The terms “identical” or “percent identity”, in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described above (or other algorithms available to persons of skill) or by visual inspection.

The term “alkyl” refers to a branched or unbranched carbon chain having, for example, about 1-20 carbon atoms, and often 1-12, 1-10, 1-8, 1-6, or 1-4 carbons. Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl (t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2 pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. The alkyl can be unsubstituted or substituted. The alkyl can also be optionally partially or fully unsaturated in certain embodiments. As such, the recitation of an alkyl group optionally includes both alkenyl and alkynyl groups. The alkyl can be a monovalent hydrocarbon radical, as described and exemplified above, or it can be a divalent hydrocarbon radical (i.e., an alkylene). In some embodiments, certain alkyl groups can be excluded from a definition. For example, in some embodiments, methyl, ethyl, propyl, butyl, or a combination thereof, can be excluded from a specific definition of alkyl in an embodiment.

The term “cycloalkyl” refers to cyclic alkyl groups of, for example, 3 to about 12, 3 to about 10, 3 to about 8, about 4 to about 8, or 5-6, carbon atoms having a single cyclic ring or multiple condensed rings. Cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantyl, and the like. The cycloalkyl can be unsubstituted or substituted. The cycloalkyl group can be monovalent or divalent, and can be optionally substituted as described for alkyl groups. The cycloalkyl group can optionally include one or more cites of unsaturation, for example, the cycloalkyl group can include one or more carbon-carbon double bonds, such as, for example, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, and the like.

As used herein, “aryl” refers to an aromatic hydrocarbon group derived from the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. The radical attachment site can be at a saturated or unsaturated carbon atom of the parent ring system. The aryl group can have from 6 to about 20 carbon atoms. The aryl group can have a single ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like. The aryl can be unsubstituted or optionally substituted, as described for alkyl groups.

The term “heteroaryl” refers to a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and that can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, as described in the definition of “substituted”. Typical heteroaryl groups contain 2-20 carbon atoms in addition to the one or more heteroatoms. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl, and xanthenyl. In one embodiment the term “heteroaryl” denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, aryl, or —(C₁-C₆)alkylaryl. In some embodiments, heteroaryl denotes an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.

The term “substituted” indicates that one or more hydrogen atoms on the group indicated in the expression using “substituted” is replaced with a “substituent.” The number referred to by “one or more” can be apparent from the moiety one which the substituents reside. For example, “one or more” can refer to, e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2, or 3; and in other embodiments 1 or 2. The substituent can be one of a selection of indicated groups, or it can be a suitable group known to those of skill in the art, provided that the substituted atom's normal valency is not exceeded, and that the substitution results in a stable compound. Suitable substituent groups include, i., alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, aroyl, (aryl)alkyl (e.g., benzyl or phenylethyl), heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, difluoromethyl, acylamino, nitro, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl, heterocyclesulfonyl, phosphate, sulfate, hydroxyl amine, hydroxyl(alkyl)amine, and cyano. Additionally, suitable substituent groups can be, e.g., —X, —R, —O—, —OR, —SR, —S—, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, N₂, —N₃, —NC(═O)R, —C(═O)R, —C(═O)NRR, —S(═O)₂O—, —S(═O)₂OH, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR, —S(C)R, —OP(═O)O₂RR, —P(═O)O₂RR, —P(═)(O—)₂, —P(═O)(OH)₂, —C(═O)R, —C(═O)X, —C(S)R, —C(O)OR, —C(O)O—, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR, or —C(NR)NRR, where each X is independently a halogen (“halo”): F, Cl, Br, or I; and each R is independently H, alkyl, aryl, (aryl)alkyl (e.g., benzyl), heteroaryl, (heteroaryl)alkyl, heterocycle, heterocycle(alkyl), or a protecting group. As would be readily understood by one skilled in the art, when a substituent is keto (═O) or thioxo (═S), or the like, then two hydrogen atoms on the substituted atom are replaced.

Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All references to singular characteristics or limitations of the disclosed method shall include the corresponding plural characteristic or limitation, and vice-versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made. The indefinite articles “a” and “an” mean “one or more.”

All combinations of method steps disclosed herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

The method disclosed herein can comprise, consist of, or consist essentially of the essential elements and steps described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in synthetic/enzymatic organic chemistry. The method disclosed herein may be practiced in the absence of any element or step which is not specifically disclosed herein.

Description of the Method

Newly developed and disclosed herein is a method of reacting an L-TA enzyme (EC 4.1.2.5) and/or an L-T-transA enzyme with a ketone substrate to yield a tertiary β-hydroxy α-amino acid product. The basic approach is illustrated in the bottom panel of FIG. 3, wherein “EWG” is an electron-withdrawing group of any description (for example, a halogen, a halo-alkyl group, a cyanate group, and alkyl carboxylate group, and the like; see the definition of EWG hereinabove). As noted above, the core of the method is to react a pyridoxal-phosphate (PLP)-dependent enzyme, either a L-threonine aldolase (L-TA; EC 4.1.2.5) or a L-threonine transaldolase, (such as C-His-TmLTA (SEQ ID NO: 1) or ObiH (SEQ ID NO: 3), respectively) with a ketone substrate and in the presence of an amino acid (glycine is shown in the bottom panel of FIG. 3) to produce an amino acid with a tertiary alcohol sidechain. It is much preferred, but not required, that the ketone substrate includes an EWG to speed the reaction. The EWG functions to activate the ketone group itself, thereby accelerating the reaction. The reaction functions with unactivated ketones, but yields are quite small without a strong driving force. This is because the reaction is reversible and subject to thermodynamic limitations. Thus, while including an EWG in the ketone substrate is not strictly required, it is strongly preferred. The reaction is very flexible in that the required enzyme(s) function to catalyze the reaction in the form of whole cells, lysates, or purified enzyme.

As shown in FIG. 4, the reaction may also be conducted using an L-T-transA enzyme (such as, but not limited to, ObiH). Here, the L-T-transA catalyzes the formation of C—C bond with threonine to yield an amino acid having a tertiary β-hydroxy sidechain. See FIG. 4, bottom panel. The native, reversible reaction of the L-T-transA enzyme is shown in FIG. 4, top panel. The bottom panel of FIG. 4 illustrates an exemplary implementation of the present method in which a ketone (any ketone) is reacted with a secondary β-hydroxy aryl serine or a secondary β-hydroxy alkyl serine to yield a tertiary β-hydroxy α-amino acid. An aldehyde is formed as a by-product.

Yield of the reaction is improved by coupling the L-T-transA reaction with an appropriate alcohol dehydrogenase (ADH), that uses NAD(P)H to reduce the aldehyde byproduct. This provides increased thermodynamic driving force to the forward reaction and significantly enhances yields.

Yield of the reaction is further improved by using a co-factor regeneration reaction that replenishes the NAD(P)+ that is formed when NAD(P)H reduces the aldehyde. That is, in reducing the aldehyde, the NAD(P)H is oxidized to NAD(P)+. Regenerating the NAD(P)H provides still greater thermodynamic driving force for the forward reaction. This is shown in FIG. 5. Across the top of FIG. 5 is shown the same reaction as in the bottom panel of FIG. 4. In the lower-right section of FIG. 5 is shown a cascading co-factor regeneration reaction that regenerates the NAD(P)H to provide additional thermodynamic driving force for the forward reaction shown at the top of FIG. 5. Here, as noted above, an ADH and NAD(P)H are used to reduce the aldehyde by-product of the forward L-T-transA reaction, thereby generating an alcohol and NAD(P)+. As shown in the bottom-right of FIG. 5, a formate dehydrogenase enzyme is used to regenerate the NAD(P)H. In this fashion, the aldehyde by-product is continuously removed by the action of ADH, and the NAD(P)H reductant is regenerated by the action of the formate dehydrogenase. The result is a massive increase in thermodynamic driving force to the forward reaction. Yields are thus increased accordingly.

The results of an exemplary implementation of the method are shown in FIG. 6. As shown across the top of the figure, 10 mM of benzyl-trifluoromethyl ketone were reacted with either threonine or glycine, in the presence of 0.5% ObiH (a L-T-transA enzyme) or TmLTA (L-TA enzyme), in the presence of KPi buffer (pH 7), PLP, 10% v/v methanol (“MeOH”), 0.1 mM NAD*, and 100 mM ammonium formate, at 37° C., and for times ranging from 2 minutes to 120 minutes. The graph shows the formation of the tertiary β-hydroxy α-amino acid product over time. As shown by the top trace, the yield is greatly improved by including the alcohol dehydrogenase/formate dehydrogenase cascade reaction. The middle trace shows the yield over time without the alcohol dehydrogenase/formate dehydrogenase cascade reaction. The bottom trace shows a very small yield when only 10 μM L-TA enzyme is used. LTA (bottom trace, 1.0 M Gly) produces the least product, corresponding to <10% yield. ObiH forms significantly more product (middle trace, 100 mM Thr) than TmLTA. That activity is increased further by adding the alcohol dehydrogenase/formate dehydrogenase cascade reaction to provide additional thermodynamic driving force.

FIG. 7 depicts an exemplary implementation of the method using ObiH or TmLTA without the alcohol dehydrogenase/formate dehydrogenase cascade reaction and without an activated ketone substrate. The substrate was 4-fluorobenzyl methyl ketone and the amino acid was again threonine or glycine. The remaining reaction conditions were as noted above for FIG. 6. Yields were significantly higher for ObiH than for TmLTA. This indicates that the thermodynamics of the L-T-transA-catalyzed reaction (ObiH) are much more favorable than the thermodynamics of the L-TA catalyzed reaction (TmLTA). Under these conditions, the unassisted reaction with TmLTA did not yield any observable product. The ObiH catalyzed reaction did yield product, even with an unactivated ketone and without the alcohol dehydrogenase/formate dehydrogenase cascade reaction (bottom trace). The best yield was realized using an L-T-transA (ObiH) as the enzyme and including in the reaction the alcohol dehydrogenase/formate dehydrogenase cascade reaction (top trace).

Table 1 below presents a comparison of the yields of an exemplary ObiH-catalyzed reaction, with and without the alcohol dehydrogenase/formate dehydrogenase cascade reaction.

TABLE 1

Comparison of ObiH alone to ObiH with acetaldehyde reductase system

[Image Omitted]

Entry
ObiH lysate concentration (% w/v)
Reduction system
Product yield (%)

1
0.1%
None
4%

(1 mg/mL)

2
0.5%
None
3%

(5 mg/mL)

3
1.0%
None
1%

(10 mg/mL)

4
0.5%
0.5% CbFDH + ScADH
24%

(5 mg/mL)

The data shows that yield is not improved simply by adding a higher concentration of the L-TA or L-T-transA enzyme; see the first two entries in Table 1. Entry 1 used 1 mg/mL ObiH lysate and yield was 4%. Entry 2 used 5 mg/mL ObiH lysate and product yield dropped to 3%. All other reaction conditions were the same.

Further efforts to optimize yields resulted in the data shown in Table 2. Here, the concentrations of Thr, ObiH, ScADH, and CbFDH were varied. Entries 10 (73%), 9 (67%), and 6 (53%) had the best yields. The abbreviation “d.r.” stands for “diastereomeric ratio” of the diastereomer shown in the header of Table 2.

TABLE 2

Yield Optimization

[Image Omitted]

entry
Thr (mM)
ObiH
ScADH (w/v)
CbFDH (w/v)
d.r.
yield (%)

1
100
0.5%
0.5%
0.5%
10:1
39%

2
100
1.0%
1.0%
1.0%
7:1
43%

3
100
1.5%
1.5%
1.5%
5:1
43%

4
100
2.0%
2.0%
2.0%
4:1
41%

5
100
2.0%
0.5%
0.5%
7:2
29%

6
100
0.5%
2.0%
0.5%
14:1
53%

7
100
0.5%
0.5%
2.0%
12:1
38%

8
100
2.0%
0.5%
2.0%
4:1
30%

9
150
0.5%
2.5%
0.5%
17:1
67%

10
150
5 μM
2.5%
0.5%
14:1
73%

FIG. 8 shows the marked improvement in yield when an L-T-transA enzyme is used in conjunction with the alcohol dehydrogenase/formate dehydrogenase cascade reaction (which is referred to as the “reduction system” in FIG. 8). The histogram clearly shows that the reduction system provides an increase in activity for diverse ketone substrates. The ketone substrates tested, from left to right in FIG. 8, were benzyl trifluoromethyl ketone, 4-fluorbenzyl methyl ketone, 2-phenyl-methyl methyl ketone, and 2-pyridinyl methyl ketone. Across the board, the yield was vastly improved by using the alcohol dehydrogenase/formate dehydrogenase cascade reaction.

FIG. 9 is a table showing the effect of buffer composition and pH on an exemplary reaction according to the present method. The reaction is shown at the top of FIG. 9. 100 mM threonine was reacted with 10 mM of 4-fluorobenzyl methyl ketone. The remaining reaction conditions were as noted in FIG. 9 itself. Yields suffered at higher pHs. At pH 8, 9, and 10, using a borate buffer, the highest yields as only 3% (borate, pH 8.0). Using “Tris” (tris(hydroxymethyl)aminomethane) buffer at pH 9.0 only bumped the yield to 9%. Yields ranged from 20% to 29% using Tris or KPi buffers, at pHs ranging from 6.0 to 8.5.

FIG. 10 is a table analogous to FIG. 9, but showing the effect of optimizing enzyme concentration. The reaction and reaction conditions are the same as shown for FIG. 9. The maximum yield, 49% was achieved using the conditions shown in Entry 6: 0.5% w/v ObiH lysate, 2.0% w/v ScADH lysate, 0.5% w/v CbFDH, and 10:1 diasteriomeric ratio (“dr”).

FIG. 11 presents a selection of the ketone substrates that have been shown to function to yield a tertiary β-hydroxy α-amino acid product using the present method. The ketones include purely aliphatic, symmetric and asymmetric ketones (e.g., methyl ethyl ketone, diethyl ketones), cyclic ketones, heterocyclic ketones, aryl ketones, heteroaryl ketones, etc. For each entry in FIG. 11, tertiary β-hydroxy amino acids corresponding to the ketone substrate have been made and observed using the method disclosed herein. These successful reactions were run using the optimized conditions found in FIG. 9, entry 6.

FIG. 12 presents a reaction and a corresponding yield curve showing that TmLTA can catalyze a transaldolase reaction using alloThr as the substrate (a diastereomer of the standard L-Thr). Yields are boosted by using the ScADH and CbFDH cascade. Here, the reaction was as follows:

[Image Omitted]

In this particular implementation of the method, any β-hydroxy-amino acid or glycine can be used for the L-TA reaction. Any β-hydroxy-amino acid can be used for the ObiH reaction, but glycine is not preferred.

Substrates that have been tested and shown to yield corresponding product include:

TmLTA

[Image Omitted]

ObiH

[Image Omitted]

FIG. 13 shows representative analytical scope for ObiH, for compounds made according to the present method, including diastereomeric ratio and total turnover number (TTN). The test reaction here was:

[Image Omitted]

As shown from the wide range of products formed, the reaction is exceedingly flexible and give acceptable yields using a host of different ketone substrates.

FIG. 14 depicts the results of an exemplary reaction to investigate the impact of substrate concentration on product yield. The corresponding yield histogram shows that the method gives good yields at high substrate concentrations. The reaction here was:

[Image Omitted]

FIG. 15 is an exemplary reaction showing that using 100 mM of ketone substrate, yields were improved by adding more Thr and formate to the reaction. Here, the reaction was the same as shown in FIG. 14. As shown in the histogram, the percent yield was very significantly improved by adding additional threonine and/or additional ADH.

FIG. 16 is an exemplary reaction showing stand-alone ScADH is sufficient for providing increase in activity, which can be improved by adding more ADH and NADH. The reaction is as follows:

[Image Omitted]

Experimental Procedure

Cloning and Expression of ObiH and ScADH:

A codon-optimized copy of each was inserted into a pET-28b(+) vector (Millipore Sigma, Burlington, Massachusetts, USA) by the Gibson Assembly method. Gibson D G, Young L, Chuang R Y, Venter J C, Hutchison C A 3rd, Smith H O (2009). “Enzymatic assembly of DNA molecules up to several hundred kilobases,” Nature Methods 6 (5): 343-345. BL21 (DE3) E. coli cells (New England Biolabs Inc. Ipswich, Massachusetts, USA) were subsequently transformed with the resulting cyclized DNA product via electroporation. After 45 min of recovery in Luria-Burtani (LB) media at 37° C., cells were plated onto LB plates with 50 μg/mL kanamycin (Kan) and incubated overnight. Single colonies were used to inoculate 5 mL LB+50 μg/mL Kan, which were grown overnight at 37° C., 200 rpm. Expression cultures, typically 1 L of Terrific Broth (TB)+50 μg/mL Kan (TB-Kan), were inoculated from these starter cultures and shaken (200 rpm) at 37° C. After 3 hours (OD₆₀₀=~0.6), the expression cultures were chilled on ice. After 30 min on ice, expression is induced with 0.5 mM IPTG, and the cultures were expressed for 16 hours at 20° C. with shaking at 200 rpm. Cells were then harvested by centrifugation at 4,300×g at 4° C. for 15 min. Cell pellets were frozen and stored at −20° C. until purification.

Cloning and Expression of TmLTA and CbFDH:

A codon-optimized copy of each was inserted into a pET-22b(+) vector (Millipore Sigma, Burlington, Massachusetts, USA) by the Gibson Assembly method. BL21 (DE3) E. coli cells were subsequently transformed with the resulting cyclized DNA product via electroporation. After 45 min of recovery in Luria-Burtani (LB) media at 37° C., cells were plated onto LB plates with either 100 μg/mL ampicillin (Amp) and incubated overnight. Single colonies were used to inoculate 5 mL LB+100 μg/mL Amp, which were grown overnight at 37° C., 200 rpm. Expression cultures, typically 1 L of Terrific Broth (TB)+50 μg/mL Amp (TB-Amp), were inoculated from these starter cultures and shaken (200 rpm) at 37° C. After 3 hours (OD₆₀₀=−0.6), the expression cultures were chilled on ice. After 30 min on ice, expression is induced with 0.5 mM IPTG, and the cultures were expressed for 16 hours at 20° C. with shaking at 200 rpm. Cells were then harvested by centrifugation at 4,300×g at 4° C. for 15 min. Cell pellets were frozen and stored at −20° C. until purification.

Purification of ObiH, TmLTA, ScADH and CbFDH:

To purify each protein, cell pellets were thawed on ice and then resuspended in lysis buffer (50 mM potassium phosphate buffer (pH=8.0), 150 mM NaCl, and (for ObiH and TmLTA only) 400 μM pyridoxal 5′-phosphate (PLP). A volume of 4 mL of lysis buffer per gram of wet cell pellet was used. After resuspension, the cell suspension was placed on ice in a metal container and subjected to lysis using a sonication device at 50% power for 5 sec on and 10 sec off for a total time of 5 min. The resulting lysate was then spun down at 50,000×g to pellet cell debris. Ni/NTA beads (Gold Biotechnology, Inc. (doing business as “GoldBio”) St. Louis, Missouri, USA) were added to the supernatant and incubated on ice for 45 min prior to purification by Ni-affinity chromatography with a gravity column. The column was washed with 5 column volumes of 20 mM imidazole, 150 mM NaCl, 50 mM potassium phosphate buffer (pH=8.0). Washing with higher concentrations of imidazole resulted in slow protein elution. Each protein was eluted with 250 mM imidazole, 150 mM NaCl, 50 mM potassium phosphate buffer, pH 8.0. For ObiH, elution of the desired protein product was monitored by the disappearance of its bright red color (resulting from the release of ObiH) from the column. FDH and ADH are colorless and their elution was monitored by the addition of 1 μL of eluent to 50 μL of Bradford reagent and evaluating the presence of protein by color change in the reagent (brown to blue). The protein products were dialyzed to <1 μM imidazole in 50 mM potassium phosphate buffer (pH 8.0) with 150 mM NaCl. Purified enzyme was flash frozen in pellet form by pipetting enzyme dropwise into a crystallization dish filled with liquid nitrogen. The enzyme was transferred to a plastic conical and stored at −80° C. until further use. Frozen pellets were thawed at room temperature and centrifuged before use. The concentration of protein was determined by Bradford assay using bovine serum albumin for a standard concentration curve. Generally, this procedure yielded 200-250 mg per L culture for ObiH, 500-600 mg per L culture for TmLTA, 100-120 mg per L culture for FDH and 30-60 mg per L culture for ADH. Protein purity was analyzed by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis using 12% polyacrylamide gels.

Enzymes

L-Threonine Aldolase

L-threonine aldolase (L-TA) includes enzymes falling under Enzyme Commission (EC) number 4.1.2.5. In its native milieu, L-TAs catalyze the cleavage of L-threonine to yield acetaldehyde and glycine. Exemplary L-TA may have an amino acid sequence of SEQ ID NO: 1 or a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical thereto. The coding sequence of SEQ ID NO: 1 is shown as SEQ ID NO: 2.

SEQ ID NO: 1 TmLTA (Thermotoga maritima)

(SEQ ID NO: 1)

MIDLRSDTVTKPTEEMRKAMAQAEVGDDVYGEDPTINELERLAAETFGKE

AALFVPSGTMGNQVSIMAHTORGDEVILEADSHIFWYEVGAMAVLSGVMP

HPVPGKNGAMDPDDVRKAIRPRNIHFPRTSLIAIENTHNRSGGRVVPLEN

IKEICTIAKEHGINVHIDGARIFNASIASGVPVKEYAGYADSVMFCLSKG

LCAPVGSVVVGDRDFIERARKARKMLGGGMRQAGVLAAAGIIALTKMVDR

LKEDHENARFLALKLKEIGYSVNPEDVKTNMVILRTDNLKVNAHGFIEAL

RNSGVLANAVSDTEIRLVTHKDVSRNDIEEALNIFEKLERKFSLEHHHHH

H

SEQ ID NO: 2 TmLTA (Thermotoga maritima)

(SEQ ID NO: 2)

ATGATCGATCTCAGGTCCGACACCGTTACAAAACCAACAGAAGAGATGAG

AAAAGCCATGGCACAGGCTGAGGTGGGAGACGATGTGTACGGAGAAGATC

CAACCATCAACGAACTCGAAAGGCTCGCCGCAGAGACCTTTGGAAAGGAA

GCGGCTCTCTTTGTACCCTCCGGCACCATGGGAAATCAAGTGAGCATAAT

GGCTCACACCCAGAGGGGCGATGAAGTGATACTGGAGGCAGACAGCCACA

TCTTCTGGTACGAGGTCGGAGCCATGGCGGTTCTCTCCGGAGTCATGCCC

CATCCTGTACCTGGAAAAAATGGAGCCATGGACCCCGATGATGTGAGGAA

GGCCATAAGACCCAGAAACATACACTTCCCCAGAACTTCGCTCATTGCCA

TCGAAAACACACACAACCGTTCCGGTGGAAGAGTGGTCCCGCTTGAAAAC

ATAAAAGAGATTTGCACGATAGCCAAAGAACACGGCATAAACGTTCACAT

AGATGGTGCGAGGATCTTCAACGCCTCAATCGCTTCAGGTGTTCCCGTGA

AGGAGTACGCCGGGTACGCCGATTCCGTGATGTTCTGTCTTTCAAAAGGT

CTCTGCGCACCCGTCGGTTCGGTGGTTGTAGGAGACAGGGACTTCATAGA

AAGAGCGAGAAAGGCGAGAAAGATGCTCGGTGGAGGGATGAGACAGGCAG

GTGTTCTCGCTGCCGCTGGAATAATCGCCTTGACAAAGATGGTAGATCGA

TTGAAAGAAGATCATGAAAACGCCAGATTTCTCGCCCTGAAGTTGAAAGA

AATAGGGTACTCCGTGAATCCCGAAGATGTGAAAACCAACATGGTGATTC

TGAGGACCGACAACCTGAAGGTGAACGCGCACGGGTTCATAGAAGCGCTC

AGAAACAGCGGGGTGCTCGCGAACGCCGTATCCGACACGGAGATCAGACT

GGTAACCCACAAAGACGTTTCAAGAAACGACATAGAAGAGGCTCTGAACA

TCTTCGAAAAACTCTTCAGAAAATTCTCCCTCGAGCACCATCACCATCAC

CATTGA

L-Threonine Transaldolase

L-threonine transaldolase (L-T-transA) includes enzymes that in their native milieu catalyze the formation of secondary β-hydroxy α-amino acids. One representative group of L T-transA includes enzymes falling under EC number 2.2.1.4. Exemplary L-T-transA may have an amino acid sequence of SEQ ID NO: 3 or a sequence at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical thereto. The coding sequence of SEQ ID NO: 3 is shown as SEQ ID NO: 4.

SEQ ID NO: 3 ObiH (Pseudomonas fluorescens

ATCC 39502)

(SEQ ID NO: 3)

MGSSHHHHHHSSMSNVKQQTAQIVDWLSSTLGKDHQYREDSLSLTANENY

PSALVRLTSGSTAGAFYHCSFPFEVPAGEWHFPEPGHMNAIADQVRDLGK

TLIGAQAFDWRPNGGSTAEQALMLAACKPGEGFVHFAHRDGGHFALESLA

QKMGIEIFHLPVNPTSLLIDVAKLDEMVRRNPHIRIVILDQSFKLRWQPL

AEIRSVLPDSCTLTYDMSHDGGLIMGGVEDSPLSCGADIVHGNTHKTIPG

PQKGYIGFKSAQHPLLVDTSLWVCPHLQSNCHAEQLPPMWVAFKEMELFG

RDYAAQIVSNAKTLARHLHELGLDVTGESFGFTQTHQVHFAVGDLQKALD

LCVNSLHAGGIRSTNIEIPGKPGVHGIRLGVQAMTRRGMKEKDFEVVARF

IADLYFKKTEPAKVAQQIKEFLQAFPLAPLAYSFDNYLDEELLAAVYQGA

QR

SEQ ID NO: 4 ObiH (Pseudomonas fluorescens

ATCC 39502)

(SEQ ID NO: 4)

ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCATGTCAAACGTGAA

GCAGCAGACGGCGCAAATCGTAGATTGGTTGTCATCAACCTTGGGGAAAG

ATCACCAATACCGCGAGGACTCCCTTTCACTTACCGCTAACGAGAACTAC

CCGTCGGCATTAGTTCGTTTGACTTCAGGTTCGACCGCCGGCGCATTCTA

CCATTGTTCTTTCCCCTTCGAGGTTCCTGCCGGGGAGTGGCACTTCCCGG

AGCCCGGTCATATGAATGCAATTGCTGACCAGGTTCGTGATTTAGGTAAA

ACCTTGATTGGTGCCCAGGCATTCGACTGGCGTCCAAATGGCGGATCAAC

CGCAGAACAGGCACTTATGCTGGCAGCATGTAAACCGGGAGAGGGGTTCG

TCCATTTTGCTCACCGCGACGGAGGCCATTTCGCTTTAGAATCTCTTGCG

CAAAAGATGGGCATCGAAATTTTCCACTTGCCTGTTAATCCGACCTCTCT

GTTAATCGATGTCGCCAAATTGGATGAAATGGTCCGCCGCAACCCGCATA

TTCGCATTGTCATTCTTGATCAGAGCTTTAAGCTGCGCTGGCAACCCCTG

GCCGAGATTCGTTCAGTTTTACCAGACTCATGCACGTTGACTTATGATAT