In a further aspect, the plasmid can include an origin of replication, allowing it to use the host cell's replication machinery to create copies of itself.

As used herein, “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one affects the function of another. For example, if sequences for multiple genes are inserted into a single plasmid, their expression may be operably linked. Alternatively, a promoter is said to be operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence.

As used herein, “expression” refers to transcription and/or accumulation of an mRNA derived from a gene or DNA fragment. Expression may also be used to refer to translation of mRNA into a peptide, polypeptide, or protein.

In one aspect, the gene that expresses cellulose synthase is isolated from plants. In a different aspect, the gene that expresses cellulose synthase is isolated from algae. In one aspect, the algal species is a red algal species such as, for example, Pyropia yezoensis (also known as Porphyra yezoensis) or Griffithsia monilis. In a further aspect, the gene that expresses cellulose synthase has SEQ ID NO. 1 or at least 70% homology thereof, at least 75% homology thereof, at least 80% homology thereof, at least 85% homology thereof, at least 90% homology thereof, or at least 95% homology thereof. In a further aspect, the cellulose synthase is able to use mannose as a substrate instead of or in addition to glucose.

Other sequences expressing cellulose synthase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 1:

TABLE 1

Cellulose Synthase Genes

Source Organism
Sequence Description
GI Number

Porphyra vezoensis
cellulose synthase
EU279853.1

Porphyra vezoensis
cellulose synthase
EU279861.1

Porphyra vezoensis
cellulose synthase
EU279857.1

pseudogene

Porphyra vezoensis
cellulose synthase
EU279858.1

pseudogene

Porphyra vezoensis
cellulose synthase
EU279854.1

pseudogene

Porphyra vezoensis
cellulose synthase
EU279855.1

pseudogene

Chondrus crispus
cellulose synthase
XM_005715532.1

family

Porphyra vezoensis
cellulose synthase
EU279859.1

pseudogene

Porphyra vezoensis
cellulose synthase
EU279856.1

pseudogene

Porphyra vezoensis
cellulose synthase
EU279860.1

pseudogene

Gelidiella liqulata
cellulose synthase
KT920245.1

catalytic subunit A

Gelidiella acerosa
cellulose synthase
KT920242.1

catalytic subunit A

Parviphycus albertanoae
cellulose synthase
KT920246.1

catalytic subunit A

Parviphycus felicinii
cellulose synthase
KT920247.1

catalytic subunit A

Gelidiella incrassata
cellulose synthase
KT920244.1

catalytic subunit A

Pterocladiella melanoidea
cellulose synthase
KT920254.1

catalytic subunit A

Griffithsia monilis
cellulose synthase A
GU563823.1

Gelidiella fanii
cellulose synthase
KT920243.1

catalytic subunit A

Pterocladia rectangularis
cellulose synthase
KT920196.1

catalytic subunit A

Ptilophora mediterranea
cellulose synthase
KT920238.1

catalytic subunit A

Gelidium pacificum
cellulose synthase
KT920227.1

catalytic subunit A

Gelidium microdon
cellulose synthase
KT920223.1

catalytic subunit A

Gelidium johnstonii
cellulose synthase
KT920222.1

catalytic subunit A

Pterocladella bartletti
cellulose synthase
KT920250.1

catalytic subunit A

Pterocladia lucida
cellulose synthase
KT920248.1

catalytic subunit A

Gelidium declerckii
cellulose synthase
KT920214.1

catalytic subunit A

Ptilophora pterocladioides
cellulose synthase
KT920240.1

catalytic subunit A

Pterocladia lucida
cellulose synthase
KT920249.1

catalytic subunit A

Gelidium madagascariense
cellulose synthase
KT920195.1

catalytic subunit A

Gelidium sp. GHB-2012
cellulose synthase
KT920237.1

catalytic subunit A

Gelidium crinate
cellulose synthase
KT920212.1

catalytic subunit A

Ptilophora scalaramosa
cellulose synthase
KT920241.1

catalytic subunit A

Gelidium robustum
cellulose synthase
KT920234.1

catalytic subunit A

Gelidium purpurascens
cellulose synthase
KT920231.1

catalytic subunit A

Gelidium pusilium
cellulose synthase
KT920232.1

catalytic subunit A

Gelidium indonesianum
cellulose synthase
KT920218.1

catalytic subunit A

Callophyllis japonica
cellulose synthase
KT920257.1

catalytic subunit A

Gelidium nudifrons
cellulose synthase
KT920225.1

catalytic subunit A

Gelidium isabelae
cellulose synthase
KT920219.1

catalytic subunit A

Pterocladiella beachiae
cellulose synthase
KT920251.1

catalytic subunit A

Gelidium spinosum
cellulose synthase
KT920235.1

catalytic subunit A

Gelidium prostratum
cellulose synthase
KT920229.1

catalytic subunit A

Geladium minimum
cellulose synthase
KT920224.1

catalytic subunit A

Gelidium sp. SMB-2011a
cellulose synthase
KT920216.1

catalytic subunit A

Gelidium elegans
cellulose synthase
KT920215.1

catalytic subunit A

Gelidium coulteri
cellulose synthase
KT920211.1

catalytic subunit A

Gracilaria textorii
cellulose synthase
KT920258.1

catalytic subunit A

Gelidium corneum
cellulose synthase
KT920210.1

catalytic subunit A

Gelidium bernabei
cellulose synthase
KT920207.1

catalytic subunit A

Gelidium abbottiorum
cellulose synthase
KT920204.1

catalytic subunit A

Grateloupia asiatica
cellulose synthase
KT920259.1

catalytic subunit A

Ptilophora prolifera
cellulose synthase
KT920239.1

catalytic subunit A

Gelidium crispum
cellulose synthase
KT920213.1

catalytic subunit A

Chondrus crispus
cellulose synthase
XM_005711895.1

family

Gelidium rex
cellulose synthase
KT920233.1

catalytic subunit A

Gelidium pulchellum
cellulose synthase
KT920230.1

catalytic subunit A

Gelidium coreanum
cellulose synthase
KT920209.1

catalytic subunit A

Gelidium capense
cellulose synthase
KT920208.1

catalytic subunit A

Rhodymenia intricata
cellulose synthase
KT920260.1

catalytic subunit A

Gelidium asperum
cellulose synthase
KT920205.1

catalytic subunit A

Aphanta pachyrrhiza
cellulose synthase
KT920193.1

catalytic subunit A

Pterocladella nana
cellulose synthase
KT920255.1

catalytic subunit A

Gelidium ornamense
cellulose synthase
KT920226.1

catalytic subunit A

Gelidium hommersandii
cellulose synthase
KT920217.1

catalytic subunit A

Gelidium australe
cellulose synthase
KT920206.1

catalytic subunit A

Pterocladiella caerulescens
cellulose synthase
KT920252.1

catalytic subunit A

Gelidium jejuense
cellulose synthase
KT920221.1

catalytic subunit A

Gelidium japonicum
cellulose synthase
KT920220.1

catalytic subunit A

Acanthopeltis
cellulose synthase
KT920200.1

longiramulosa
catalytic subunit A

Capreolia implexa
cellulose synthase
KT920201.1

catalytic subunit A

Acanthopeltis japonica
cellulose synthase
KT920199.1

catalytic subunit A

Acanthopeltis hirsuta
cellulose synthase
KT920198.1

catalytic subunit A

Acanthopeltis hirsuta
cellulose synthase
KT920197.1

catalytic subunit A

Gelidium vagum
cellulose synthase
KT920236.1

catalytic subunit A

Aphanta sp. GHB-2016
cellulose synthase
KT920194.1

catalytic subunit A

Pterocladelia capillacea
cellulose synthase
KT920253.1

catalytic subunit A

Gelidium pristoides
cellulose synthase
KT920228.1

catalytic subunit A

Pterocladelia tenuis
cellulose synthase
KT920256.1

catalytic subunit A

Gelidium divaricatum
cellulose synthase
KT920202.1

catalytic subunit A

Aphanomyces astaci
hypothetical protein
XM_009832014.1

Aphanomyces invadans
hypothetical protein
XM_008862896.1

Acanthamoeba castellanii
putative cellulose
XM_004335119.1

str. Neff
synthase

Phytophthora sojae
hypothetical protein
XM_009526171.1

Phytophthora parasitica
hypothetical protein
XM_008915779.1

Aphanomyces invadans
hypothetical protein
XM_008862899.1

Saprolegnia diclina
hypothetical protein
XM_008616683.1

Phytophthora infestans
putative cellulose
XM_002897169.1

synthase catalytic

subunit

Phytophthora sojae
cellulose synthase 1
EF563997.1

Phytophthora infestans
cellulose synthase 3
EF563995.1

Jatropha curcas
sucrose transport
XM_012212213.2

protein

Jatropah curcas
sucrose transport
XM_012212212.2

protein

Jatropha curcas
sucrose transport
XM_012212211.2

protein

Jatropha curcas
sucrose transport
XM_012212210.2

protein

Jatropha curcas
sucrose transport
NM_001319920.1

protein

Saprolegnia parasitica
hypothetical protein
XM_012347851.1

Protopolystoma xenopodis
unidentified
LM730806.1

Aphanomyces invadans
hypothetical protein
XM_008869048.1

Uncultured bacterium
unidentified
JX649872.1

A1Q1

Plasmopara viticola
cellulose synthase 1
GQ258973.1

Pyrus × bretschneideri
H2 finger protein
XM_009348167.2

ATL20-like

In one aspect, the gene that expresses galactomannan galactosyltransferase is isolated from a plant. In one aspect, the galactomannan galactosyltransferase is able to catalyze the synthesis of bonds between an oligo- or poly-mannose backbone and pendant galactose moieties to produce carbo sugars. In a further aspect, the plant is Oryza japonica, Medicago truncatula, Glycine max, Trigonella foenum-graecum, Lotus japonicus, Senna occidentalis, Cucumis sativus, Fragaria vesca, or Cyamopsis tetragonoloba. In a further aspect, the galactomannan galactosyltransferase has SEQ ID NOs. 2-4 or at least 70% homology thereof. In a further aspect, the gene that expresses galactomannan galactosyltransferase has SEQ ID NO. 4 or at least 70% homology thereof, at least 75% homology thereof, at least 80% homology thereof, at least 85% homology thereof, at least 90% homology thereof, or at least 95% homology thereof.

Other sequences expressing galactomannan galactosyltransferase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 2:

TABLE 2

Galactomannan Galactosyltransferase Genes

Source Organism
Sequence Description
GI Number

Cyamopsis tetragonoloba
galactomannan
AJ938067.1

galactosyltransferase

Glycine max
galactomannan
XM_003525821.3

galactosyltransferase

1-like

Medicago truncatula
unidentified
AC140720.21

Medicago truncatula
galactosyl transferase
XM_003608493.2

Lotus japonicus
galactomannan
AJ567668.1

galactosyltransferase

Lupinus angustifolius
galactomannan
XM_019567307.1

galactosyltransferase

1-like

Lupinus angustifolius
galactomannan
XM_019567302.1

galactosyltransferase

1-like

Trigonella foenum-graecum
alpha
AJ245478.1

galactosyltransferase

Arachis ipaensis
galactomannan
XM_016346122.2

galactosyltransferase 1

Arachis ipaensis
galactomannan
XM_016346121.2

galactosyltransferase 1

Ziziphus jujuba
galactomannan
XM_016034856.1

galactosyltransferase

1-like

Ziziphus jujuba
galactomannan
XM_016034855.1

galactosyltransferase

1-like

Arachis duranensis
galactomannan
XM_016108311.2

galactosyltransferase 1

Arachis duranensis
galactomannan
XM_016108310.2

galactosyltransferase 1

Glycine max
galactomannan
XM_003539215.3

galactosyltransferase

1-like

Glycine max
galactomannan
XM_006590559.2

galactosyltransferase

1-like

Glycine max
unidentified
AC235306.1

Glycine max
unidentified
AK245471.1

Senna occidentalis
galactomannan
AJ938068.1

galactosyltransferase

Phaseolus vulgaris
hypothetical protein
XM_007156638.1

Glycine max
galactomannan
XM_003517306.3

galactosyltransferase

Juglans regia
putative
XM_018958015.1

glycosyltransferase 7

Prunus mume
galactomannan
XM_008239498.2

galactosyltransferase

1-like

Prunus mume
galactomannan
XM_008239489.2

galactosyltransferase

1-like

Prunus persica
galactomannan
XM_007205133.2

galactosyltransferase 1

Iponoea nil
glycosyltransferase
XM_019317795.1

6-like

Vigna radiata var.
galactomannan
XM_014665934.1

radiata
galactosyltransferase

1-like

Lupinus angustifolius
galactomannan
XM_019589398.1

galactosyltransferase

1-like

Lupinus angustifolius
galactomannan
XM_019589395.1

galactosyltransferase

1-like

Lupinus angustifolius
galactomannan
XM_019589394.1

galactosyltransferase

1-like

Nelumbo nucifera
putative
XM_010274767.2

glycosyltransferase 7

Vigna angularis
galactomannan
XM_017570547.1

galactosyltransferase

1-like

Vigna angularis var.
unidentified
AP015043.1

angularis

Pyrus × bretschneideri
galactomannan
XM_009364320.2

galactosyltransferase

Vitis vinifera
unidentified
AM447747.2

Morus notabilis
galactomannan
XM_010108622.1

galactosyltransferase 1

Malus domestica
galactomannan
NM_001328775.1

galactosyltransferase

1-like

Malus × domestica
galactomannan
XM_008363913.2

galactosyltransferase

1-like

Malus × domestica
unidentified
AB627270.1

Malus × domestica
unidentified
HM122522.1

Cucumis sativus
galactomannan
XM_004141806.2

galactosyltransferase

1-like

Malus × domestica
galactomannan
XM_008349663.1

galactosyltransferase

1-like

Cajanus cajan
galactomannan
XM_020374857.1

galactosyltransferase

1-like

Cajanus cajan
galactomannan
XM_020374856.1

galactosyltransferase

1-like

Capsicum annum
putative
XM_016705445.1

glycosyltransferase 7

Malus × domestica
unidentified
HM122524.1

Fragaria vesca
galactomannan
XM_004288217.2

galactosyltransferase

1-like

Solanum tuberosum
putative
XM_006363751.2

glycosyltransferase 7

Solanum tuberosum
putative
XM_006363750.2

glycosyltransferase 7

Medicago truncatula
galactosyl transferase
XM_003611508.2

Medicago truncatula
unidentified
CT573500.2

Solanum lycopersicum
putative
XM_004231889.3

glycosyltransferase 7

Solanum lycopersicum
unidentified
AC226502.1

Lycopersicon esculentum
unidentified
BT013963.1

Sesamum indicum
glycosyltransferase
XM_011101097.2

6-like

Cucumis melo
galactomannan
XM_008442195.2

galactosyltransferase

1-like

Cucumis melo
unidentified
LN713262.1

Cucumis melo
unidentified
LN681875.1

Solanum lycopersicum
unidentified
HG975514.1

Solanum pennellii
putative
XM_015208067.1

glycosyltransferase 7

Solanum pennellii
unidentified
HG975441.1

Sesamum indicum
glycosyltransferase 6
XM_011075379.2

Coffea arabica
galactomannan
EU568117.1

galactosyltransferase

Cicer arietinum
galactomannan
XM_012718954.1

galactosyltransferase

1-like

Theobroma cacao
putative
XM_007047507.2

glycosyltransferase 7

Theobroma cacao
unidentified
LT594788.1

Erythranthe guttatus
glycosyltransferase
XM_012998642.1

6-like

Populus trichocarpa
galactosyltransferase
XM_002310854.2

family protein

Prunus persica
glycosyltransferase 6
XM_007208119.2

Nicotiana attenuata
putative
XM_019380314.1

glycosyltransferase 7

Gossypium arboreum
putative
XM_017765398.1

glycosyltransferase 7

Gossypium arboreum
putative
XM_017765397.1

glycosyltransferase 7

Gossypium hirsutum
putative
XM_016879483.1

glycosyltransferase 7

Gossypium hirsutum
putative
XM_016879482.1

glycosyltransferase 7

Gossypium raimondii
putative
XM_012581704.1

glycosyltransferase 7

Gossypium raimondii
putative
XM_012581703.1

glycosyltransferase 7

Nicotiana tomentosiformis
putative
XM_009626576.2

glycosyltransferase 7

Prunus mume
glycosyltransferase
XM_008239940.2

6-like

Populus euphratica
putative
XM_011033007.1

glycosyltransferase 7

Populus euphratica
putative
XM_011017963.1

glycosyltransferase 7

Musa acuminata subsp.
probable
XM_009396086.2

malaccensis
glycosyltransferase 7

Daucus carota subsp.
putative
XM_017387245.1

sativus
glycosyltransferase 7

Daucus carota subsp.
putative
XM_017387244.1

sativus
glycosyltransferase 7

Gossypium hirsutum
putative
XM_016818092.1

glycosyltransferase 7

Gossypium raimondii
putative
XM_012614197.1

glycosyltransferase 7

Arabis alpina
unidentified
LT669794.1

Eucalyptus grandis
putative
XM_010029779.2

glycosyltransferase 7

Nicotiana tabacum
putative
XM_016593700.1

glycosyltransferase 7

Nicotiana sylvestris
putative
XM_009763295.1

glycosyltransferase 7

Gossypium hirsutum
putative
XM_016864575.1

glycosyltransferase 7

Gossypium arboreum
galactomannan
XM_017774502.1

galactosyltransferase 1

Gossypium hirsutum
putative
XM_016850999.1

glycosyltransferase 7

Gossypium hirsutum
glycosyltransferase
XM_016841848.1

6-like

Gossypium raimondii
glycosyltransferase
XM_012636540.1

6-like

Gossypium arboreum
putative
XM_017768866.1

glycosyltransferase 7

Raphanus sativus
glycosyltransferase
XM_018611230.1

6-like

Jatropha curcas
galactomannan
XM_012232035.2

galactosyltransferase 1

Ricinus communis
galactomannan
XM_002513376.2

galactosyltransferase 1

Raphanus sativus
glycosyltransferase 6
XM_018626243.1

Prunus mume
putative
XM_008227859.1

glycosyltransferase 7

A lipase is an esterase that catalyzes the hydrolysis of fats, oils, and lipids. In one aspect, the gene that expresses lipase is isolated from a bacterium. In a further aspect, the bacterium is a Micrococcus species, a Pseudomonas species, a Moraxella species, or an Acinetobacter species. In a further aspect, the gene that expresses lipase has SEQ ID NO. 9 or at least 70% homology thereof, at least 75% homology thereof, at least 80% homology thereof, at least 85% homology thereof, at least 90% homology thereof, or at least 95% homology thereof. In a further aspect, the cellulose synthase is able to use mannose as a substrate instead of or in addition to glucose. In one aspect, the gene that expresses lipase can be positioned anywhere in the DNA construct disclosed herein. In one aspect, the gene that expresses lipase is positioned 5′ (i.e., prior) to the gene that expresses cellulose synthase.

Other sequences expressing lipase or related or homologous genes can be identified in a database such as, for example, GenBank. In one aspect, sequences useful herein include those with the GI numbers listed in Table 3:

TABLE 3

Lipase Genes

Source Organism
Sequence Description
GI Number

Micrococcus sp. HL-2003
lipase gene
AY268069.1

Pseudomonas sp.
esterase gene
M68491.1

Moraxella L1
lipase 1
X53053.1

A. calcoaceticus
carboxylesterase and
X74839.1

peptidyl prolyl-cis-

trans-isomerase

Acinetobacter sp. ADP1
genomic DNA
CR543861.1

A. calcoaceticus
esterase
X71598.1

Pseudomonas trivialis
genomic DNA
CP011507.1

Pseudomonas azotoformans
genomic DNA
CP019856.1

Pseudomonas
genomic DNA
LT629689.1

extremaustralis

Pseudomonas fluorescens
genomic DNA
CP005975.1

Pseudomonas fluorescens
genomic DNA
CP010896.1

Pseudomonas fluorescens
genomic DNA
AF228666.1

Pseudomonas simiae
genomic DNA
CP007637.1

Pseudomonas fluorescens
genomic DNA
AM181176.4

Pseudomonas Antarctica
genomic DNA
CP015600.1

Pseudomonas fluorescens
genomic DNA
CP015639.1

Pseudomonas fluorescens
genomic DNA
LT907842.1

Pseudomonas sp. NS1
genomic DNA
CP022960.1

Pseudomonas poae
genomic DNA
LT629706.1

Pseudomonas poae
genomic DNA
CP004045.1

Pseudomonas rhodesiae
genomic DNA
LT629801.1

Pseudomonas trivialis
genomic DNA
LT629760.1

Pseudomonas azotoformans
genomic DNA
LT629702.1

Pseudomonas Antarctica
genomic DNA
LT629704.1

Pseudomonas fluorescens
genomic DNA
CP012400.1

Pseudomonas azotoformans
genomic DNA
CP014546.1

Pseudomonas yamanorum
genomic DNA
LT629793.1

Pseudomonas prosekii
genomic DNA
LT629762.1

Pseudomonas koreensis
genomic DNA
CP014947.1

Pseudomonas libanensis
genomic DNA
LT629699.1

Pseudomonas sp. GR 6-02
genomic DNA
CP011567.1

Pseudomonas fluorescens
genomic DNA
CP014868.1

Pseudomonas fluorescens
genomic DNA
CP011117.1

Pseudomonas fluorescens
genomic DNA
S69066.1

Pseudomonas cedrina
genomic DNA
LT629753.1

Pseudomonas sp. bs2935
genomic DNA
LT629744.1

Pseudomonas fluorescens
genomic DNA
CP017296.1

Pseudomonas sp. WCS374
genomic DNA
CP007638.1

Pseudomonas fluorescens
genomic DNA
CP003041.1

Pseudomonas corrugate
genomic DNA
LT629798.1

Pseudomonas corrugate
genomic DNA
CP014262.1

Pseudomonas mediterranea
genomic DNA
LT629790.1

Pseudomonas tolaasii
genomic DNA
CP020369.1

Pseudomonas fluorescens
genomic DNA
CP015638.1

Pseudomonas fluorescens
genomic DNA
CP015637.1

Pseudomonas sp. TKP
genomic DNA
CP006852.1

Synthetic construct
carboxylesterase
HM212419.1

Synthetic construct
carboxylesterase
FJ213454.1

Pseudomonas sp. FDAARGOS
genomic DNA
CP023969.1

380

Pseudomonas synxantha
genomic DNA
LT629786.1

Pseudomonas orientalis
genomic DNA
LT629782.1

Pseudomonas sp.
genomic DNA
LN854573.1

URMO17WK12:I11

In another aspect, said construct further includes d) a promoter, e) a terminator or stop sequence, f) a gene that confers resistance to an antibiotic (a “selective marker”), g) a reporter protein, or a combination thereof.

In another aspect, the DNA construct has the following genetic components: (1) a promoter, (2) a gene that expresses cellulose synthase, (3) a gene that expresses galactomannan galactosyltransferase, and (4) a terminator or stop sequence.

In an alternative aspect, the DNA construct has the following genetic components: (1) a promoter, (2) a gene that expresses lipase, (3) a gene that expresses cellulose synthase, (4) a gene that expresses galactomannan galactosyltransferase, and (5) a terminator or stop sequence.

In one aspect, the regulatory sequence is already incorporated into a vector such as, for example, a plasmid, prior to genetic manipulation of the vector. In another aspect, the regulatory sequence can be incorporated into the vector through the use of restriction enzymes or any other technique known in the art.

In one aspect, the regulatory sequence is a promoter. The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence. In one aspect, the coding sequence to be controlled is located 3′ to the promoter. In another aspect, the promoter is derived from a native gene. In an alternative aspect, the promoter is composed of multiple elements derived from different genes and/or promoters. A promoter can be assembled from elements found in nature, from artificial and/or synthetic elements, or from a combination thereof. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, at different stages of development, in response to different environmental or physiological conditions, and/or in different species. In one aspect, the promoter functions as a switch to activate the expression of a gene.

In one aspect, the promoter is “constitutive.” A constitutive promoter is a promoter that causes a gene to be expressed in most cell types at most times. In another aspect, the promoter is “regulated.” A regulated promoter is a promoter that becomes active in response to a specific stimulus. A promoter may be regulated chemically, such as, for example, in response to the presence or absence of a particular metabolite (e.g., lactose or tryptophan), a metal ion, a molecule secreted by a pathogen, or the like. A promoter also may be regulated physically, such as, for example, in response to heat, cold, water stress, salt stress, oxygen concentration, illumination, wounding, or the like.

Promoters that are useful to drive expression of the nucleotide sequences described herein are numerous and familiar to those skilled in the art. Suitable promoters include, but are not limited to, the following: T3 promoter, T7 promoter, an iron promoter, and GAL1 promoter. In one aspect, the promoter has SEQ ID NO. 5 or at least 70% homology thereof of the plasmid pYES2. Variants of these promoters are also contemplated. The skilled artisan will be able to use site-directed mutagenesis and/or other mutagenesis techniques to modify the promoters to promote more efficient function. The promoter may be positioned, for example, from 10-100 nucleotides of a ribosomal binding site. In one aspect, the promoter may be native to the vectors described herein. In another aspect, the promoter is positioned before the gene that expresses cellulose synthase, galactomannan galactosyltransferase, or a combination thereof.

In one aspect, the promoter is a GAL1 promoter. In another aspect, the GAL1 promoter is native to the plasmid used to create the vector. In another aspect, a GAL1 promoter is positioned before the gene that expresses cellulose synthase and the gene that expresses galactomannan galactosyltransferase. In another aspect, the promoter is a GAL1 promoter obtained from or native to the pYES2 plasmid.

In another aspect, the regulatory sequence is a terminator or stop sequence. As used herein, a terminator is a sequence of DNA that marks the end of a gene or operon to be transcribed. In a further aspect, the terminator is an intrinsic terminator or a Rho-dependent transcription terminator. As used herein, an intrinsic terminator is a sequence wherein a hairpin structure can form in the nascent transcript that disrupts the mRNA/DNA/RNA polymerase complex. As used herein, a Rho-dependent transcription terminator requires a Rho factor protein complex to disrupt the mRNA/DNA/RNA polymerase complex. In one aspect, the terminator is a T7 terminator. In an alternative aspect, the terminator is a CYC1 terminator obtained from or native to the pYES2 plasmid.

In a further aspect, the regulatory sequence includes both a promoter and a terminator or stop sequence. In a still further aspect, the regulatory sequence can include multiple promoters or terminators. Other regulatory elements, such as enhancers, are also contemplated. Enhancers may be located from about 1 to about 2000 nucleotides in the 5′ direction from the start codon of the DNA to be transcribed, or may be located 3′ to the DNA to be transcribed. Enhancers may be “cis-acting,” that is, located on the same molecule of DNA as the gene whose expression they affect.

In one aspect, when the vector is a plasmid, the plasmid can also contain a multiple cloning site or polylinker. In a further aspect, the polylinker contains recognition sites for multiple restriction enzymes. The polylinker can contain up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 recognition sites for restriction enzymes. Further, restriction sites may be added, disabled, or removed as required, using techniques known in the art. In one aspect, the plasmid contains restriction sites for any known restriction enzyme such as, for example, HindIII, KpnI, SacI, BamHI, BstXI, EcoRI, BsaBI, NotI, XhoI, SphI, XbaI, ApaI, SaII, ClaI, EcoRV, PstI, SmaI, XmaI, SpeI, EagI, SacII, or any combination thereof. In a further aspect, the plasmid contains more than one recognition site for the same restriction enzyme.

In one aspect, the restriction enzyme can cleave DNA at a palindromic or an asymmetrical restriction site. In a further aspect, the restriction enzyme cleaves DNA to leave blunt ends; in an alternative aspect, the restriction enzyme cleaves DNA to leave “sticky” or overhanging ends. In another aspect, the enzyme can cleave DNA a distance of from 20 bases to over 1000 bases away from the restriction site. A variety of restriction enzymes are commercially available and their recognition sequences, as well as instructions for use (e.g. amount of DNA needed, precise volumes of reagents, purification techniques, as well as information about salt concentration, pH, optimum temperature, incubation time, and the like) are provided by enzyme manufacturers.

In one aspect, a plasmid with a polylinker containing one or more restriction sites can be digested with one restriction enzyme and a nucleotide sequence of interested can be ligated into the plasmid using a commercially-available DNA ligase enzyme. Several such enzymes are available, often as kits containing all reagents and instructions required for use. In another aspect, a plasmid with a polylinker containing two or more restriction sites can be simultaneously digested with two restriction enzymes and a nucleotide sequence of interest can be ligated into the plasmid using a DNA ligase enzyme. Using two restriction enzymes provides an asymmetric cut in the DNA, allowing for insertion of a nucleotide sequence of interest in a particular direction and/or on a particular strand of the double-stranded plasmid. Since RNA synthesis from a DNA template proceeds from 5′ to 3′, usually starting just after a promoter, the order and direction of elements inserted into a plasmid can be especially important. If a plasmid is to be simultaneously digested with multiple restriction enzymes, these enzymes must be compatible in terms of buffer, salt concentration, and other incubation parameters.

In some aspects, prior to ligation using a ligase enzyme, a plasmid that has been digested with a restriction enzyme is treated with an alkaline phosphatase enzyme to remove 5′ terminal phosphate groups. This prevents self-ligation of the plasmid and thus facilitates ligation of heterologous nucleotide fragments into the plasmid.

In one aspect, different genes can be ligated into a plasmid in one pot. In this aspect, the genes will first be digested with restriction enzymes. In certain aspects, the digestion of genes with restriction enzymes provides multiple pairs of matching 5′ and 3′ overhangs that will spontaneously assemble the genes in the desired order. In another aspect, the genes and components to be incorporated into a plasmid can be assembled into a single insert sequence prior to insertion into the plasmid. In a further aspect, a DNA ligase enzyme can be used to assist in the ligation process.

In another aspect, the ligation mix may be incubated in an electromagnetic chamber. In one aspect, this incubation lasts for about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or about 1 hour.

The DNA construct described herein can be part of a vector. In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with the hosts. The vector ordinarily carries a replication site as well as marking sequences that are capable of performing phenotypic selection in transformed cells. Plasmid vectors are well known and are commercially available. Such vectors include, but are not limited to, pWLneo, pSV2cat, pOG44, pXT1, pSG (Strategene), pSVK3, pBSK, pBR322, pYES, pYES2, pBSKII, pUC, and pUC19 vectors.

Plasmids are double-stranded, autonomously-replicating, genetic elements that are not integrated into host cell chromosomes. Further, these genetic elements are usually not part of the host cell's central metabolism. In bacteria, plasmids may range from 1 kilobase (kb) to over 200 kb. Plasmids can be engineered to encode a number of useful traits including the production of secondary metabolites, antibiotic resistance, the production of useful proteins, degradation of complex molecules and/or environmental toxins, and others. Plasmids have been the subject of much research in the field of genetic engineering, as plasmids are convenient expression vectors for foreign DNA in, for example, microorganisms. Plasmids generally contain regulatory elements such as promoters and terminators and also usually have independent replication origins. Ideally, plasmids will be present in multiple copies per host cell and will contain selectable markers (such as genes for antibiotic resistance) to allow the skilled artisan to select host cells that have been successfully transfected with the plasmids (for example, by growing the host cells in a medium containing the antibiotic).

In certain aspects, the DNA construct includes a ribosomal binding site. In one aspect, the ribosomal binding site in the DNA construct is AGGAGG or a derivative or variant thereof. In one aspect, the ribosomal binding site is native to the vector used herein. In certain aspects, when the DNA construct further includes a ribosomal switch. In one aspect, the ribosomal switch is SEQ ID NO. 6 or at least 70% homology thereof. The ribosomal binding site and optional ribosomal switch are positioned after the gene for galactomannan galactosyltransferase from 5′ to 3′.

In another aspect, the DNA construct includes a terminator. In one aspect, the terminator in the DNA construct is a known stop codon (TAA, TAG, TGA) or a derivative or variant thereof. In a further aspect, the terminator is native to the vector in which the DNA construct is incorporated. The terminator is positioned after the gene for galactomannan galactosyltransferase from 5′ to 3′.

In one aspect, the vector encodes a selection marker. In a further aspect, the selection marker is a gene that confers resistance to an antibiotic. In certain aspects, during fermentation of host cells transformed with the vector, the cells are contacted with the antibiotic. For example, the antibiotic may be included in the culture medium. Cells that have not been successfully transformed cannot survive in the presence of the antibiotic; only cells containing the vector which confers antibiotic resistance can survive. Optimally, only cells containing the vector to be expressed will be cultured, as this will result in the highest production efficiency of the desired gene products (e.g., peptides). Cells that do not contain the vector would otherwise compete with transformed cells for resources. In one aspect, the antibiotic is tetracycline, neomycin, kanamycin, ampicillin, hygromycin, chloramphenicol, amphotericin B, bacitracin, carbapenam, cephalosporin, ethambutol, fluoroquinolones, isonizid, methicillin, oxacillin, vancomycin, streptomycin, quinolines, rifampin, rifampicin, sulfonamides, cephalothin, erythromycin, streptomycin, gentamycin, penicillin, other commonly-used antibiotics, or a combination thereof.

In certain aspects, the DNA construct can include a gene that expresses a reporter protein. The selection of the reporter protein can vary. For example, the reporter protein can be a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In one aspect, the reporter protein is a yellow fluorescent protein and the gene that expresses the reporter protein has SEQ ID NO. 7 or at least 70% homology thereof. The amount of fluorescence that is produced by the biological device can be correlated to the amount of DNA incorporated into the plant cells. The fluorescence produced by the device can be detected and quantified using techniques known in the art. For example, spectrofluorometers are typically used to measure fluorescence. The Examples provide exemplary procedures for measuring the amount of fluorescence as a result of the expression of DNA.

FIGS. 1-3B provide non-limiting examples of DNA constructs described herein. In one aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a T7 promoter; (b) a gene that expresses cellulose synthase; (c) a gene that expresses galactomannan galactosyltransferase; (d) a T7 terminator or stop codon. In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a Gall promoter; (b) a gene that expresses cellulose synthase; (c) a gene that expresses galactomannan galactosyltransferase; (d) a CYC1 terminator or stop codon.

In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a Gall promoter; (b) a gene that expresses cellulose synthase; (c) a CYC1 terminator; (d) a Gall promoter; (e) a gene that expresses galactomannan galactosyltransferase; and (f) a CYC1 terminator.

In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a Gall promoter; (b) a gene that expresses lipase; (c) a CYC1 terminator; (d) a Gall promoter; (b) a gene that expresses cellulose synthase; (e) a CYC1 terminator; (f) a Gall promoter; (g) a gene that expresses galactomannan galactosyltransferase; and (h) a CYC1 terminator.

In one aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a T7 promoter; (b) a gene that expresses lipase, (c) a gene that expresses cellulose synthase; (d) a gene that expresses galactomannan galactosyltransferase; (e) a T7 terminator or stop codon. In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses lipase, (c) a gene that expresses cellulose synthase; (d) a gene that expresses galactomannan galactosyltransferase; (e) a CYC1 terminator or stop codon.

In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses lipase, (c) a gene that expresses cellulose synthase; (d) a CYC1 terminator; (e) a Gall promoter; (f) a gene that expresses galactomannan galactosyltransferase; and (g) a CYC1 terminator.

In one aspect, the construct comprises from 5′ to 3′ the following genetic components in the following order: (a) a T7 promoter having SEQ ID NO 5 or at least 70% homology thereof; (b) a gene that expresses cellulose synthase having SEQ ID NO 1 or at least 70% homology thereof; (c) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO 4 or at least 70% homology thereof; (d) a ribosomal binding site; and (e) a terminator or stop codon.

In another aspect, the construct is a plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a T7 promoter having SEQ ID NO 5 or at least 70% homology thereof; (b) a gene that expresses cellulose synthase having SEQ ID NO 1 or at least 70% homology thereof; (c) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO 4 or at least 70% homology thereof; (d) a ribosomal binding site; and (e) a terminator or stop codon.

In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a T7 promoter having SEQ ID NO 5 or at least 70% homology thereof; (b) a gene that expresses cellulose synthase having SEQ ID NO 1 or at least 70% homology thereof; (c) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO 4 or at least 70% homology thereof; (d) a ribosomal binding site; and (e) a terminator or stop codon.

In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses cellulose synthase having SEQ ID NO 1 or at least 70% homology thereof; (c) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO 4 or at least 70% homology thereof; (d) a ribosomal binding site; and (e) a terminator or stop codon.

In one aspect, the construct comprises from 5′ to 3′ the following genetic components in the following order: (a) a T7 promoter having SEQ ID NO 5 or at least 70% homology thereof; (b) a gene that expresses lipase having SEQ ID NO. 9 or at least 70% homology thereof, (c) a gene that expresses cellulose synthase having SEQ ID NO 1 or at least 70% homology thereof; (d) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO 4 or at least 70% homology thereof; (e) a ribosomal binding site; and (f) a terminator or stop codon.

In another aspect, the construct is a plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a T7 promoter having SEQ ID NO 5 or at least 70% homology thereof; (b) a gene that expresses lipase having SEQ ID NO. 9 or at least 70% homology thereof; (c) a gene that expresses cellulose synthase having SEQ ID NO 1 or at least 70% homology thereof; (d) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO 4 or at least 70% homology thereof; (e) a ribosomal binding site; and (f) a terminator or stop codon.

In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a T7 promoter having SEQ ID NO 5 or at least 70% homology thereof; (b) a gene that expresses lipase having SEQ ID NO. 9 or at least 70% homology thereof; (c) a gene that expresses cellulose synthase having SEQ ID NO 1 or at least 70% homology thereof; (d) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO 4 or at least 70% homology thereof; (e) a ribosomal binding site; and (f) a terminator or stop codon.

In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses lipase having SEQ ID NO. 9 or at least 70% homology thereto; (c) a gene that expresses cellulose synthase having SEQ ID NO 1 or at least 70% homology thereof; (d) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO 4 or at least 70% homology thereof; (e) a ribosomal binding site; and (f) a terminator or stop codon.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses cellulose synthase and (b) a gene that expresses galactomannan galactosyltransferase.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses cellulose synthase; (c) a gene that expresses galactomannan galactosyltransferase; and (d) a CYC1 terminator or stop codon.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses lipase, (b) a gene that expresses cellulose synthase, and (c) a gene that expresses galactomannan galactosyltransferase.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses lipase, (c) a gene that expresses cellulose synthase; (d) a gene that expresses galactomannan galactosyltransferase; and (e) a CYC1 terminator or stop codon.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses cellulose synthase; (c) a CYC1 terminator; (d) a Gall promoter; (e) a gene that expresses galactomannan galactosyltransferase; and (f) a CYC1 terminator.

In another aspect, the construct includes the following genetic components: (a) a T7 promoter; (b) a gene that expresses cellulose synthase having SEQ ID NO. 1 or at least 70% homology thereof; and (c) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO. 4 or at least 70% homology thereof.

In another aspect, the construct includes the following genetic components: (a) a Gal1 promoter; (b) a gene that expresses cellulose synthase having SEQ ID NO. 1 or at least 70% homology thereof; and (c) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO. 4 or at least 70% homology thereof.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses cellulose synthase having SEQ ID NO. 1 or at least 70% homology thereof; and (c) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO. 4 or at least 70% homology thereof.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses cellulose synthase; (c) a CYC1 terminator; (d) a Gal1 promoter; (e) a gene that expresses galactomannan galactosyltransferase; and (f) a CYC1 terminator.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses cellulose synthase having SEQ ID NO. 4 or at least 70% homology thereof; (c) a CYC1 terminator; (d) a Gall promoter; (e) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO. 1 or at least 70% homology thereof; and (f) a CYC1 terminator.

In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses cellulose synthase or at least 70% homology thereof; (c) a CYC1 terminator; (d) a Gal1 promoter; (e) a gene that expresses galactomannan galactosyltransferase or at least 70% homology thereof; and (f) a CYC1 terminator.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a Gall promoter; (b) a gene that expresses cellulose synthase; (c) a CYC1 terminator; (d) a Gall promoter; (e) a gene that expresses galactomannan galactosyltransferase; and (f) a CYC1 terminator.

In another aspect, the construct includes the following genetic components: (a) a T7 promoter; (b) a gene that expresses cellulose synthase having SEQ ID NO. 1 or at least 70% homology thereof; and (c) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO. 4 or at least 70% homology thereof.

In another aspect, the construct includes the following genetic components: (a) a Gall promoter; (b) a gene that expresses lipase having SEQ ID NO 9 or at least 70% homology thereof; (c) a gene that expresses cellulose synthase having SEQ ID NO. 1 or at least 70% homology thereof; and (d) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO. 4 or at least 70% homology thereof.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses lipase having SEQ ID NO. 9 or at least 70% homology thereof, (c) a gene that expresses cellulose synthase having SEQ ID NO. 1 or at least 70% homology thereof; and (d) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO. 4 or at least 70% homology thereof.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses lipase, (c) a gene that expresses cellulose synthase; (d) a CYC1 terminator; (e) a Gall promoter; (f) a gene that expresses galactomannan galactosyltransferase; and (g) a CYC1 terminator.

In another aspect, the construct includes from 5′ to 3′ the following genetic components in the following order: (a) a Gal1 promoter; (b) a gene that expresses lipase having SEQ ID NO. 9 or at least 70% homology thereof; (c) a gene that expresses cellulose synthase having SEQ ID NO. 4 or at least 70% homology thereof; (d) a CYC1 terminator; (e) a Gall promoter; (f) a gene that expresses galactomannan galactosyltransferase having SEQ ID NO. 1 or at least 70% homology thereof; and (g) a CYC1 terminator.

In another aspect, the construct is a pYES2 plasmid having from 5′ to 3′ the following genetic components in the following order: (a) a Gall promoter; (b) a gene that expresses lipase or at least 70% homology thereof; (c) a gene that expresses cellulose synthase or at least 70% homology thereof; (d) a CYC1 terminator; (e) a Gall promoter; (f) a gene that expresses galactomannan galactosyltransferase or at least 70% homology thereof; and (g) a CYC1 terminator.

In one aspect, the construct has SEQ ID NO 8, which is depicted in FIGS. 3A and 3B.

Exemplary methods for producing the DNA constructs described herein are provided in the Examples. Restriction enzymes and purification techniques known in the art can be used to assemble the DNA constructs. Backbone plasmids and synthetic inserts can be mixed together for ligation purposes at different ratios ranging from 1:1, 1:2, 1:3, 1:4, and up to 1:5. In one aspect, the ratio of backbone plasmid to synthetic insert is 1:4. After the vector comprising the DNA construct has been produced, the resulting vector can be incorporated into the host cells using the methods described below.

II. Biological Devices

In one aspect, a “biological device” is formed when a microbial cell is transfected with the DNA construct described herein. The biological devices are generally composed of microbial host cells, where the host cells are transformed with a DNA construct described herein.

In one aspect, the DNA construct is carried by the expression vector into the cell and is separate from the host cell's genome. In another aspect, the DNA construct is incorporated into the host cell's genome. In still another aspect, incorporation of the DNA construct into the host cell enables the host cell to produce carbo sugars. “Heterologous” genes and proteins are genes and proteins that have been experimentally inserted into a cell that are not normally expressed by that cell. A heterologous gene may be cloned or derived from a different cell type or species than the recipient cell or organism. Heterologous genes may be introduced into cells by transduction or transformation.

An “isolated” nucleic acid is one that has been separated from other nucleic acid molecules and/or cellular material (peptides, proteins, lipids, saccharides, and the like) normally present in the natural source of the nucleic acid. An “isolated” nucleic acid may optionally be free of the flanking sequences found on either side of the nucleic acid as it naturally occurs. An isolated nucleic acid can be naturally occurring, can be chemically synthesized, or can be a cDNA molecule (i.e., is synthesized from an mRNA template using reverse transcriptase and DNA polymerase enzymes).

“Transformation” or “transfection” as used herein refers to a process for introducing heterologous DNA into a host cell. Transformation can occur under natural conditions or may be induced using various methods known in the art. Many methods for transformation are known in the art and the skilled practitioner will know how to choose the best transformation method based on the type of cells being transformed. Methods for transformation include, for example, viral infection, electroporation, lipofection, chemical transformation, and particle bombardment. Cells may be stably transformed (i.e., the heterologous DNA is capable of replicating as an autonomous plasmid or as part of the host chromosome) or may be transiently transformed (i.e., the heterologous DNA is expressed only for a limited period of time).

“Competent cells” refers to microbial cells capable of taking up heterologous DNA. Competent cells can be purchased from a commercial source, or cells can be made competent using procedures known in the art. Exemplary procedures for producing competent cells are provided in the Examples.

The host cells as referred to herein include their progeny, which are any and all subsequent generations formed by cell division. It is understood that not all progeny may be identical due to deliberate or inadvertent mutations. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.

A transformed cell includes the primary subject cell and its progeny. The host cells can be naturally-occurring cells or “recombinant” cells. Recombinant cells are distinguishable from naturally-occurring cells in that naturally-occurring cells do not contain heterologous DNA introduced through molecular cloning procedures. In one aspect, the host cell is a prokaryotic cell such as, for example, Escherichia coli. In other aspects, the host cell is a eukaryotic cell such as, for example, the yeast Saccharomyces cerevisiae. Host cells transformed with the DNA construct described herein are referred to as “biological devices.”

The DNA construct is first delivered into the host cell. In one aspect, the host cells are naturally competent (i.e., able to take up exogenous DNA from the surrounding environment). In another aspect, cells must be treated to induce artificial competence. This delivery may be accomplished in vitro, using well-developed laboratory procedures for transforming cell lines. Transformation of bacterial cell lines can be achieved using a variety of techniques. One method involves calcium chloride. The exposure to the calcium ions renders the cells able to take up the DNA construct. Another method is electroporation. In this technique, a high-voltage electric field is applied briefly to cells, producing transient holes in the membranes of the cells through which the vector containing the DNA construct enters. Another method involves exposing intact yeast cells to alkali cations such as, for example, lithium. In one aspect, this method includes exposing yeast to lithium acetate, polyethylene glycol, and single-stranded DNA such as, for example, salmon sperm DNA. Without wishing to be bound by theory, the single-stranded DNA is thought to bind to the cell wall of the yeast, thereby blocking plasmids from binding. The plasmids are then free to enter the yeast cell. Enzymatic and/or electromagnetic techniques can also be used alone, or in combination with other methods, to transform microbial cells. Exemplary procedures for transforming yeast and bacteria with specific DNA constructs are provided in the Examples. In certain aspects, two or more types of DNA can be incorporated into the host cells. Thus, different metabolites can be produced from the same host cells at enhanced rates.

III. Preparation of Carbo Sugars

The biological devices described herein are useful in the production of carbo sugars. Once the DNA construct has been incorporated into the host cell, the cells are cultured such that the cells multiply. A satisfactory microbiological culture contains available sources of hydrogen donors and acceptors, carbon, nitrogen, sulfur, phosphorus, inorganic salts, and, in certain cases, vitamins or other growth-promoting substances. For example, the addition of peptone provides a readily-available source of nitrogen and carbon. Furthermore, the use of different types of media results in different growth rates and different stationary phase densities; stationary phase is where secondary metabolite production occurs most frequently. A rich media results in a short doubling time and higher cell density at a stationary phase. Minimal media results in slow growth and low final cell densities. Efficient agitation and aeration increase final cell densities.

In one aspect, host cells may be cultured or fermented by any method known in the art. The skilled practitioner will be able to select a culture medium based on the species and/or strain of host cell selected. In certain aspects, the culture medium will contain a carbon source. A variety of carbon sources are contemplated, including, but not limited to: monosaccharides such as glucose and fructose, disaccharides such as lactose or sucrose, oligosaccharides, polysaccharides such as starch, or mixtures thereof. Unpurified mixtures extracted from feedstocks are also contemplated and include molasses, barley malt, and related compounds and compositions. Other glycolytic and tricarboxylic acid cycle intermediates are also contemplated as carbon sources, as are one-carbon substrates such as carbon dioxide and/or methanol in the cases of compatible organisms. The carbon source utilized is limited only by the particular organism being cultured.

Culturing or fermenting of host cells may be accomplished by any technique known in the art. In one aspect, batch fermentation may be conducted. In batch fermentation, the composition of the culture medium is set at the beginning and the system is closed to future artificial alterations. In some aspects, a limited form of batch fermentation may be carried out, wherein factors such as oxygen concentration and pH are manipulated, but additional carbon is not added. Continuous fermentation methods are also contemplated. In continuous fermentation, equal amounts of a defined medium are continuously added to and removed from a bioreactor. In other aspects, microbial host cells are immobilized on a substrate. Fermentation may be carried out on any scale and may include methods in which literal “fermentation” is carried out as well as other culture methods which are non-fermentative.

In one aspect, the method involves growing the biological devices described herein for a sufficient time to produce carbo sugars. The ordinary artisan will be able to choose a culture medium and optimum culture conditions based on the biological identity of the host cells. In a further aspect, a salt or electrolyte can optionally be added to the culture medium. Without wishing to be bound by theory, the salt can help to solubilize the carbo sugar as it is produced. In one aspect, the salt is present in solution at about a 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, or 5% concentration. In a still further aspect, the salt is KCl. In this aspect, the presence of KCl can cause the appearance of a transient, transparent blue color when placed in solution with the carbo sugar. Exemplary procedures for producing carbo sugars using the biological devices described herein are provided in the Examples. In one aspect, the amount of carbo sugar produced by the microbial host cells as described above is from 1.1 to 10-fold greater than the amount of carbo sugar produced by nontransformed (control) microbial cells of the same species and strain. In a further aspect, the carbo sugar produced by the microbial host cells as described above is free from lignin.

In certain aspects, after culturing the biological device to produce the carbo sugar, the host cells of the device can be lysed with one or more enzymes. For example, when the host cells are yeast, the yeast cells can be lysed with lyticase. In one aspect, the lyticase concentration can be 500, 600, 700, 800, 900, or 1000 μL per liter of culture, where any value can be the lower and upper end-point of a range (e.g., 500 to 900 μL, 600 to 800 μL, etc.).

In addition to enzymes, other components can be used to facilitate lysis of the host cells. In one aspect, chitosan can be used in combination with an enzyme to lyse the host cells. Chitosan is generally composed of glucosamine units and N-acetylglucosamine units and can be chemically or enzymatically extracted from chitin, which is a component of arthropod exoskeletons and fungal and microbial cell walls. In certain aspects, the chitosan can be acetylated to a specific degree of acetylation in order to enhance tissue growth during culturing as well as metabolite production. In one aspect, the chitosan is from 60% to about 100%, 70% to 90%, 75% to 85%, or about 80% acetylated. The molecular weight of the chitosan can vary, as well. For example, the chitosan comprises about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 glucosamine units and/or N-acetylglucosamine units. In another aspect, the chitosan includes 5 to 7 glucosamine units and/or N-acetylglucosamine units. In one aspect, chitosan can be added until a concentration of 0.0015, 0.0025, 0.0050, 0.0075, 0.01, 0.015, 0.02, 0.03, 0.04, or 0.05, where any value can be a lower and upper end-point of a range (e.g., 0.005 to 0.02, 0.0075 to 0.015, etc.) is achieved in the culture. Still further in this aspect, the chitosan is present at a concentration of 0.01%.

In a further aspect, the carbo sugar can be collected, separated from the microbial cells (lysed or intact), and/or purified through any technique known in the art such as, for example, precipitation, centrifugation, filtration, and the like. In one aspect, the carbo sugar can be purified via microfiltration to remove impurities. In one aspect, the microfilter has a pore size of 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.35 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, or 0.8 μm, where any value can be a lower and upper end-point of a range (e.g., 0.3 μm to 0.5 μm). The Examples provide an exemplary procedure for producing and purifying the carbo sugars described herein.

In another aspect, the carbo sugar can be chemically-modified to produce additional desirable properties. Alternatively compositions composed of the carbo sugar with lysed and/or intact host cells (e.g., yeast) can be used herein, where it is not necessary to separate the host cells and other components from the carbo sugar.

In one aspect, the carbo sugars can be produced in 24-48 hours from transformed host cells at a cost of approximately $0.50 per kilogram. In another aspect, the carbo sugar is provided in solution. In an alternative aspect, the carbo sugar is provided in powdered or dried form.

IV. Applications of the Carbo Sugars

In certain aspects, the carbo sugars can be dissolved in an aqueous electrolyte solution. The electrolyte solution can be, for example, aqueous potassium chloride, sodium chloride, or calcium chloride. In one aspect, the electrolyte solution is a 2% solution of potassium chloride in water at pH 7-8. The pH of the electrolyte solution may be adjusted using any of a variety of acids, bases, or buffer systems known in the art.

In other aspects, the presence of metal ions, crosslinking, temperature, solution pH, and polymer concentration can also affect the viscosity of solutions containing the carbo sugars described herein. In one aspect, crosslinking reactions can be performed to link the side chains of the carbo sugars to one another.

In one aspect, the carbo sugars disclosed herein are free from impurities such as soy impurities and pesticide residues that are known to cause adverse reactions such as allergies or illnesses in some human subjects.

In one aspect, the carbo sugars disclosed herein can be used in the petroleum industry. In a further aspect, the carbo sugars can be included as a component of a hydraulic fracturing fluid, as a component of a drilling fluid, or to alter the viscosity of a crude petroleum product. For example, the carbo sugar as an aqueous solution can be used alone or in combination with other components as a fracturing fluid typically used in hydraulic fracturing.

In another aspect, the carbo sugars disclosed herein can be used in thickening textile and carpet dyes, facilitating paper processing, waterproofing and/or gelling in explosive compositions, binding pharmaceutical tablets, thickening cosmetics and toiletry preparations, controlling the viscosity of fire retardants, drug delivery, flocculating and/or flotation in metallurgy and/or mining applications, or as an additive in the food industry. In a further aspect, the carbo sugars disclosed herein can be used as a thickener, stabilizer, binder, or texturizing agent in food products such as baked goods, dairy products, meat, condiments, canned goods, ice cream, hot cereals, and the like.

In some aspects, compositions including carbo sugars can include other ingredients such as, for example, electrolyte solutions, fillers, particulates, surfactants, and the like. In certain aspects, compositions including the carbo sugars can be used as fluids for hydraulic fracturing. In some aspects, the carbo sugars used for hydraulic fracturing can further include petroleum oil.

In one aspect, surfactants can act in concert with the carbo sugar to alter the viscosity and friction properties of petroleum samples and compositions. The surfactant can be of any type including, for example, a cationic surfactant, an anionic surfactant, a nonionic surfactant, an amphiphilic surfactant, a zwitterionic surfactant, or a combination thereof. In one aspect, the surfactant is a nonionic surfactant such as, for example, ethoxylated alcohols, polysorbate 20, or polysorbate 80. In another aspect, the surfactant is a zwitterionic surfactant such as, for example, soy lecithin. In another aspect, the fillers and particulates can be used as proppants during hydraulic fracturing. As used herein, “proppant” refers to any material that can keep a hydraulic fracture open. Proppants may exert their effects either during or after hydraulic fracturing. Proppants include materials such as sand, ceramic particles, glass, bauxite, and combinations thereof.

In certain aspects, the carbo sugars described and disclosed herein can be used to alter the viscosity of petroleum oil. In one aspect, the carbo sugars can be directly mixed with petroleum oil. In another aspect, the carbo sugars can first be diluted with a solution of an electrolyte, then mixed with petroleum oil. In another aspect, the carbo sugars can be mixed with guar derived from plant-based sources prior to being placed into contact with petroleum oil. In a further aspect, compositions composed of the carbo sugars described herein can additionally include a surfactant.

In one aspect, contacting petroleum oil with carbo sugar compositions disclosed herein can alter the API gravity of the petroleum oil. “API gravity” as used herein refers to a set of standards developed by the American Petroleum Institute to classify the density of petroleum liquids compared to water. API gravity corresponds to grades of oil; light crude oil typically has API gravity of 31.1° API, medium crude oil between 22.3 and 31.1° API, and heavy crude oil below 22.3° API. API gravity can be derived from density which can be measured by methods such as, for example, ASTM D1298 or ASTM D4052.

In a further aspect, the altered API gravity can be increased; that is, the viscosity of the petroleum oil is reduced through contact with the carbo sugar compositions. In certain aspects, alteration of the viscosity of the petroleum oil facilitates downstream processing of the petroleum oil.

In another aspect, the carbo sugars described herein can be used for the bioremediation of polluted water and soil. For example, the carbo sugars can be used for the biodegradation of petroleum products that have contaminated the environment. In another aspect, the carbo sugars described herein can be used for the biodegradation of petroleum products present in soil and the surface water as well as for the bioremediation and enhancement of soil condition to support healthy plant growth.

Pollution of the environment, particularly contamination of water and soil, results in extraordinary damage to the environment and loss of vitality and productivity. Such pollution and general degradation also causes enormous economic loss to fishing, farming and tourist industries. The occurrence of large scale petroleum pollution is a frequent occurrence, and a number of potential solutions for bioremediation of petroleum pollution have been tried or proposed. For example, current cleanup practices include: 1) physical removal of the polluting petroleum by use of absorption media, 2) dispersal using detergents, 3) burning, 4) microbial degradation under ideal certain conditions, 5) agglomeration of oil on water and sinking to the floor of the water body, and 6) use of organic chemicals, such as kerosene based dispersants, to dissolve and disperse the oil. All of these methodologies are expensive to implement and have very limited usefulness. Further, even under ideal conditions these methods are often hazardous to carry out and extremely difficult to use to effectively clean up the pollution. In general therefore, bioremediation of soil and water are extremely difficult and expensive tasks. The carbo sugars described herein provide a viable alternative for the bioremediation of environmental contaminants.

In one aspect, the carbo sugars described herein can be used to degrease water mixed with petroleum oil or another fatty substance. In another aspect, the carbo sugars described herein can be used to degrease a surface coated with petroleum oil or another fatty substance. The term “degrease” as used herein is the ability of the carbo sugars described herein to remove petroleum oil or fatty substances from water or a surface. In one aspect, the carbo sugar removes up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 95%, or up to 99% of the petroleum oil or fatty substances from water or the surface. In one aspect, biological devices expressing lipase can catalyze the hydrolysis of fatty substances contaminating water or a surface.

In one aspect, the carbo sugar as an aqueous solution is added to the petroleum-containing water. In one aspect, the concentration of the carbo sugar is 0.1, 0.5%, 0.75%, 1%, 2%, 3%, 4%, or 5% by weight, where any value can be the lower and upper end-point of a range (e.g., 0.1 to 3%, 0.5 to 2%, etc.). In another aspect, the volume ratio of petroleum-containing water to carbo sugar (1% concentration) is1: 1, 1:2, 1:3, 1:4, or 1:5, where any ratio can be the lower and upper end-point of a range (e.g., 1:1 to 1:4, 1:2 to 1:3, etc.). As demonstrated in the examples, the carbo sugar when added to the petroleum-containing water separates the oil from the water, which then can be easily removed from the water using techniques known in the art.

In one aspect, the water to be decontaminated is from the ocean, ground water, a lake, a river, a stream, or another natural water supply. In an alternative aspect, the water to be treated is waste water from a petroleum drilling operation or is from a reservoir or municipal water supply.

In another aspect, the surface to be degreased is a natural surface such as stone, wood, compacted dirt, animal fur or feathers or skin, or the like. In still another aspect, the surface to be degreased is a manmade surface such as, for example, glass or glassware, metal, concrete, asphalt, a building, a vehicle, oil drilling equipment, a road or path, a household object, or the like. In a further aspect, the fatty substance to be removed from the water or surface is petroleum or petroleum derived, is a plant fat or animal fat such as, for example, food waste or cooking oil. In the food industry, the carbo sugars produced herein can be used to degrease surfaces coated with cooking oils and fats. For example, frying pans and other utensils coated with cooking oils and grease can be degreased with the carbo sugars. In other aspects, grease traps for collecting and storing cooking oil and grease can be degreased with the carbo sugars herein.

In another aspect, the carbo sugars produced herein can be used to produce polyurethane compositions that have numerous applications.

In one aspect, the polyurethane composition is produced by:

- a. admixing the carbo sugar produced herein and a natural oil polyol to produce a first admixture; and
- b. reacting the first admixture with a polyisocyanate to produce the polyurethane composition.

A “natural oil” as used herein is any oil extracted from a living organism. In one aspect, the living organism is a plant or alga. In a further aspect, the plant is the castor bean or castor oil plant (Ricinus communis). In another aspect, the living organism is an animal. In an alternative aspect, the living organism is a fungus. Natural oils can additionally contain triglycerides, fatty acids, fatty acid esters, sterols, isoprenoid or terpenoid compounds, alkaloids, phenols, and other metabolites.

“Natural oil polyols” are compounds derived from or present in natural oils that include at least one free hydroxyl group. A natural oil polyol may be naturally occurring, as with the ricinoleic acid in castor oil, or it may be chemically synthesized from an oil or fat containing one or more carbon-carbon double bonds. In one aspect, a natural fatty acid or triglyceride containing a carbon-carbon double bond is subjected to ozonolysis to cleave the double bond, followed by treatment with another molecule such as, for example, ethylene glycol, to form an alcohol. In another aspect, a natural fatty acid or triglyceride containing a carbon-carbon double bond can be epoxidized and treated with a nucleophile to generate an alcohol. In still another aspect, a natural fatty acid or triglyceride containing a carbon-carbon double bond can be formylated in the presence of carbon monoxide and hydrogen gas, followed by hydrogenation to generate a hydroxyl group. Other methods of producing natural oil polyols are also contemplated. Natural oils can be used as extracted or can optionally be purified. In one aspect, the natural oil polyol is or is derived from soy, a chemically-modified vegetable oil, a carbohydrate, lignin, cork, cashew nutshell liquid, Lesquerella oil, or a combination thereof. In one aspect, the natural oil polyol is castor oil. In another aspect, the natural oil polyol is ricinoleic acid. In still another aspect, the natural oil polyol is coriolic acid or a chemically-modified fatty acid.

“Castor oil” can optionally be extracted from the seeds of the castor oil plant. The primary component of castor oil is ricinoleic acid; minor components include oleic acid, linoleic acid, linolenic acid, stearic acid, palmitic acid, dihydroxystearic acid, and other trace fatty acids.

In one aspect, the natural polyol can include one or more hydroxy fatty acids, which is defined herein as a fatty acid having at least at least one free hydroxyl group. The hydroxy fatty acid has the general formula R′C(O)OH, wherein R′ is a saturated or unsaturated hydrocarbon chain having from 10 to 25 carbon atoms, and at least one hydroxyl group is covalently attached to a carbon atom of the hydrocarbon chain. The hydrocarbon can be linear or branched. In the case when the hydrocarbon is unsaturated, the hydrocarbon can have one carbon-carbon double bond or multiple carbon-carbon double bonds. Examples of monohydroxy fatty acids (i.e., one hydroxyl group present on the fatty acid) include, but are not limited to, hydroxynervonic acid, cerebronic acid, 10-hydroxy-20 decenoic acid, hydrox-2-decenoic acid 10-phosphate, strophantus acid, lesquerolic acid, densipolic acid, auricolic acid, a-dimorphecolic acid, kamlolenic acid, 8-hydroxyoctadeca-9.11-diynoic acid, 8-hydroxyoctadeca-17-en-9.11-diynoic acid (isanolic), or 8-hydroxyoctadeca-13.17-dien-9.11-diynoic acid. Examples of polyhydroxy fatty acids (i.e., two or more hydroxyl groups) include, but are not limited to, axillarenic acid, tetrapedic acids, byrsonic acid, 9,10-dihydroxyoctadecanoic acid, phaseolic acid, phloionolic acid, Resolvin D1, 10,17S-docosatriene, or Resolvin E1. The hydroxy fatty acids can be sued as is in the natural oil (e.g., castor oil), isolated from a natural oil, or synthesized accordingly.

In certain aspects, chitosan can be used to produce the polyurethane compositions described herein, where it is admixed with the carbo sugar and a natural oil polyol to produce the first admixture. “Chitosan” as used herein is a linear polymer of randomly-distributed GlcN and NAG residues. Chitosan may be obtained from chitin using alkali extraction or by any other technique known in the art. In one aspect, chitin is “deacetylated” using alkali to produce chitosan. In one aspect, deacetylation can remove some or all of the acetyl groups from the NAG residues of chitin. In one aspect, the chitin is from about 50% to about 100% acetylated. In a further aspect, the chitin is from about 50% to about 80% acetylated, 60% to about 80% acetylated, or 70% to about 80% acetylated. In a still further aspect, the chitosan is about 50% acetylated, about 60% acetylated, or about 77% acetylated. The molecular weight of the chitosan can vary. For example, the chitosan can contain about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any range thereof, of GlcN and/or NAG units. In another aspect, the chitosan can include 5 to 7 GlcN and/or NAG units. In certain aspects, the inclusion of chitosan can impart anti-microbial properties to the polyurethane composition.

In certain aspects, a surfactant can be used to produce the polyurethane compositions described herein, where it is admixed with the carbo sugar and a natural oil polyol to produce the first admixture. A “surfactant” is an organic compound that may be derived from a natural product, or may result from chemical modification of a natural product, or may be completely chemically synthesized. Surfactants typically contain hydrophilic head groups and hydrophobic tails. In one aspect, the head group is anionic, cationic, non-ionic, or zwitterionic. In another aspect, the tail is composed of a hydrocarbon or a glucoside. Surfactants alter the surface tension of liquids and may form micelles or bilayers in aqueous solution. Many applications of surfactants are known in the art. Surfactants are, for example, commonly employed as emulsifiers, detergents, wetting agents, and in other related uses.

Numerous cationic surfactants can be used in the compositions described herein. In one aspect, the cationic surfactant can be a quaternary ammonium salt.

Numerous zwitterionic surfactants can be used in the compositions described herein. In one aspect, the zwitterionic surfactant can be a lecithin such as soy lecithin; in another aspect, the zwitterionic surfactant can be a hydroxysultaine, a betaine, a sulfobetaine, or a mixture thereof. Among betaines, surfactants may be selected from the group comprising high alkyl betaines such as cetyl dimethyl carboxymethyl betaine, cocamidopropyl betaine, cocobetaine, coco dimethyl carboxymethyl betaine, lauryl amidopropyl betaine, lauryl bis-(2-hydroxyethyl) carboxymethyl betaine, lauryl bis-(2-hydroxypropyl) alpha-carboxyethyl betaine, lauryl dimethyl alphacarboxyethyl betaine, lauryl dimethyl carboxymethyl betaine, oleyl betaine, oleyl dimethyl gamma-carboxypropyl betaine, stearyl bis-(2-hydroxypropyl) carboxymethyl betaine, and mixtures thereof. Among sulfobetaines, surfactants may be selected from the group comprising coco dimethyl sulfopropyl betaine, lauryl bis-(2-hydroxyethyl) sulfopropyl betaine, lauryl dimethyl sulfoethyl betaine, stearyl dimethyl sulfopropyl betaine, and mixtures thereof. Amidobetaines and amidosulfobetaines are also contemplated.

Numerous nonionic surfactants can be used in the compositions described herein. Nonionic surfactants useful in the compositions described herein include alkoxylated fatty acid esters, alkyl glucosides, alkyl polyglucosides, amine oxides, alcohol ethoxylates, cocoamine oxide, glyceryl monohydroxystearate, glyceryl stearate, hydroxy stearic acid, lauramine oxide, laureth-2, polyhydroxy fatty acid amides, polyoxyalkylene stearates, propylene glycol stearate, sorbitan monostearate, sucrose cocoate, sucrose esters, sucrose laurate, steareth-2, PEG-40 hydrogenated castor oil, and mixtures thereof. Preferred nonionic surfactants include those based on polyethoxylated sorbitan and oleic acid such as, for example, polysorbate 80 and polysorbate 20, both of which are available under a variety of trade names.

Further nonionic surfactants contemplated herein include, in one aspect, the nonionic surfactants include the condensation products of a higher aliphatic alcohol, such as a fatty alcohol, containing about 8 to about 20 carbon atoms, in a straight or branched chain configuration, condensed with about 3 to about 100 moles, preferably about 5 to about 40 moles, most preferably about 5 to about 20 moles of ethylene oxide. Examples of such nonionic ethoxylated fatty alcohol surfactants are the Tergitol™ 15-S series from Union Carbide and Brij™ surfactants from ICI. Tergitol™ 15-S Surfactants include C₁₁-C₁₅secondary alcohol polyethylene glycol ethers. Brij™ 97 surfactant is Polyoxyethylene(10) oleyl ether; Brij™ 58 surfactant is polyoxyethylene(20) cetyl ether; and Brij™ 76 surfactant is polyoxyethylene(10) stearyl ether.

In another aspect, a useful class of nonionic surfactants includes the polyethylene oxide condensates of one mole of alkyl phenol containing from about 6 to 12 carbon atoms in a straight or branched chain configuration, with about 3 to about 100 moles, preferably about 5 to about 40 moles, most preferably about 5 to about 20 moles of ethylene oxide to achieve the above defined HLB. Examples of nonreactive nonionic surfactants are the Igepal™ CO and CA series from Rhone-Poulenc. Igepal™ CO surfactants include nonylphenoxy poly(ethyleneoxy)ethanols. Igepal™ CA surfactants include octylphenoxy poly(ethyleneoxy)ethanols. Still another useful class of hydrocarbon nonionic surfactants includes block copolymers of ethylene oxide and propylene oxide or butylene oxide with HLB values of about 6to about 19, preferably about 9 to about 18, and most preferably about 10 to about 16. Examples of such nonionic block copolymer surfactants are the Pluronic™ and Tetronic™ series of surfactants from BASF. Pluronic™ surfactants include ethylene oxide-propylene oxide block copolymers. Tetronic™ surfactants include ethylene oxide-propylene oxide block copolymers. In other aspects, the nonionic surfactants include sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters and polyoxyethylene stearates having HLBs of about 6 to about 19, about 9 to about 18, and about 10 to about 16. Examples of such fatty acid ester nonionic surfactants are the Span™, Tween™, and Myj™ surfactants from ICI. Span™ surfactants include C12-C18 sorbitan monoesters. Tween™ surfactants include poly(ethylene oxide) C₁₂-C₁₈sorbitan monoesters. Myj™ surfactants include poly(ethylene oxide) stearates. In one aspect, the nonionic surfactant can include polyoxyethylene alkyl ethers, polyoxyethylene alkyl-phenyl ethers, polyoxyethylene acyl esters, sorbitan fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkylamides, polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene octylphenyl ether, polyoxyethylene nonylphenyl ether, polyethylene glycol laurate, polyethylene glycol stearate, polyethylene glycol distearate, polyethylene glycol oleate, oxyethylene-oxypropylene block copolymer, sorbitan laurate, sorbitan stearate, sorbitan distearate, sorbitan oleate, sorbitan sesquioleate, sorbitan trioleate, polyoxyethylene sorbitan laurate, polyoxyethylene sorbitan stearate, polyoxyethylene sorbitan oleate, polyoxyethylene laurylamine, polyoxyethylene laurylamide, laurylamine acetate, hard beef tallow propylenediamine dioleate, ethoxylated tetramethyldecynediol, fluoroaliphatic polymeric ester, polyether-polysiloxane copolymer, and the like.

Numerous anionic surfactants can be used herein. In one aspect, the anionic surfactant can be selected from the group comprising alcohol phosphates and phosphonates, alkyl alkoxy carboxylates, alkyl aryl sulfates, alkyl aryl sulfonates, alkyl carboxylates, alkyl ether carboxylates, alkyl ether sulfates, alkyl ether sulfonates, alkyl phosphates, alkyl polyethoxy carboxylates, alkyl polyglucosides, alkyl polyglucoside sulfates, alkyl polyglucoside sulfonates, alkyl succinamates, alkyl sulfates, alkyl sulfonates, aryl sulfates, aryl sulfonates, fatty taurides, isethionates, N-acyl taurates, nonoxynol phosphates, octoxynol phosphates, sarcosinates, sulfated fatty acid esters, taurates, and mixtures thereof. Useful anionic surfactants include, but are not limited to, alkali metal and (alkyl) ammonium salts of: 1) alkyl sulfates and sulfonates such as sodium dodecyl sulfate, sodium 2-ethylhexyl sulfate, and potassium dodecanesulfonate; 2) sulfates of polyethoxylated derivatives of straight or branched chain aliphatic alcohols and carboxylic acids; 3) alkylbenzene or alkylnaphthalene sulfonates and sulfates such as sodium laurylbenzene-4-sulfonate and ethoxylated and polyethoxylated alkyl and aralkyl alcohol carboxylates; 5) glycinates such as alkyl sarcosinates and alkyl glycinates; 6) sulfosuccinates including dialkyl sulfosuccinates; 7) isothionate derivatives; 8) N-acyltaurine derivatives such as sodium N methyl-N-oleyltaurate); 9) amine oxides including alkyl and alkylamidoalkyldialkylamine oxides; and 10) alkyl phosphate mono or di-esters such as ethoxylated dodecyl alcohol phosphate ester, sodium salt. Representative commercial examples of suitable anionic sulfonate surfactants include, for example, sodium lauryl sulfate, available as TEXAPON™ L-100 from Henkel Inc., Wilmington, Del., or as POLYSTEP™ B-3 from Stepan Chemical Co, Northfield, Ill.; sodium 25 lauryl ether sulfate, available as POLYSTEPTM B-12 from Stepan

Chemical Co., Northfield, Ill.; ammonium lauryl sulfate, available as STANDAPOL™. A from Henkel Inc., Wilmington, Del.; and sodium dodecyl benzene sulfonate, available as SIPONATE™ DS-10 from Rhone-Poulenc, Inc., Cranberry, N.J., dialkyl sulfosuccinates, having the trade name AEROSOL™ OT, commercially available from Cytec Industries, West Paterson, N.J.; sodium methyl taurate (available under the trade designation NIKKOL™ CMT30 from Nikko Chemicals Co., Tokyo, Japan); secondary alkane sulfonates such as Hostapur™ SAS which is a Sodium (C14-C17) secondary alkane sulfonates (alpha-olefin sulfonates) available from Clariant Corp., Charlotte, N.C.; methyl-2-sulfoalkyl esters such as sodium methyl-2-sulfo(C12-16) ester and disodium 2-sulfo(C12-C16) fatty acid available from Stepan Company under the trade designation ALPHASTE™ PC48 ; alkylsulfoacetates and alkylsulfosuccinates available as sodium laurylsulfoacetate (under the trade designation LANTHANOL™ LAL) and disodiumlaurethsulfosuccinate (STEPANMILD™ SL3), both from Stepan Company; alkylsulfates such as ammoniumlauryl sulfate commercially available under the trade designation STEPANOL™ AM from Stepan Company, and/or dodecylbenzenesulfonic acid sold under BIO-SOFT® AS-100 from Stepan Chemical Co. In one aspect, the surfactant can be a disodium alpha olefin sulfonate, which contains a mixture of C₁₂to C₁₆sulfonates. In one aspect, CALSOFT™ AOS-40 manufactured by Pilot Corp. can be used herein as the surfactant. In another aspect, the surfactant is DOWFAX 2A1 or 2G manufactured by Dow Chemical, which are alkyl diphenyl oxide disulfonates. Representative commercial examples of suitable anionic phosphate surfactants include a mixture of mono-, di- and tri-(alkyltetraglycolether)-o-phosphoric acid esters generally referred to as trilaureth-4-phosphate commercially available under the trade designation HOSTAPHAT™ 340KL from Clariant Corp., as well as PPG-5 cetyl 10 phosphate available under the trade designation CRODAPHOS™ SG from Croda Inc., Parsipanny, N.J. Representative commercial examples of suitable anionic amine oxide surfactants those commercially available under the trade designations AMMONYX™ LO, LMDO, and CO, which are lauryldimethylamine oxide, laurylamidopropyldimethylamine oxide, and cetyl amine oxide, all from Stepan Company.

In one aspect, a surfactant is chosen based on its ability to form a stable emulsion containing an acidic aqueous solution of a polysaccharide and a natural oil polyol. In a further aspect, the concentration of surfactant can be from 0.001% to 1% (v/v), or is about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.35%, 0.5%, or 1% (v/v) with respect to the final emulsion volume. In another aspect, 0.35% of polysorbate 80 is used. In a further aspect, emulsion formation can be evaluated as function of stirring time (e.g., about 1 minute, about 2 minutes, about 4 minutes, about 6 minutes, about 8 minutes, or about 10 minutes) and/or stirring speed (e.g., about 2,000 rpm, about 5,000 rpm, about 10,000 rpm, or about 20,000 rpm).

The order in which the carbo sugar and natural oil polyol can be admixed with one another to produce the first admixture can vary. In one aspect, the natural oil polyol can be added to a solution of the carbo sugar. In one aspect, the natural oil polyol is added over time (e.g., 2 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, or 10 minutes) with stirring (2,000 rpm, 5,000 rpm, 10,000 rpm, or 20,000 rpm) to create a final admixture that also incorporates the carbo sugar. In one aspect, the natural oil polyol is castor oil and stirring is conducted at 10,000 rpm for 5 minutes.

In one aspect, wherein the carbo sugar is from 0.1 to 1% by weight of the first admixture. In another aspect, the amount of carbo sugar is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 wt % of the first admixture, where any value can be a lower or upper endpoint of a range (e.g., 0.2 to 0.7, etc.). In another aspect, the carbo sugar can be prepared and used as a solution. In one aspect, the carbo sugar is an aqueous solution of 1 to 5% w/v, where the first admixture includes 20% to 80% (v/v) of the aqueous solution of carbo sugar.

In one aspect, the natural oil polyol is from 20% to 80% (v/v) of the first admixture. In another aspect, the natural oil polyol is from 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% (v/v) of the first admixture, where any value can be a lower or upper endpoint of a range (e.g., 40% to 60%, etc.).

Prior to the addition of the polyisocyanate, additional components can be added to the first admixture of carbo sugar and natural oil polyol. In one aspect, a catalyst can be added to the first admixture. A “catalyst” as used herein is any substance that can increase the rate of a chemical reaction. In one aspect, the catalyst is not consumed in the reaction. A single molecule of a catalyst can assist with multiple chemical reactions. Catalysts useful herein include, but are not limited to, tertiary amines such as dimethylethanolamine (DMAE), triethylenediamine (DABCO), 3-aminopropyldimethylamine (DMAPA), dimethylcyclohexylamine (DMCHA); compounds containing hydroxyl groups or secondary amines such as, for example, propylene glycol; metallic compounds including metal carboxylates such as, for example, dibutyltin dilaurate (DBTDL) as well as mercury, lead, bismuth, and zinc carboxylates; and other alkyl tin carboxylates, oxides, and mercaptides. In one aspect, the catalyst is added to an emulsion containing the carbo sugar and natural oil polyol at from about 0.05% to about 2% (v/v) with respect to the volume of the emulsion. In another aspect, about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.4%, 0.6%, 0.7%, 0.8%, 1%, 1.2%, 1.5%, or 2% catalyst is used. In some aspects, a combination of catalysts is used. In one aspect, 0.5% (v/v) dibutyltin dilaurate and 1% (v/v) dimethylethanolamine were used in combination. In a further aspect, stirring is used to incorporate the catalyst throughout an emulsion containing the carbo sugar and natural oil polyol. In one aspect, different stirring times (e.g. about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 8 minutes, or about minutes) and different stirring speeds (about 100 rpm, about 200 rpm, about 300 rpm, about 400 rpm, about 500 rpm, about 600 rpm, or about 700 rpm) are evaluated to determine the minimum stirring time and speed required to fully incorporate the catalyst into the emulsion. In one aspect, the emulsion and added catalyst are stirred 10 at 300 rpm for 3 minutes.

In another aspect, a clay can be added to the first admixture. “Clay” and “clay minerals” as used herein refer to hydrous aluminum phylosilicates. Clays can optionally include oxides and/or chelates of other metals and semimetals such as, for example, silicon, iron, calcium, magnesium, sodium, potassium, and other alkali and alkaline earth metals. “Bentonite” is a category of impure clay that can consist of montmorillonite, kaolinite, and other species; and that can include potassium, sodium, calcium, aluminum, as well as other metals. “Zeolites” are microporous aluminosilicates that can accommodate a variety of cations, including, but not limited to, sodium, potassium, calcium, and magnesium. The cations in zeolites can be exchanged in aqueous solutions. Clays, bentonites, and zeolites can be used as sources of trace oxides and/or ions in the practice of the present invention. An “oxide” as used herein refers to a molecule, a network solid, or an ionic compound containing at least one oxygen atom and one other element. In one aspect, clays, bentonites, and zeolites contain chelated metal and semimetal ions. Not wishing to be bound by theory, the inclusion of the clay can be used to vary the pore size of the final biofoam product produced.

In one aspect, a metal or semimetal oxide or a chelated metal ion can be incorporated into the first admixture. In one aspect, the metal or semimetal oxide includes, for example, Al₂O₃, Fe₂O₃, MgO, CaO, Na₂O, K₂O, SiO₂, or a combination thereof. In this aspect, the metal or semimetal oxide can be introduced into the polyurethane compositions as a pure compound. In an alternative aspect, ions such as, for example, aluminum, iron (III), magnesium, calcium, sodium, potassium, silicon, and combinations thereof, can be incorporated into the polyurethane compositions described herein through the inclusion of clays or clay minerals. In one aspect, the metal or semimetal oxides or chelated metals are incorporated at concentrations of from about 0.02 nM to about 1.2 mM, or at 0.2 nM, 0.04 nM, 0.06 nM, 0.08 nM, 0.1 nM, 0.15 nM, 0.2 nM, 0.25 nM, 0.3 nM, 0.35 nM, 0.4 nM, 0.45 nM, 0.5 nM, 0.55 nM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, or 1.2 mM.

In another aspect, one or more water-soluble metal salts can be incorporated into the first admixture. In one aspect, the water-soluble metal salts can include, for example, gallium (III) nitrate hydrate, zinc sulfate, zinc acetate, or a combination thereof. In one aspect, 50 mg/L of gallium (III) nitrate hydrate is incorporated into the emulsion containing the carbo sugar and natural oil polyol. In another aspect, 100 mg/L of zinc sulfate is incorporated into the emulsion containing the carbo sugar and natural oil polyol.

After preparation of the first admixture as described above, a polyisocyanate is added to the first admixture. “Polyisocyanates” as used herein are compounds with two or more —N═C═O groups. In one aspect, the polyisocyanate is an aliphatic diisocyanate, a cycloaliphatic diisocyanate, an aromatic diisocyanate, or an isomer thereof. In another aspect, the isocyanate or polyisocyanate is 2,4-toluenediisocyanate, 2,6-toluenediisocyanate, 4,4′-methylene diphenyl diisocyanate (MDI), 4,4′-methylenebis (cyclohexylisocyanate) (H12-MDI), 1-isocyanato-3-isocyanato-methyl-3,5,5-trimethylcyclohexane (isophorone diisocyanate), 2,4,4-trimethylhexamethylenediisocyanate, ethylidenediisocyanate, butylenediisocyanate, hexamethylenediisocyanate, cyclohexylene-1,4-diisocyanate, cyclohexylene-1,2-diisocyanate, xylylene diisocyanate, dichlorohexamethylene diisocyanate, dicyclohexyl-4,4′-diisocyanate, 1-methyl-2,4-diisocyanato-cyclohexane, 1-methyl-2,6-diisocyanato-cyclohexane, naphthalene-1,5-diisocyanate, p-phenylendiisocyanate, tetramethyl-xylylenediisocyanate (TMXDI), or any combination thereof. The isocyanate or polyisocyanate can exist as one or more structural isomers. Alternatively, the isocyanate or polyisocyanate can be a dimer, trimer, or oligomer. In other aspects, the isocyanate or polyisocyanate can exist as one or more positional isomers. For example, the polyisocyanate can be a mixture of 2,4-toluenediisocyanate and 2,6-toluenediisocyanate. In a further aspect, the polyisocyanate can be a 65:35 mixture of 2,4-TDI and 2,6-TDI (i.e., TDI 65). In a different aspect, the polyisocyanate can be an 80:20 mixture of 2,4-TDI and 2,6-TDI (i.e., TDI 80). In an alternative aspect, the polyisocyanate is a modified MDI or polyphenylmethane polyisocyanate such as one of those sold by Yantai Wanhua Polyurethanes Co. under the trade name WANNATE®.

In one aspect, the polyisocyanate is added to the first admixture at different ratios such as, for example, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, or about 1:8 with respect to the total emulsion volume, or any range thereof (e.g., 1: to 1:8, 1:3 to 1:5, etc.). In this aspect, polymerization reactions can then be carried out. Different reaction times (e.g. 8 minutes, 10 minutes, 12 minutes, 15 minutes, or 20 minutes) and stirring speeds (e.g., 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, or 1000 rpm) can be evaluated to determine the optimum reaction time and stirring speed. In one aspect, the first admixture is admixed with the polyisocyanate for 10 minutes at 500 rpm. In another aspect, the reaction is conducted at room temperature.

Upon admixing the components in the first admixture with the polyisocyanate, isocyanate-reactive functional groups present on the carbo sugar and/or natural oil polyol react with the isocyanate groups on the polyisocyanate to produce a polyurethane. Here, a polymer composed of organic residues joined by urethane linkages is produced. Although the components in the first admixture include hydroxyl groups, other components may be present that include other isocyanate-reactive functional groups an amine groups, a thiol groups, or other nucleophilic groups capable of reacting with isocyanate groups.

The amount of the carbo sugar present in the final biofoam product can vary. In one the amount of carbo sugar present in the biofoam is from 0.005% to 0.1% by weight of the biofoam. In another aspect, the amount of carbo sugar present in the biofoam is from 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% by weight of the biofoam, where any value can be a lower and upper end-point of a range (e.g., 0.01% to 0.05%). When used to prepare the biofoams, the carbo sugar can be prepared as a stock solution. For example, the carbo sugar in powder form (0.05 g, 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g or 1 g) can be added to water (100 mL to 1 L) to produce a stock solution. The pH of the stock solution can be adjusted accordingly. In one aspect, the pH of the carbo sugar stock solution is from 1 to 5, 1.5 to 4, or 2 to 3.

The polyurethane compositions produced herein can be used to produce biofoams that have numerous applications. The term “biofoam” as used herein is any substance formed when pockets of gas have been trapped in a solid or liquid. In one aspect, the biofoams produced herein can exist as an emulsion or dispersion at room temperature. In other aspects, he biofoams produced herein are solid materials at room temperature.

The selection and amounts of reactants as well as processing conditions will determine the physical state of the biofoams. For example, the density of the biofoam can be modulated by varying the relative amount of the components used to produce the biofoam. In one aspect, the density of the biofoam is from 0.1 to 05 g/cm³. In another aspect, the density of the biofoam is from 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 05 g/cm³, where any value can be a lower or upper end-point of a range (e.g., 0.1 to 0.3 g/cm³.

In one aspect, when the polyisocyanate is admixed with the first admixture, a solid biofoam is produced. In one aspect, the biofoams produced herein are solid, rigid materials that have numerous applications in the construction and building industry. The polyurethane compositions produced herein can be poured into a mold of any desired shape. If necessary, the mold containing the polyurethane composition can be placed in an oven to remove residual solvent and produce the final biofoam.

In other aspects, one or more blowing agents can be incorporated into the polyurethane compositions to produce the biofoams. A blowing agent can be physical or chemical in nature. A “physical blowing agent” is a gas or low boiling point liquid which expands due to heat generated by the polyurethane-forming reaction, thus forming bubbles and creating foam. A “chemical blowing agent” is a compound or substance that reacts to form a gas. In one aspect, the blowing agent is a physical blowing agent. Physical blowing agents include compounds such as, for example, hydrofluorocarbons (HFCs), hydrocarbons (HCs), hydrofluoroolefins, liquid CO₂, and other low boiling point liquids. In one aspect, the physical blowing agent is HFC-134a (1,1,1,2-tetrafluoroethane), HFC-245fa (pentafluoropropane), HFC-365mfc (1,1,1,3,3-pentafluorobutane), HFC-152a (1,1-difluoroethane), formic acid, methyl formate, HFO-1234ze (1,3,3,3-tetrafluoropropene), cyclopentane, n-pentane, iso-pentane, iso-butane, acetone, dichloromethane, or a mixture thereof. In another aspect, the blowing agent is a chemical blowing agent. In one aspect, the chemical blowing agent is carbon dioxide produced by the reaction of isocyanate groups with water. In a further aspect, both chemical and physical blowing agents can be used.

In other aspects, the biofoams include additional additives not already described above such as, for example, flame retardants, color additives, release agents, biocides, other additives, or a combination thereof. The additional components can be admixed with a dispersion or emulsion of polyurethane composition in order incorporate the additives throughout the biofoam. In the alternative, the additives can be applied to the surface of the solid biofoam.

In another aspect, after the preparation of the biofoam, the biofoam can contain residual solvent (e.g., water). In certain aspects, it is desirable to remove all or substantially all (e.g., greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99%, or 100%) of the solvent in the biofoam. In one aspect, drying of the biofoams can be accomplished in an oven at about 20° C., 30° C., 40° C., 50° C., 60° C., or about 70° C. In one aspect, the biofoams are dried in an oven at 50° C. In a further aspect, the biofoams can be dried for from about 0.5 to about 100 hours, or for about 72 hours. In one aspect, removal of water from biofoams is assessed by periodically removing the biofoams from the oven and weighing them. When the biofoams have the same weight at, for example, at least 2 or 3 successive weighings separated by several hours, the biofoams can be considered to be dry and can be removed from the oven.

In one aspect, the biofoams disclosed herein can be used in the construction industry in applications such as, for example, extruded building materials of any size (e.g., 2 by 4, etc.), alternatives to gypsum wall boarding, alternatives to gypsum wall “mud,” substitutes for wall paneling, substitutes for 4 by 8 plywood sheets, rigid precast structures such as columns, as a substitute for Gunnite in swimming pools, bulletproofing for walls in homes, insulation, poured flooring, roofing tiles, I-beams, trusses, rebar, and the like. In an alternative aspect, the biofoams disclosed herein can be used in the transportation industry in applications such as, for example, as a substitute for fiberglass in boat hulls or bulletproofing for cars. In still another aspect, additional uses for the biofoams disclosed herein are contemplated including, but not limited to, ammunition (e.g., pelletized biofoams as shotgun shell shot or rifle ammunition slugs), silverware or cutlery, lead-free microwave safe dishware, conical antenna dishes, aircraft drone propellers, and related applications.

In another aspect, the biofoams disclosed herein can be used in the medical industry. In one aspect, the biofoam can be used where it is desirable to reduce or minimize blunt force or trauma to a subject. For example, the polyurethane composition can be injected between the skin of the subject and a cast to produce a biofoam that can further prevent any applied force to the broken bone of the subject. In certain aspects, the polyurethane composition can include anti-microbial agents in order to prevent odor. In other aspects, the biofoams can be used to manufacture casts, braces, helmets, or any other medical article that can be used to reduce or prevent applied force or trauma to a subject.

In other aspects, the polyurethane compositions described herein can be used as adhesives. For example, the polyurethane composition can be in a sufficient amount of solvent so that is can readiliy be applied to the surface of a substrate (e.g., spray coating, dipping, brushing). Upon removal of the solvent a biofoam is produced, which results in the formation of a strong bond between to substrates. In other aspects, the polyurethane compositions can be used to seal cracks and holes. Here, the polyurethane composition is sprayed in a hole or crack then forms a biofoam.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods described and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric. Numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction ranges and conditions can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such processes and conditions.

Preparation of DNA Construct for Production of Carbo Sugars

The following DNA sequences were synthesized: a gene for expressing galactomannan galactosyltransferase gene from Cyamopsis tetragonoloba (SEQ ID NO. 4), a gene for expressing cellulose synthase from Pyropia yezoensis (SEQ ID NO. 1), a gene for expressing lipase from Micrococcus sp. HL-2003 (SEQ ID NO. 9), and yellow fluorescent reporter protein (SEQ ID NO. 7). Lipase was included in some constructs and was functional at any position in the construct. However, a position 5′ of the gene for expressing cellulose synthase was preferable when the lipase gene was included.

A pYES2 plasmid vector was used to assemble a DNA construct, which also contained a T7 promoter, and a terminator. The cloning of the DNA construct into the biological devices was performed as follows. Overlapping oligonucleotides were amplified by polymerase chain reaction. These oligonucleotides were then ligated using standard protocols to form an insert. The insert was cloned into cloning vector pBSK. Individual clones were sequenced and site-directed mutagenesis was used to correct mutations in the clones. Following site-directed mutagenesis, clones were sequenced again to verify the DNA sequence of the insert. A pYES2 plasmid was then digested with HindIII restriction enzyme according to directions provided by the enzyme's supplier (Promega). The complete insert, containing HindIII recognition sites on each end, was then excised from the cloning vector and ligated into the pYES2 plasmid using the protocol described below. Successful construction of the insert and ligation of the insert into the plasmid were confirmed using gel electrophoresis. Backbone plasmids at different ratios ranging from 1:1, 1:2, 1:3, 1:4, and up to 1:5 were evaluated.

Ligation of the insert occurred in one pot and was carried out using a kit and enzymes from Promega (Madison, WI). A 1:2 molar ratio of vector/insert was used for most ligation reactions; a typical ligation reaction used 100-200 ng of vector DNA. To this mixture were added 8 μL of T4 DNA ligase and 12 μL of ligase buffer. In some reactions, the ligation mixture was incubated at room temperature for 5 minutes. In other reactions, the ligation mixture was introduced into an electromagnetic chamber for 15 minutes.

A schematic of the DNA construct is depicted in FIGS. 3A and 3B. DNA quantification was performed using a UV-Vis spectrophotometer and recording the ratio of the absorbance at 260 nm to the absorbance at 280 nm. Plasmids were then electrophoresed to verify the insertion of genes (FIG. 4), then purified prior to further use.

Selection of Microorganisms

Carbo sugars was produced using transfected yeasts (Saccharomyces cerevisiae, ATCC® 200892™) and/or bacteria (Escherichia coli, ONESHOT® Top 10 competent cells from Life Technologies™).

Development of Competent Yeast Cells

Yeast cells were made competent by subjecting them to an electrochemical process adapted from Gietz and Schiestl (Nature Protocols, 2007, 2:35-37). Briefly, a single yeast colony was inoculated into 100 mL YPD (yeast extract peptone dextrose) growth media. Yeast was grown overnight on a shaker at 30° C. to OD₆₀₀=1.0. (Acceptable results were obtained with OD₆₀₀values ranging from 0.6 to 1.8.) Cells were centrifuged at 2,000 rpm in a tabletop centrifuge and resuspended in 10 mL TEL buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1 M LiAc, pH=7.5) and shaken vigorously overnight at room temperature. Cells were again centrifuged and resuspended in 1 mL TEL buffer. Cells prepared in this manner could be stored in the refrigerator for up to one month.

Transformation of Yeast Cells to Produce Carbo Sugar Device

Competent cells were stored in the freezer until needed. Cells were thawed on ice and 100 μL of competent cells in TEL buffer were placed in a sterile 1.5 mL microcentrifuge tube. To this was added 5 μL of a 10 mg/mL solution of salmon sperm DNA (carrier DNA). Transforming DNA was added in various amounts. From 1 to 5 μg was sufficient for plasmids from commercial sources, but more DNA was required when transforming yeast with artificial DNA constructs. 10 μL of the DNA device were added to the microcentrifuge tube containing the competent yeast cells and the contents of the tube were mixed. The DNA-yeast suspension was incubated for 30 min at room temperature.

A PLATE solution (consisting of 40% PEG-3350 in 1× TEL buffer) was prepared. 0.7 mL of PLATE solution was added to the DNA-yeast suspension and the contents were mixed thoroughly and incubated for 1 h at room temperature. The mixture was placed in an electromagnetic chamber for 30 minutes. Cells were then heated at 42° C. for 5-10 minutes and 250 μL aliquots were plated on yeast malt agar to which selective growth compounds had been added. Plates were incubated overnight at 30° C.

DNA expression and effectiveness of transformation were determined by fluorescence of the transformed cells expressed in fluorescence units (FSUs) using a 20/20 Luminometer (Promega) according to a protocol provided by the manufacturer. Plasmid DNA extraction, purification, PCR, and gel electrophoresis were also used to confirm transformation. Different transformed devices were obtained. Different types of fluorescent reporter proteins were used (e.g., yellow, red, green, and blue) for all transformed cells and/or constructs. However, the yellow fluorescent protein was preferred. When no fluorescent reporter protein was assembled, no fluorescence was observed.

S. cerevisiae cells were subjected to transformation with the modified pYES2 plasmid as described above. Transformed yeast cells were incubated for 30 min at 28-30° C. Colonies of transformed yeast cells were selected, their DNA isolated and subjected to PCR amplification. Two control treatments were also carried out: (1) a negative control involving competent yeast and nuclease free water instead of plasmid and (2) a positive control involving competent yeast with unmodified pYES2 plasmid.