm
(mg · L⁻¹)

YA4613-7
—
74
<20

5 g · L−1
70
<20

YA5212-1
—
37
<20

5 g · L−1
51
44

YA5214-1
—
40
26

1 g · L−1
40
58

2 g · L−1
49
90

5 g · L−1
46
178

YA5214-4
—
37
31

5 g · L−1
41
159

YA5215-1
—
40
26

1 g · L−1
50
43

2 g · L−1
51
76

5 g · L−1
46
159

YA5215-2
—
38
27

5 g · L−1
50
162

Example 8: Replacement of Yeast Native GAPDH Genes by Gdp1 from Kluyveromyces lactis to Favor Reaching a Neutral Redox Balance of a 1-Propanol and Acetone Co-Production Pathway

As described in the [208] there are metabolic engineering ways to generate anaerobic strains for the co-production of 1-propanol and acetone with neutral redox balance. It is described here an example, which is the replacement of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Saccharomyces cerevisiae, encoding by 3 genes (TDH1, TDH2 and TDH3), an enzyme with strict requirement for the oxidized NAD+ cofactor by GAPDH (encoded by GDP1.K1) from crabtree negative yeast Kluyveromyces lactis, which accepts either NAD+ or NADP+ and also displays a similar affinity for both compounds.

To determine the minimal GAPDH activity required to sustain an adequate carbon flux towards PEP, the contribution of each of the three yeast GAPDH isozymes was assayed in both a wild type and mutant strains. As the TDH1A, TDH2A, TDH3A triple mutant yeast cells were not viable, strains bearing either single or double TDH deletions were engineered and the GAPDH activity was assayed within crude extract of the resulting strains.

TABLE 43

Strains constructed to test TDHs activity.

Strain
Relevant Genotype

YA4693-1A
ade2, his3, leu2, tdh1::URA3.Sba-loxP,

tdh3::LEU2.K1-loxP, trp1, ura3

YA4693-1B
ade2, his3, leu2, tdh1::URA3.Sba-loxP,

tdh2::HIS5.Sp-loxP, trp1, ura3

YA4693-1D
ade2, his3, leu2, tdh2::HIS5.Sp-loxP,

trp1, ura3

YA4693-2D
ade2, his3, leu2, tdh1::URA3.Sba-loxP,

trp1, ura3

YA4693-3D
ade2, his3, leu2, tdh2::HIS5.Sp-loxP,

tdh3::LEU2.K1-loxP, trp1, ura3

YA4693-4C
ade2, his3, leu2, tdh3::LEU2.K1-loxP,

trp1, ura3

TDH3 activity assay is as described in Nakajima H, Itakura M, Kubo T, Kaneshige A, Harada N, Izawa T, Azuma Y T, Kuwamura M, Yamaji R, Takeuchi T. (2017) J Biol Chem 292(11):4727-4742

TABLE 44

Activity of GAPDH isozymes to NAD consumption.

Activity

Strain
Active genes
(nmol · min⁻¹· mg⁻¹)

NAD

Wild type
TDH1, TDH2,
24000

TDH3

YA4693-2D
TDH2, TDH3
24000

YA4693-1D
TDH1, TDH3
24000

YA4693-4C
TDH1, TDH2
6000

YA4693-1B
TDH3
24000

YA4693-1A
TDH2
6000

YA4693-3D
TDH1
3500

Deletion of TDH3 leads to a 4-fold decrease of GAPDH activity. TDH1, TDH2 or both TDH1 and TDH2 had no effect on GAPDH activity and deletion of both TDH2 and TDH3 led to an 8-fold activity decrease. It is also important to note that TDH3 gene is responsible for most of the GAPDH activity in yeast cells.

To measure the activity of the Kluyveromyces lactis GAPDH enzyme, GDP1.K1 was expressed under the control of a strong promoter in a TDH1 deleted strain and compared with wild type cells.

TABLE 45

Strain constructed to measure the GDP1.Kl activity.

Strain
Relevant Genotype

YA4807-1
MAT-a, ade2, his3, leu2, tdh2::loxP, tdh3::loxP,

trp1, ura3

YA4857-3
ade2, his3, jlp1::[ADE2.Sba-loxP-GDP1.Kl],

leu, tdh2::loxP, thd3::loxP, trp1, ura3

YA4915-25C
tdh1::loxP, tdh2::loxP, tdh3::loxP,

ura3::[GDP1.Kl-URA3]x8

YA4918-67C
tdh1::loxP, tdh2::loxP, tdh3::loxP,

ura3::[GDP1.Kl-URA3]x11

TABLE 46

Activity of GDP1.Kl to co-factor consumption

Activity

(nmol · min⁻¹· mg⁻¹)

Strain
Active genes
NAD
NADP

Wild type
TDH1, TDH2, TDH3
24000
0

YA4807-1
TDH1
3200
Not detected

YA4857-3
TDH1 + pStrong-GDP1.Kl
7500
2800

YA4915-25C
[pStrong-GDP1.Kl] x8
25000
32000

YA4918-67C
[pStrong-GDP1.Kl] x11
35000
47000

Expressed in Saccharomyces cerevisiae cells, GDP1.K1 consumed either NAD+ or NADP+. This activity appears to be strong to complement the deletion of the three S. cerevisiae TDH genes with the increment of the copy number of GDP1.K1.

Besides, the replacement of GAPDH in combination with the use of HPD1 enzyme (Candida albicans), that catalyzes the conversion of malonate semialdehyde to 3-hydroxypropionic acid (3-HP), can be used to reaching neutral redox balance.

Example 9: Co-Production of 1-Propanol and Acetone

As described on previous examples, the in-vivo production of 3-HP, MSA and acetone have been demonstrated from glucose through the B-alanine route. Besides, the in-vivo conversion of 3HP into 1-propanol has been successfully demonstrated too supplementing culture media with 1-5 g/L 3HP. Considering engineered variants of oxaloacetate decarboxylase have increased activity for the conversion of oxaloacetate into malonate semialdehyde, it is expected that such enhanced oxaloacetate decarboxylase engineered variants are able to sustain the production of malonate semialdehyde and malonate semialdehyde-derivatives such as 1-propanol from glucose fermentation. For example, one of the best engineered oxaloacetate decarboxylase variants showed to be 4-5× lower only compared to PAND enzyme (panD, Tribolium castaneum), that catalyzes the aspartate conversion to β-alanine, under in-vitro enzymatic experiment, indicating such engineered variant would sustain in-vivo production of 3-HP from glucose (data not shown).

Based on the information here disclosed, an engineered microbial strain can be generated to co-produce 1-propanol and acetone by introducing for example the target 1-propanol pathway enzymes into an already acetone-producing engineered strain such as the ones previously described. Besides, it's been successfully demonstrated previously the in-vivo production of 3-HP from glucose through the β-alanine pathway under anaerobic fermentation conditions. So, it is expected that such 3HPA-producing strain starts to co-produce 1-propanol and acetone after introducing both target enzymes of the 1-propanol and acetone pathways.

For example, such 1-propanol and acetone co-producing engineered strains could be grown in 25 mL of rich medium in the presence of 4% glucose in Erlenmeyer flasks with or without silicon cap plus two 1 mL pipette tip with filter. After 24 h of culture, another amount of 4% glucose is added. Stirring is maintained at 135 rpm (50 mm shaking diameter). The presence of 1-propanol and acetone into the culture medium is measured by standard GC/MS-MS head space analysis.

Example 10: Co-Production of 1-Propanol and 2-Propanol

As described on Example 9, an engineered microbial strain can be generated to co-produce 1-propanol and acetone by introducing for example the target 1-propanol pathway enzymes into an already acetone-producing engineered strain such as the ones previously described. Besides, it is been successfully demonstrated previously the in-vivo production of 3-HP from glucose through the β-alanine pathway under anaerobic fermentation conditions. So, it is expected that such 3HPA-producing strain starts to co-produce 1-propanol and acetone after introducing both target enzymes of the 1-propanol and acetone pathways. Then, to co-produce 1-propanol and 2-propanol, the 2-propanol dehydrogenase, for example as listed in Table 15, can be introduced on strain described on example 9.

To evaluate 1-propanol and 2-propanol co-production, the strains could be grown in 25 mL of rich medium in the presence of 4% glucose in Erlenmeyer flasks with or without silicon cap plus two 1 mL pipette tip with filter. After 24 h of culture, another amount of 4% glucose is added. Stirring is maintained at 135 rpm (50 mm shaking diameter). The presence of 1-propanol and 2-propanol into the culture medium is measured by standard GC/MS-MS head space analysis.

NUMBERED EMBODIMENTS
Embodiment 1

A recombinant microorganism capable of co-producing 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof, from a feedstock comprising one or more carbon sources, wherein the recombinant microorganism comprises one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionic acid dehydrogenase that catalyzes the production of 3-HP from malonate semialdehyde;
- (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a
  - (i) malonate semialdehyde dehydrogenase that catalyzes the production of acetyl-CoA from malonate semialdehyde, and/or
  - (ii) malonyl-CoA reductase and/or 2-keto acid decarboxylase that catalyzes the conversion of malonate semialdehyde into malonyl-CoA, and malonyl-CoA decarboxylase that catalyzes the production of acetyl-CoA from malonyl-CoA;
- (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an enzyme that is able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde.

Embodiment 2

The recombinant microorganism of embodiment 1, wherein the recombinant microorganism is capable of producing 1-propanol.

Embodiment 3

The recombinant microorganism of embodiment 2, wherein the recombinant microorganism comprises one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a propionyl-CoA synthase that catalyzes the conversion of 3-HP into propionyl-CoA;
- (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionyl-CoA synthetase/CoA transferase, 3-hydroxypropionyl-CoA dehydratase, and acrylyl-CoA reductase that catalyzes the conversion of 3-HP into propionyl-CoA;
- (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an alcohol/aldehyde dehydrogenase that catalyzes the conversion of propionyl-CoA from (a) or (b) into 1-propanol, or an aldehyde dehydrogenase (acetylating) and alcohol dehydrogenase that catalyzes together the production of 1-propanol from propionyl-CoA.

Embodiment 4

The recombinant microorganism of embodiment 1, wherein the recombinant microorganism is capable of producing acetone.

Embodiment 5

The recombinant microorganism of embodiment 4, wherein the recombinant microorganism comprises one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase that catalyzes the production of acetoacetyl-CoA from acetyl-CoA;
- (b) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA synthase that catalyzes the production of acetoacetyl-CoA from malonyl-CoA;
- (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA transferase that catalyzes the production of acetoacetate from acetoacetyl-CoA;
- (d) at least one endogenous and/or exogenous nucleic acid molecule encoding a
  - (i) hydroxymethylglutaryl-CoA synthase that catalyzes the production of HMG-CoA from acetoacetyl-CoA, and
  - (ii) hydroxymethylglutaryl-CoA lyase that catalyzes the production of acetoacetate from HMG-CoA; and
- (e) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetate decarboxylase that catalyzes the production of acetone from acetoacetate.

Embodiment 6

The recombinant microorganism of embodiment 5, wherein the endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase encodes an amino acid sequence comprising ERG10 (SEQ ID NO: 209), thlA (SEQ ID NO: 210), atoB (SEQ ID NO: 211), H16_B0759 (SEQ ID NO: 212), Msed_0656 (SEQ ID NO: 213), or AAT1 (SEQ ID NO: 214).

Embodiment 7

The recombinant microorganism of embodiment 6, wherein the endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase encode an amino acid sequence comprising ERG10 (SEQ ID NO: 209).

Embodiment 8

The recombinant microorganism of embodiment 5, wherein the endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA synthase encodes an amino acid sequence comprising nphT7 (SEQ ID NO: 285).

Embodiment 9

The recombinant microorganism of embodiment 5, wherein the endogenous and/or exogenous nucleic acid molecule encoding a acetoacetyl-CoA transferase encodes an amino acid sequence comprising atoA/atoD (SEQ ID NO: 215 and 216), ctfA/ctfB (SEQ ID NO: 219 and 220), ctfA/ctfB (SEQ ID NO:221 and 222) or ctfA/ctfB (SEQ ID NO:223 and 224).

Embodiment 10

The recombinant microorganism of embodiment 9, wherein the endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA transferase encodes an amino acid sequence comprising atoA/atoD (SEQ ID NO: 215 and 216)

Embodiment 11

The recombinant microorganism of embodiment 5, wherein the endogenous and/or exogenous nucleic acid molecule encoding a hydroxymethylglutaryl-CoA synthase and a hydroxymethylglutaryl-CoA lyase encode an amino acid sequence comprising ERG13 (SEQ ID NO: 283) and yngG (SEQ ID NO: 284).

Embodiment 12

The recombinant microorganism of embodiment 5, wherein the endogenous and/or exogenous nucleic acid molecule encoding a acetoacetate decarboxylase encodes an amino acid sequence comprising Adc.Ca (SEQ ID NO: 225), Adc.Cbe (SEQ ID NO: 226), Adc (SEQ ID NO: 227), Adc (SEQ ID NO: 228), Adc (SEQ ID NO: 229) or Adc.Pp (SEQ ID NO: 230).

Embodiment 13

The recombinant microorganism of embodiment 12, wherein the endogenous and/or exogenous nucleic acid molecule encoding a acetoacetate decarboxylase encodes an amino acid sequence comprising Adc.Pp (SEQ ID NO: 230).

Embodiment 14

The recombinant microorganism of embodiment 1, wherein the recombinant microorganism catalyzes malonate semialdehyde for the production of both 3-HP and acetyl-CoA, and/or derivatives thereof.

Embodiment 15

The recombinant microorganism of embodiment 1, wherein the derivatives of 3-HP are selected from the group consisting of: acrylic acid, 1-propanol, propene, and polypropylene.

Embodiment 16

The recombinant microorganism of embodiment 1, wherein the derivatives of acetyl-CoA are selected from the group consisting of: acetone, 2-propanol, propene, and polypropylene.

Embodiment 17

The recombinant microorganism of embodiment 1, wherein the microorganism is selected from a bacterium, a fungus, or a yeast.

Embodiment 18

The recombinant microorganism of embodiment 1, wherein the recombinant microorganism is derived from a parental microorganism selected from the group consisting of: Clostridium sp., Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillus sp, Corynebacterium sp., Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacter glycolicus.

Embodiment 19

The recombinant microorganism of embodiment 17, wherein the recombinant microorganism is a yeast.

Embodiment 20

The recombinant microorganism of embodiment 19, wherein the yeast is Saccharomyces cerevisiae.

Embodiment 21

The recombinant microorganism of embodiment 19, wherein the yeast is capable of aerobic and anaerobic production.

Embodiment 22

The recombinant microorganism of embodiment 14, wherein the recombinant microorganism comprises a decarboxylase capable of acting on oxaloacetate to produce malonate semialdehyde.

Embodiment 23

A method of producing 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof, the method comprising culturing the recombinant microorganism in a culture medium containing a feedstock comprising a carbon source until the 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof, are produced.

Embodiment 24

A method of producing a recombinant microorganism capable of co-producing 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof, from a feedstock comprising one or more carbon sources, the method comprising introducing into and/or overexpressing in the recombinant microorganism one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionic acid dehydrogenase that catalyzes the production of 3-HP from malonate semialdehyde;
- (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a
  - (i) malonate semialdehyde dehydrogenase that catalyzes the production of acetyl-CoA from malonate semialdehyde, and/or
  - (ii) malonyl-CoA reductase and/or 2-keto acid decarboxylase that catalyzes the conversion of malonate semialdehyde into malonyl-CoA, and malonyl-CoA decarboxylase that catalyzes the production of acetyl-CoA from malonyl-CoA;
- (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an enzyme that is able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde.

Embodiment 25

The method of embodiment 24, wherein the recombinant microorganism is capable of producing 1-propanol.

Embodiment 26

The method of embodiment 24, wherein the method further comprises introducing into and/or overexpressing in the recombinant microorganism one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a propionyl-CoA synthase that catalyzes the conversion of 3-HP into propionyl-CoA;
- (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionyl-CoA synthetase/CoA transferase, 3-hydroxypropionyl-CoA dehydratase, and acrylyl-CoA reductase that catalyzes the conversion of 3-HP into propionyl-CoA;
- (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an alcohol/aldehyde dehydrogenase that catalyzes the conversion of propionyl-CoA from (a) or (b) into 1-propanol, or an aldehyde dehydrogenase (acetylating) and alcohol dehydrogenase that catalyzes together the production of 1-propanol from propionyl-CoA.

Embodiment 27

The method of embodiment 24, wherein the recombinant microorganism is capable of producing acetone.

Embodiment 28

The method of embodiment 27, wherein the method further comprises introducing into and/or overexpressing in the recombinant microorganism one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase that catalyzes the production of acetoacetyl-CoA from acetyl-CoA;
- (b) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA synthase that catalyzes the production of acetoacetyl-CoA from malonyl-CoA;
- (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA transferase that catalyzes the production of acetoacetate from acetoacetyl-CoA;
- (d) at least one endogenous and/or exogenous nucleic acid molecule encoding a
  - (i) hydroxymethylglutaryl-CoA synthase that catalyzes the production of HMG-CoA from acetoacetyl-CoA, and
  - (ii) hydroxymethylglutaryl-CoA lyase that catalyzes the production of acetoacetate from HMG-CoA; and
- (e) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetate decarboxylase that catalyzes the production of acetone from acetoacetate.

Embodiment 29

The method of embodiment 24, wherein the recombinant microorganism catalyzes malonate semialdehyde for the production of both 3-HP and acetyl-CoA, and/or derivatives thereof.

Embodiment 30

The method of embodiment 24, wherein the derivatives of 3-HP are selected from the group consisting of: acrylic acid, 1-propanol, propene, and polypropylene.

Embodiment 31

The method of embodiment 24, wherein the derivatives of acetyl-CoA are selected from the group consisting of: acetone, 2-propanol, propene, and polypropylene.

Embodiment 32

The method of embodiment 24, wherein the microorganism is selected from a bacterium, a fungus, or a yeast.

Embodiment 33

The method of embodiment 24, wherein the recombinant microorganism is derived from a parental microorganism selected from the group consisting of: Clostridium sp., Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillus sp, Corynebacterium sp., Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacter glycolicus.

Embodiment 34

The method of embodiment 32, wherein the recombinant microorganism is a yeast.

Embodiment 35

The method of embodiment 34, wherein the yeast is Saccharomyces cerevisiae.

Embodiment 36

The method of embodiment 34, wherein the recombinant microorganism co-produces 3-HP, and/or derivatives and Acetyl-CoA and/or derivatives, in an aerobic, microanaerobic or anaerobic production process, preferably an anaerobic process.

Embodiment 37

The method of embodiment 29, wherein the recombinant microorganism comprises a decarboxylase capable of acting on oxaloacetate to produce malonate semialdehyde.

Embodiment 38

The method of embodiment 24, wherein at least a portion of excess NAD(P)H produced in the production of acetyl-CoA is utilized as a source of reducing equivalents in the production of 3-HP and/or derivatives thereof.

Embodiment 39

The recombinant microorganism of embodiment 1, wherein at least a portion of excess NAD(P)H produced in the production of acetyl-CoA is utilized as a source of reducing equivalents in the production of 3-HP and/or derivatives thereof.

Embodiment 40

A recombinant microorganism capable of co-producing 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and/or derivatives thereof, from a feedstock comprising one or more carbon sources, wherein the recombinant microorganism comprises one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding an 3-hydroxypropionic acid dehydrogenase that catalyzes the production of 3-HP from malonate semialdehyde;
- (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a
  - (i) malonate semialdehyde dehydrogenase that catalyzes the production of acetyl-CoA from malonate semialdehyde, and/or
  - (ii) malonyl-CoA reductase and/or 2-keto acid decarboxylase that catalyzes the conversion of malonate semialdehyde into malonyl-CoA, and malonyl-CoA decarboxylase that catalyzes the production of acetyl-CoA from malonyl-CoA; and
- (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an aspartate amino transferase, aspartate decarboxylase, β-alanine pyruvate amino transferase and/or β-alanine transaminase that catalyze the production of malonate semialdehyde from oxaloacetate, having beta-alanine as an intermediate.

Embodiment 41

The recombinant microorganism of embodiment 40, wherein the recombinant microorganism is capable of producing 1-propanol wherein the recombinant microorganism comprises one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a propionyl-CoA synthase that catalyzes the production of propionyl-CoA from 3-HP;
- (b) at least one endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionyl-CoA synthetase, 3-hydroxypropionyl-CoA dehydratase, and acrylyl-CoA reductase that catalyzes the production of propionyl-CoA from 3-HP; and
- (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an alcohol/aldehyde dehydrogenase that catalyzes the production of 1-propanol from propionyl-CoA.

Embodiment 42

The recombinant microorganism of embodiment 40, wherein the recombinant microorganism is capable of producing acetone wherein the recombinant microorganism comprises one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase that catalyzes the production of acetoacetyl-CoA from acetyl-CoA;
- (b) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA synthase that catalyzes the production of acetoacetyl-CoA from malonyl-CoA;
- (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA transferase that catalyzes the production of acetoacetate from acetoacetyl-CoA;
- (d) at least one endogenous and/or exogenous nucleic acid molecule encoding a
  - (i) hydroxymethylglutaryl-CoA synthase that catalyzes the production of HMG-CoA from acetoacetyl-CoA, and
  - (ii) hydroxymethylglutaryl-CoA lyase that catalyzes the production of acetoacetate from HMG-CoA; and
- (e) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetate decarboxylase that catalyzes the production of acetone from acetoacetate.

Embodiment 43

The recombinant microorganism of embodiments 40 through 42, wherein the derivatives of 3-HP are selected from the group consisting of: acrylic acid, 1-propanol, propene, and polypropylene.

Embodiment 44

The recombinant microorganism of embodiments 40 through 42, wherein the derivatives of acetyl-CoA are selected from the group consisting of: acetone, 2-propanol, propene, and polypropylene.

Embodiment 45

A recombinant microorganism capable of producing acetone and/or derivatives thereof, from a feedstock comprising one or more carbon sources, wherein the recombinant microorganism comprises one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding:
  - (i) at least one endogenous and/or exogenous nucleic acid molecule encoding an enzyme that is able to catalyze the decarboxylation of oxaloacetate into malonate semialdehyde or
  - (ii) at least one endogenous and/or exogenous nucleic acid molecule encoding an aspartate amino transferase, aspartate decarboxylase, β-alanine pyruvate amino transferase and/or β-alanine transaminase that catalyze the production of malonate semialdehyde from oxaloacetate, having beta-alanine as an intermediate,
  - (iii) malonate semialdehyde dehydrogenase that catalyzes the production of acetyl-CoA from malonate semialdehyde from (i) or (ii), or
  - (iv) malonyl-CoA reductase and/or 2-keto decarboxylase that catalyzes the conversion of malonate semialdehyde from (i) or (ii) into malonyl-CoA, and malonyl-CoA decarboxylase that catalyzes the production of acetyl-CoA from malonyl-CoA
- (b) at least one or more endogenous and/or exogenous nucleic acid molecules capable of catalyze the conversion of acetyl-CoA to acetone.

Embodiment 46

The recombinant microorganism of embodiment 45, wherein the recombinant microorganism further comprises one or more of the following:

- (a) at least one endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase that catalyzes the production of acetoacetyl-CoA from acetyl-CoA;
- (b) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA synthase that catalyzes the production of acetoacetyl-CoA from malonyl-CoA;
- (c) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA transferase that catalyzes the production of acetoacetate from acetoacetyl-CoA;
- (d) at least one endogenous and/or exogenous nucleic acid molecule encoding a
  - (i) hydroxymethylglutaryl-CoA synthase that catalyzes the production of HMG-CoA from acetoacetyl-CoA, and
  - (ii) hydroxymethylglutaryl-CoA lyase that catalyzes the production of acetoacetate from HMG-CoA; and
- (e) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetate decarboxylase that catalyzes the production of acetone from acetoacetate.

Embodiment 47

The recombinant microorganism of embodiment 46, wherein the derivatives of acetone are selected from the group consisting of: 2-propanol, propene, and polypropylene.

Embodiment 48

The recombinant microorganism of embodiment 46, wherein at least a portion of excess NAD(P)H produced in the production of acetone is utilized as a source of reducing equivalents on a coproducing pathway.

Embodiment 49

The recombinant microorganism of embodiment 48, wherein a co-producing pathway is a 3-HP pathway.

Embodiment 50

The recombinant microorganism of embodiments 40-48, wherein the recombinant microorganism produces acetone in an aerobic, microaerobic or anaerobic production process.

Embodiment 51

A method of co-producing 3-HP, and/or derivatives thereof and Acetyl-CoA and/or derivatives thereof by contacting the recombinant microorganism of any of the embodiments 1 with a fermentable carbon source under conditions and for a sufficient period of time to produce 3-HP, or derivatives and Acetyl-CoA or derivatives.

Embodiment 52

A method of producing Acetone or derivatives by contacting the recombinant microorganism of embodiment with a fermentable carbon source under conditions and for a sufficient period of time to produce acetone or derivatives.

Embodiment 53

The methods of embodiments 51 or 52, wherein the carbon source is selected from sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, lignocellulose, proteins, carbon dioxide, and carbon monoxide.

Embodiment 54

The recombinant microorganism of embodiments 51 or 52, wherein the recombinant microorganism produces acetone in an aerobic, microaerobic or anaerobic production process.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. Further, the following references are hereby incorporated by reference: