# Patent

## Title
Strain for butanol production with increased membrane unsaturated trans fatty acids

## Abstract

Bacteria that are not natural butanol producers were found to have increased tolerance to butanol when the membrane content of unsaturated trans fatty acids was increased. Feeding cells with unsaturated trans fatty acids increased their concentration in the membrane, which may also be accomplished by expressing a fatty acid cistrans isomerase.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is related to and claims the benefit of priority to U.S. Provisional Application No. 61/119,451 filed Dec. 3, 2008, the entirety of which is herein incorporated by reference.


FIELD OF THE INVENTION
The invention relates to the field of microbiology and tolerance of microorganisms to butanol. More specifically, increased membrane trans fatty acid composition was found to play a role in butanol tolerance in bacteria which are not natural butanol producers.
BACKGROUND OF THE INVENTION
Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.
Butanol may be made through chemical synthesis or by fermentation. The most popular fermentation process produces a mixture of acetone, 1-butanol and ethanol and is referred to as the ABE processes (Blaschek et al., U.S. Pat. No. 6,358,717). Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations, and the pathways and genes responsible for the production of these solvents have been reported (Girbal et al., Trends in Biotechnology 16:11-16 (1998)). Additionally, recombinant microbial production hosts expressing a 1-butanol biosynthetic pathway (U.S. Patent Application Publication No. US20080182308A1), a 2-butanol biosynthetic pathway (U.S. Patent Application Publication Nos. US20070259410A1 and US 20070292927A1), and an isobutanol biosynthetic pathway (U.S. Patent Application Publication No. US 20070092957) have been described. However, biological production of butanols is believed to be limited by butanol toxicity to the host microorganism used in the fermentation.
Bacteria of the genus Clostridium naturally produce butanol. Strains of Clostridium with increased tolerance to 1-butanol have been isolated by chemical mutagenesis (U.S. Pat. No. 5,192,673; and U.S. Pat. No. 6,358,717), overexpression of certain classes of genes such as those that express stress response proteins (U.S. Pat. No. 6,960,465; and Tomas et al., Appl. Environ. Microbiol. 69(8):4951-4965 (2003)), and by serial enrichment (Quratulain et al., Folia Microbiologica (Prague) 40(5):467-471 (1995); and Soucaille et al., Current Microbiology 14(5):295-299 (1987)). Additionally, the isolation of 1-butanol tolerant strains from estuary sediment (Sardessai et al., Current Science 82(6):622-623 (2002)) and from activated sludge (Bieszkiewicz et al., Acta Microbiologica Polonica 36(3):259-265 (1987)) has been described.
It has been reported that in Pseudomonas putida, that cis unsaturated fatty acids are converted to the trans confirmation when cells are stressed with chemicals such as toluene. The increased trans fatty acid in the cell membrane plays a role in the toluene tolerance of P. putida (Junker and Ramos (1999) J. Bacteriol. 181:5693-5700).). In contrast, it has been reported that feeding a trans fatty acid to Clostridium acetobutylicum did not lead to improved butanol tolerance (Kuhn and Linden, Biotechnology and Bioengineering Symposium 17(Symp. Biotechnol. Fuels Chem., 8th, 1986), 197-207).
There is a need, therefore, for bacterial host strains which do not naturally produce butanol but can be engineered to express a butanol biosynthetic pathway to be more tolerant to these chemicals. In addition there is a need for methods of producing butanols using bacterial host strains engineered for butanol production that are more tolerant to these chemicals.
SUMMARY OF THE INVENTION
Provided herein are butanol tolerant bacterial cells comprising an engineered butanol biosynthetic pathway and having an increased concentration of membrane unsaturated trans fatty acids as compared with a wildtype cell. In some embodiments, the concentration of at least one unsaturated trans fatty acid selected from the group consisting of elaidic acid, vaccenic acid, and C16:1 trans fatty acid is increased. In some embodiments, the cell is a member of a genus selected from the group consisting of Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Leuconostoc, and Brevibacterium. In some embodiments, the cell is a member of the genus Lactobacillus and the growth yield of the cell is at least about 1.6 to about 3.5-fold higher in 2.5% isobutanol than when the cell does not have an increased concentration of membrane unsaturated trans fatty acids. In some embodiments, the cell is a member of the genus Lactobacillus and the growth yield of the cell is at least about 1.6 to about 3.0-fold higher in 2.25% 1-butanol than when the cell does not have an increased concentration of membrane unsaturated trans fatty acids. In some embodiments, the cell is a member of the genus Lactobacillus the growth yield of the cell is at least about 2.2 to about 4-fold higher in 4.0% 2-butanol than when the cell does not have an increased concentration of membrane unsaturated trans fatty acids. In some embodiments, the membrane content of at least one unsaturated trans fatty acid is about 44 fold higher as compared with a wildtype cell.
In some embodiments, the butanol tolerant bacterial cells comprise at least one gene encoding fatty acid cistrans isomerase. In some embodiments, the at least one gene encoding cistrans isomerase has an amino acid sequence which is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
In some embodiments, the butanol biosynthetic pathway is selected from the group consisting of: a) 1-butanol biosynthetic pathway; b) a 2-butanol biosynthetic pathway; and c) an isobutanol biosynthetic pathway.
Further, provided herein are methods for the production of a butanol producing butanol tolerant bacterial cell comprising: a) providing a bacterial cell comprising an engineered butanol biosynthetic pathway; and b) feeding the bacterial cell of step (a) at least one trans fatty acid under conditions wherein the concentration of trans unsaturated fatty acids in the membrane of the cell are increased. In one embodiment, the at least one fatty acid is selected from the group consisting of elaidic acid, vaccenic acid and C16:1 trans fatty acid.
Provided herein are methods for the production of a butanol producing butanol tolerant bacterial cell comprising: a) providing a bacterial cell comprising an engineered butanol biosynthetic pathway and at least one gene encoding a fatty acid cistrans isomerase; and b) expressing the at least one gene encoding a fatty acid cistrans isomerase whereby the concentration of unsaturated trans fatty acids in the membrane of the cell are increased. In some embodiments, the at least one gene encoding cistrans isomerase has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
Provided herein are methods for the production of isobutanol comprising: a) providing a bacterial cell comprising an engineered isobutanol biosynthetic pathway; b) feeding the bacterial cell of step (a) at least one trans fatty acid under conditions wherein the concentration of unsaturated trans fatty acids in the membrane of the cell are increased; and c) growing the bacterial cell of step (b) under conditions wherein isobutanol is produced.
Provided herein are methods for the production of isobutanol comprising: a) providing a bacterial cell comprising an engineered isobutanol biosynthetic pathway and at least one gene encoding cistrans isomerase; b) expressing the at least one gene encoding fatty acid cistrans isomerase whereby the concentration of unsaturated trans fatty acids in the membrane of the cell are increased; and c) growing the bacterial cell of step (b) under conditions wherein isobutanol is produced.
In some embodiments, methods provided herein for the production of isobutanol comprise an isobutanol pathway wherein the isobutanol biosynthetic pathway comprises: a) at least one gene encoding acetolactate synthase; b) at least one gene encoding acetohydroxy acid isomeroreductase; c) at least one gene encoding acetohydroxy acid dehydratase; d) at least one gene encoding a branched-chain keto acid decarboxylase; and e) at least one gene encoding branched-chain alcohol dehydrogenase
Sequence Descriptions
The various embodiments of the invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.
The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.







TABLE 1







SEQ ID Numbers for Examples of Coding Regions and Proteins


for 1-Butanol Biosynthetic Pathway











SEQ



SEQ ID NO:
ID NO:


Description
Nucleic acid
Peptide












Acetyl-CoA acetyltransferase thlA from
1
2


Clostridium acetobutylicum ATCC 824




Acetyl-CoA acetyltransferase thlB from
3
4


Clostridium acetobutylicum ATCC 824




3-Hydroxybutyryl-CoA dehydrogenase from
5
6


Clostridium acetobutylicum ATCC 824




Crotonase from Clostridium acetobutylicum
7
8


ATCC 824




Putative trans-enoyl CoA reductase from
9
10


Clostridium acetobutylicum ATCC 824




Euglena gracilis butyryl-CoA dehydrogenase/
39
40


trans-2-enoyl-CoA reductase codon optimized




lacking mitochondrial presequence.




Butyraldehyde dehydrogenase from
11
12


Clostridium beijerinckii NRRL B594




1-Butanol dehydrogenase bdhB from
13
14


Clostridium acetobutylicum ATCC 824




1-Butanol dehydrogenase
15
16


bdhA from Clostridium acetobutylicum




ATCC 824
















TABLE 2







SEQ ID Numbers for Examples of Coding Regions and Proteins


for 2-Butanol Biosynthetic Pathway










SEQ ID NO:
SEQ ID NO:


Description
Nucleic acid
Peptide





budA, acetolactate decarboxylase from
17
18


Klebsiella pneumoniae ATCC 25955




budB, acetolactate synthase from Klebsiella
19
20


pneumoniae ATCC 25955




budC, butanediol dehydrogenase from
21
22


Klebsiella pneumoniae IAM1063




pddA, butanediol dehydratase alpha subunit
23
24


from Klebsiella oxytoca ATCC 8724




pddB, butanediol dehydratase beta subunit
25
26


from Klebsiella oxytoca ATCC 8724




pddC, butanediol dehydratase gamma subunit
27
28


from Klebsiella oxytoca ATCC 8724




sadH, 2-butanol dehydrogenase from
29
30


Rhodococcus ruber 219
















TABLE 3







SEQ ID Numbers for Examples of Coding Regions and Proteins


for Isobutanol Biosynthetic Pathway










SEQ ID NO:
SEQ ID NO:


Description
Nucleic acid
Peptide





Klebsiella pneumoniae budB (acetolactate
19
20


synthase)




E. coli ilvC (acetohydroxy acid
31
32


reductoisomerase)




B. subtilis ilvC (acetohydroxy acid
41
42


reductoisomerase)




E. coli ilvD (acetohydroxy acid dehydratase)
33
34


Lactococcus lactis kivD (branched-chain
35
36


α-keto acid decarboxylase), codon optimized




E. coli yqhD (branched-chain alcohol
37
38


dehydrogenase)
















TABLE 4







Representative fatty acid cistrans isomerase coding regions and


encoded proteins










SEQ ID NO:
SEQ ID NO:


Organism
nucleic acid
amino acid












Shewanella sp. MR-4
43
44


Shewanella sp. MR-7
45
46


Vibrio vulnificus YJ016
47
48


Colwellia psychrerythraea 34H
49
50


Saccharophagus degradans 2-40
51
52


Pseudomonas fluorescens Pf-5
53
54


Pseudomonas aeruginosa PAO1
55
56


Vibrio vulnificus CMCP6
57
58


Pseudomonas aeruginosa UCBPP-PA14
59
60


Pseudomonas fluorescens PfO-1
61
62


Methylococcus capsulatus str. Bath
63
64


Pseudomonas syringae pv. tomato str.
65
66


DC3000




Vibrio parahaemolyticus RIMD 2210633
67
68


Nitrosomonas europaea ATCC 19718
69
70


Vibrio cholerae O1 biovar eltor str. N16961
71
72


Pseudomonas syringae pv. phaseolicola
73
74


1448A




Bdellovibrio bacteriovorus HD100
75
76


Vibrio fischeri ES114
77
78


Photobacterium profundum SS9
79
80


Pseudoalteromonas haloplanktis TAC125
81
82


Pseudoalteromonas atlantica T6c
83
84


Azotobacter vinelandii AvOP
85
86


Pseudomonas entomophila L48
87
88


Alcanivorax borkumensis SK2
89
90


Vibrio cholerae V51
91
92


Vibrio cholerae MO10
93
94


Vibrio cholerae O395
95
96


Shewanella baltica OS155
97
98


Vibrio cholerae RC385
99
100


Pelobacter propionicus DSM 2379
101
102


Pseudomonas aeruginosa C3719
103
104


Pseudomonas aeruginosa 2192
105
106


Vibrio sp. Ex25
107
108


Vibrio cholerae V52
109
110


Shewanella sp. ANA-3
111
112


Pseudomonas putida F1
113
114


Vibrio splendidus 12B01
115
116


Congregibacter litoralis KT71
117
118


Pseudoalteromonas tunicata D2
119
120


Vibrio sp. MED222
121
122


Vibrio alginolyticus 12G01
123
124


Photobacterium profundum 3TCK
125
126


Pseudomonas aeruginosa PA7
127
128


Oceanobacter sp. RED65
129
130


Shewanella baltica OS195
131
132


Pseudomonas aeruginosa PACS2
133
134


Pseudomonas putida KT2440
135
136









SEQ ID NO:137 is the nucleotide sequence of the L. Plantarum atpB promoter.
SEQ ID NOs:138 and 139 are primers for PCR amplification of the L. Plantarum atpB promoter.
SEQ ID NOs:140 and 141 are primers for PCR amplification of a DNA fragment from Lactobacillus plantarum (Genbank NC—004567) with homology to IdhL.
SEQ ID NO:142 is the integration vector pFP988.
SEQ ID NOs:143 and 144 are primers for PCR amplification of the Cm resistance gene with its promoter from pC194 (GenBank NC—002013).
SEQ ID NOs:145 and 146 are oligonucleotides for constructing the P11 promoter.
SEQ ID NOs:147 and 148 are primers for PCR amplification of the L. plantarum IdhL promoter.
SEQ ID NOs:149 and 150 are oligonucleotides for constructing the P11 promoter.
SEQ ID NOs:151 and 152 are primers for PCR amplification of the L. plantarum IdhL promoter.
SEQ ID NOs:153 and 154 are primers for PCR amplification of the fatty acid cistrans isomerase coding region from P. putida KT2440 (ATCC#47054 D-5).
SEQ ID NOs:155 and 156 are primers for PCR amplification of a trc promoter-cti gene fragment.


DETAILED DESCRIPTION
The invention provides a recombinant bacterial cell which does not naturally produce butanol at detectable levels, but which is engineered to express a butanol biosynthetic pathway, that is modified to have increased concentration of unsaturated trans fatty acid in the cell membrane fatty acid composition as compared with a corresponding membrane fatty acid unmodified bacterial cell. Such cells have an increased tolerance to butanol as compared with cells that lack the membrane fatty acid modification. Increase in membrane unsaturated trans fatty acid may be accomplished by feeding the cell with an unsaturated trans fatty acid or by genetically modifying the cell to increase expression of at least one gene involved in unsaturated trans fatty acid synthesis, such as one encoding fatty acid cistrans isomerase. The present cells may be used to produce butanol, which may be used as an alternative energy source to fossil fuels.
The following abbreviations and definitions will be used for the interpretation of the specification and the claims.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.
The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.
As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like.
The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.
The term “butanol” as used herein, refers to 1-butanol, 2-butanol, isobutanol, or mixtures thereof.
The terms “butanol tolerant bacterial strain” and “tolerant” when used to describe a modified bacterial strain of the invention, refers to a modified bacterium that shows better growth in the presence of butanol than the parent strain from which it is derived.
The term “wildtype” as it applies to a butanol tolerant bacterial cell of the invention refers to a cell which has not been modified or altered to increase butanol tolerance with respect to the concentration of unsaturated fatty acids in the membrane.
The term “butanol biosynthetic pathway” refers to an enzyme pathway to produce 1-butanol, 2-butanol, or isobutanol.
The term “1-butanol biosynthetic pathway” refers to an enzyme pathway to produce 1-butanol from acetyl-coenzyme A (acetyl-CoA).
The term “2-butanol biosynthetic pathway” refers to an enzyme pathway to produce 2-butanol from pyruvate.
The term “isobutanol biosynthetic pathway” refers to an enzyme pathway to produce isobutanol from pyruvate.
The term “acetyl-CoA acetyltransferase” refers to an enzyme that catalyzes the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA). Preferred acetyl-CoA acetyltransferases are acetyl-CoA acetyltransferases with substrate preferences (reaction in the forward direction) for a short chain acyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego]; although, enzymes with a broader substrate range (E.C. 2.3.1.16) will be functional as well. Acetyl-CoA acetyltransferases are available from a number of sources, for example, Escherichia coli (GenBank Nos: NP—416728, NC—000913; NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence), Clostridium acetobutylicum (GenBank Nos: NP—349476.1 (SEQ ID NO:2), NC—003030; NP—149242 (SEQ ID NO:4), NC—001988), Bacillus subtilis (GenBank Nos: NP—390297, NC—000964), and Saccharomyces cerevisiae (GenBank Nos: NP—015297, NC—001148).
The term “3-hydroxybutyryl-CoA dehydrogenase” refers to an enzyme that catalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. 3-Hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adenine dinucleotide (NADH)-dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally, 3-hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 1.1.1.157 and E.C. 1.1.1.36, respectively. 3-Hydroxybutyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP—349314 (SEQ ID NO:6), NC—003030), B. subtilis (GenBank NOs: AAB09614, U29084), Ralstonia eutropha (GenBank NOs: ZP—0017144, NZ_AADY01000001, Alcaligenes eutrophus (GenBank NOs: YP—294481, NC—007347), and A. eutrophus (GenBank NOs: P14697, J04987).
The term “crotonase” refers to an enzyme that catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA and H2O. Crotonases may have a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 4.2.1.17 and E.C. 4.2.1.55, respectively. Crotonases are available from a number of sources, for example, E. coli (GenBank NOs: NP—415911 (SEQ ID NO:8), NC—000913), C. acetobutylicum (GenBank NOs: NP—349318, NC—003030), B. subtilis (GenBank NOs: CAB13705, Z99113), and Aeromonas caviae (GenBank NOs: BAA21816, D88825).
The term “butyryl-CoA dehydrogenase”, also called trans-enoyl CoA reductase, refers to an enzyme that catalyzes the conversion of crotonyl-CoA to butyryl-CoA. Butyryl-CoA dehydrogenases may be NADH-dependent or NADPH-dependent and are classified as E.C. 1.3.1.44 and E.C. 1.3.1.38, respectively. Butyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP—347102 (SEQ ID NO:10), NC—003030), Euglena gracilis (GenBank NOs: Q5EU90, AY741582), Streptomyces collinus (Gen Bank NOs: AAA92890, U37135), and Streptomyces coelicolor (GenBank NOs: CAA22721, AL939127).
The term “butyraldehyde dehydrogenase” refers to an enzyme that catalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH or NADPH as cofactor. Butyraldehyde dehydrogenases with a preference for NADH are known as E.C. 1.2.1.57 and are available from, for example, Clostridium beijerinckii (GenBank NOs: AAD31841 (SEQ ID NO:12), AF157306) and C. acetobutylicum (GenBank NOs: NP—149325, NC—001988).
The term “1-butanol dehydrogenase” refers to an enzyme that catalyzes the conversion of butyraldehyde to 1-butanol. 1-butanol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. 1-butanol dehydrogenase may be NADH- or NADPH-dependent. 1-butanol dehydrogenases are available from, for example, C. acetobutylicum (GenBank NOs: NP—149325, NC—001988; NP—349891 (SEQ ID NO:14), NC—003030; and NP—349892 (SEQ ID NO:16), NC—003030) and E. coli (GenBank NOs: NP—417-484, NC—000913).
The term “acetolactate synthase”, also known as “acetohydroxy acid synthase”, refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of two molecules of pyruvic acid to one molecule of alpha-acetolactate. Acetolactate synthase, known as EC 2.2.1.6 [formerly 4.1.3.18] (Enzyme Nomenclature 1992, Academic Press, San Diego) may be dependent on the cofactor thiamin pyrophosphate for its activity. Suitable acetolactate synthase enzymes are available from a number of sources, for example, Bacillus subtilis (GenBank Nos: AAA22222 NCBI (National Center for Biotechnology Information) amino acid sequence, L04470 NCBI nucleotide sequence), Klebsiella terrigena (Gen Bank Nos: AAA25055, L04507), and Klebsiella pneumoniae (GenBank Nos: AAA25079 (SEQ ID NO:20), M73842 (SEQ ID NO:19).
The term “acetolactate decarboxylase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of alpha-acetolactate to acetoin. Acetolactate decarboxylases are known as EC 4.1.1.5 and are available, for example, from Bacillus subtilis (GenBank Nos: AAA22223, L04470), Klebsiella terrigena (GenBank Nos: AAA25054, L04507) and Klebsiella pneumoniae (SEQ ID NO:18 (amino acid) SEQ ID NO:17 (nucleotide)).
The term “butanediol dehydrogenase” also known as “acetoin reductase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 2,3-butanediol. Butanediol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes may have specificity for production of R- or S-stereochemistry in the alcohol product. S-specific butanediol dehydrogenases are known as EC 1.1.1.76 and are available, for example, from Klebsiella pneumoniae (GenBank Nos: BBA13085 (SEQ ID NO:22), D86412. R-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus (GenBank Nos. NP—830481, NC—004722; AAP07682, AE017000), and Lactococcus lactis (GenBank Nos. AAK04995, AE006323).
The term “butanediol dehydratase”, also known as “diol dehydratase” or “propanediol dehydratase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2,3-butanediol to 2-butanone, also known as methyl ethyl ketone (MEK). Butanediol dehydratase may utilize the cofactor adenosyl cobalamin. Adenosyl cobalamin-dependent enzymes are known as EC 4.2.1.28 and are available, for example, from Klebsiella oxytoca (GenBank Nos: BAA08099 (alpha subunit) (SEQ ID NO:24), BAA08100 (beta subunit) (SEQ ID NO:26), and BBA08101 (gamma subunit) (SEQ ID NO:28), (Note all three subunits are required for activity), D45071).
The term “2-butanol dehydrogenase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2-butanone to 2-butanol. 2-butanol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. 2-butanol dehydrogenase may be NADH- or NADPH-dependent. The NADH-dependent enzymes are known as EC 1.1.1.1 and are available, for example, from Rhodococcus ruber (GenBank Nos: CAD36475 (SEQ ID NO:30), AJ491307 (SEQ ID NO:29)). The NADPH-dependent enzymes are known as EC 1.1.1.2 and are available, for example, from Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169).
The term “acetohydroxy acid isomeroreductase” or “acetohydroxy acid reductoisomerase” refers to an enzyme that catalyzes the conversion of acetolactate to 2,3-dihydroxyisovalerate using NADPH (reduced nicotinamide adenine dinucleotide phosphate) as an electron donor. Preferred acetohydroxy acid isomeroreductases are known by the EC number 1.1.1.86 and sequences are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: NP—418222 (SEQ ID NO:32), NC—000913 (SEQ ID NO:31)), Saccharomyces cerevisiae (GenBank Nos: NP—013459, NC—001144), Methanococcus maripaludis (GenBank Nos: CAF30210, BX957220), and Bacillus subtilis (GenBank Nos: CAB14789, Z99118).
The term “acetohydroxy acid dehydratase” refers to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate. Preferred acetohydroxy acid dehydratases are known by the EC number 4.2.1.9. These enzymes are available from a vast array of microorganisms, including, but not limited to, E. coli (GenBank Nos: YP—026248 (SEQ ID NO:34), NC—000913 (SEQ ID NO:33)), S. cerevisiae (GenBank Nos: NP—012550, NC—001142), M. maripaludis (GenBank Nos: CAF29874, BX957219), and B. subtilis (GenBank Nos: CAB14105, Z99115).
The term “branched-chain α-keto acid decarboxylase” refers to an enzyme that catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde and CO2. Preferred branched-chain α-keto acid decarboxylases are known by the EC number 4.1.1.72 and are available from a number of sources, including, but not limited to, Lactococcus lactis (GenBank Nos: AAS49166, AY548760; CAG34226 (SEQ ID NO:36), AJ746364, Salmonella typhimurium (GenBank Nos: NP—461346, NC—003197), and Clostridium acetobutylicum (GenBank Nos: NP—149189, NC—001988).
The term “branched-chain alcohol dehydrogenase” refers to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol. Preferred branched-chain alcohol dehydrogenases are known by the EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases (specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize NADH (reduced nicotinamide adenine dinucleotide) and/or NADPH as electron donor and are available from a number of sources, including, but not limited to, S. cerevisiae (GenBank Nos: NP—010656, NC—001136; NP—014051, NC—001145), E. coli (GenBank Nos: NP—417-484 (SEQ ID NO:38), NC—000913 (SEQ ID NO:37)), and C. acetobutylicum (GenBank Nos: NP—349892, NC—003030).
The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.
As used herein the term “coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.
The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The terms “plasmid” and “vector” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation vector” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell.
As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.
A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to identify putatively a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.
As used herein, “substantially similar” enzymes will refer to enzymes belonging to a family of proteins in the art known to share similar structures and function. It is well within the skill of one in the art to identify substantially similar proteins given a known structure. Typical methods to identify substantially similar structures will rely upon known sequence information (nucleotide sequence and/or amino acid sequences) and may include PCR amplification, nucleic acid hybridization, and/or sequence identity/similarity analysis (e.g., sequence alignments between partial and/or complete sequences and/or known functional motifs associated with the desired activity).
A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Given the nucleic acid sequences described herein, one of skill in the art can identify substantially similar nucleic acid fragments that may encode proteins having similar activity. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (2001), particularly Chapter 11 and Table 11.1 therein. The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS at 65° C. followed by 0.1×SSC, 0.1% SDS at 65° C., for example.
In one aspect, suitable nucleic acid fragments encode polypeptides that are at least about 70% identical to the amino acid sequences reported herein. In another aspect, the nucleic acid fragments encode amino acid sequences that are at least about 85-90% identical to the amino acid sequences reported herein. In a further aspect, the nucleic acid fragments encode amino acid sequences that are at least about 90-100% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least about 50 amino acids, preferably at least about 100 amino acids, more preferably at least about 150 amino acids, still more preferably at least about 200 amino acids, and most preferably at least about 250 amino acids.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the Clustal method of alignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10), unless otherwise specified. Default parameters for pairwise alignments using the Clustal method are: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 75% identical, and more preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are at least about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.
The term “homology” refers to the relationship among sequences whereby there is some extent of likeness, typically due to descent from a common ancestral sequence. Homologous sequences can share homology based on genic, structural, functional and/or behavioral properties. The term “ortholog” or “orthologous sequences” refers herein to a relationship where sequence divergence follows speciation (i.e., homologous sequences in different species arose from a common ancestral gene during speciation). In contrast, the term “paralogous” refers to homologous sequences within a single species that arose by gene duplication. One skilled in the art will be familiar with techniques required to identify homologous, orthologous and paralogous sequences.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein, “default values” will mean any set of values or parameters (as set by the software manufacturer) which originally load with the software when first initialized
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W. Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. et al., In Current Protocols in Molecular Biology, published by Greene Publishing and Wiley-Interscience, 1987.
Butanol Tolerance in Butanol Non-Producing Bacteria—Membrane Composition
The invention relates to the discovery that an increase in the unsaturated trans fatty acid content of the membrane of a bacterial cell that does not naturally produce butanol increases butanol tolerance of the cell. The discovery came from results of studies on feeding butanol non-producing bacterial cells with different fatty acids followed by analysis of butanol tolerance. Any bacteria that does not naturally produce butanol may have butanol tolerance increased through increase in membrane unsaturated trans fatty acid composition. Examples include, but are not limited to, bacterial cells of Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Leuconostoc, and Brevibacterium. Examples of specific bacterial cells include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, and Bacillus subtilis. 
Increasing Membrane Unsaturated Trans Fatty Acids
In the bacterial cells of the present invention, the amount of unsaturated trans fatty acids in the membrane may be increased with respect to the amounts of other types of fatty acids by any method. Examples of methods that may be used include feeding the cells a fatty acid that will result in an increase in membrane unsaturated trans fatty acid and making a genetic modification that results in increasing the membrane unsaturated trans fatty acid composition. Fatty acids that may be fed to cells to increase membrane unsaturated fatty acid composition include, for example, elaidic acid (C18:1 trans-9; IUPAC name: (E)-octadec-9-enoic acid), vaccenic acid (18:1 trans-11; IUPAC name: (E)-11-octadecenoic acid) and C16:1 trans fatty acid.
Genetic modifications that increase membrane unsaturated fatty acid composition include expression of at least one gene whose encoded enzyme is able to convert unsaturated cis fatty acids to unsaturated trans fatty acids. One example is the enzyme fatty acid cistrans isomerase. Modification of any bacterial cell, that does not naturally make butanol, for expression of any fatty acid cistrans isomerase may be used to prepare cells of the present invention. Examples of amino acid sequences and the encoding DNA sequences for representative fatty acid cistrans isomerases are given in Table 4 as SEQ ID NOs: 43-136. Additional fatty acid cistrans isomerases that may be used in the present bacterial cells may be identified by one skilled in the art through bioinformatics methods as described above. Additional proteins that have at least about 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or at least about 98% sequence identity to any of SEQ ID NOs:even numbers 44-136 and having fatty acid cistrans isomerase activity may be used. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
In addition to using protein or coding region sequence and bioinformatics methods to identify additional fatty acid cistrans isomerases, the sequences described herein or those recited in the art may be used to experimentally identify other homologs in nature. For example each of the fatty acid cistrans isomerase encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.
For example, genes encoding similar proteins or polypeptides to the fatty acid cistrans isomerase encoding genes described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired organism using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the disclosed nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments by hybridization under conditions of appropriate stringency.
Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, “The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders”, in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).
Generally two short segments of the described sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the described nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding microbial genes.
Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (e.g., BRL, Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).
Alternatively, the described fatty acid cistrans isomerase encoding sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. Probes are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are “hybridizable” to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).
Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).
Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.
For expression of a fatty acid citrans isomerase, a coding region for a fatty acid cistrans isomerase is introduced into a bacterial cell and is expressed from a plasmid or is integrated into the cell genome. Typically the coding region is operably linked to regulatory sequences, which may be native to the gene including the coding region or heterologous to the coding region. More typically, a promoter that is not native to the gene and known to be active in the host bacterial cell is operably linked to the fatty acid cistrans isomerase coding region for expression. Examples of promoters and plasmids (vectors) that may be used for transfer and expression of fatty acid cistrans isomerase genes in bacteria such as E. coli, Lactobacillus, and Pseudomonas are the same as those described below for expression of butanol pathway genes.
It may be desirable to codon-optimize a heterologous coding region for optimal expression in a particular bacterial cell. Methods for codon-optimization are well known in the art.
Butanol Tolerance of Increased Membrane Unsaturated Trans Fatty Acid Strain
A bacterial cell of the present invention modified for increased membrane unsaturated trans fatty acid composition has improved tolerance to butanol. The increased tolerance may be assessed by assaying growth in concentrations of butanol that are detrimental to growth of the parental strain (prior to modification for increased membrane unsaturated trans fatty acid composition). Improved tolerance is to butanol compounds including 1-butanol, isobutanol, and 2-butanol. The amount of tolerance improvement will vary depending on the inhibiting chemical and its concentration, growth conditions and the specific modified cell. For example, as shown in Example 2 herein, cells of L. plantarum having increased membrane unsaturated trans fatty acid composition had a growth yield in 2.5% to 3.5% (w.v) isobutanol that was between 1.6 and 3.5-fold higher than L. plantarum cells without increased membrane unsaturated trans fatty acid composition. In Example 3 herein is shown that cells of L. plantarum having increased membrane unsaturated trans fatty acid composition had a growth yield in 2.25% to 3.0% (w/v) 1-butanol that was between 1.6 and 3-fold higher than L. plantarum cells without increased membrane unsaturated trans fatty acid composition. In Example 4 herein is shown that cells of L. plantarum having increased membrane unsaturated trans fatty acid composition had a growth yield in 4.0% to 4.9% (w/v) 2-butanol that was between 2.2 and 4-fold higher than L. plantarum cells without increased membrane unsaturated trans fatty acid composition.
Butanol Biosynthetic Pathway
In the present invention, a modification conferring increased unsaturated trans fatty acid in the membrane is made in a bacterial cell that does not naturally produce butanol, but that has an engineered butanol biosynthetic pathway. Either modification may take place prior to the other.
The butanol biosynthetic pathway may be a 1-butanol, 2-butanol, or isobutanol biosynthetic pathway. Particularly suitable bacterial hosts for the production of butanol and modification for increased butanol tolerance include, but are not limited to, members of the genera Escherichia, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, and Enterococcus. Preferred hosts include: Escherichia coli, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, and Enterococcus faecalis. 
1-Butanol Biosynthetic Pathway
A suitable biosynthetic pathway for the production of 1-butanol is described by Donaldson et al. in U.S. Patent Application Publication No. US20080182308A1 incorporated herein by reference. This biosynthetic pathway comprises the following substrate to product conversions:
a) acetyl-CoA to acetoacetyl-CoA, as catalyzed for example by acetyl-CoA acetyltransferase (which may be encoded, for example, by the genes given as SEQ ID NO:1 or 3);
b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, as catalyzed for example by 3-hydroxybutyryl-CoA dehydrogenase (which may be encoded, for example, by the gene given as SEQ ID NO:5);
c) 3-hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed for example by crotonase (which may be encoded, for example, by the gene given as SEQ ID NO:7);
d) crotonyl-CoA to butyryl-CoA, as catalyzed for example by butyryl-CoA dehydrogenase (which may be encoded, for example, by the gene given as SEQ ID NO:9);
e) butyryl-CoA to butyraldehyde, as catalyzed for example by butyraldehyde dehydrogenase (which may be encoded, for example, by the gene given as SEQ ID NO:11); and
f) butyraldehyde to 1-butanol, as catalyzed for example by 1-butanol dehydrogenase (which may be encoded, for example, by the genes given as SEQ ID NO:13 or 15).
The pathway requires no ATP and generates NAD+ and/or NADP+, thus, it balances with the central, metabolic routes that generate acetyl-CoA.
Other suitable biosynthetic pathways for the production of 1-butanol will be apparent to those of skill in the art.
2-Butanol Biosynthetic Pathway
Suitable biosynthetic pathways for the production of 2-butanol are described by Donaldson et al. in U.S. Patent Application Publication Nos. US20070259410A1 and US 20070292927A1, both incorporated herein by reference. One 2-butanol biosynthetic pathway comprises the following substrate to product conversions:
a) pyruvate to alpha-acetolactate, as catalyzed for example by acetolactate synthase (which may be encoded, for example, by the gene given as SEQ ID NO:19);
b) alpha-acetolactate to acetoin, as catalyzed for example by acetolactate decarboxylase (which may be encoded, for example, by the gene given as SEQ ID NO:17);
c) acetoin to 2,3-butanediol, as catalyzed for example by butanediol dehydrogenase (which may be encoded, for example, by the gene given as SEQ ID NO:21);
d) 2,3-butanediol to 2-butanone, catalyzed for example by butanediol dehydratase (which may be encoded, for example, by genes given as SEQ ID NOs:23, 25, and 27); and
e) 2-butanone to 2-butanol, as catalyzed for example by 2-butanol dehydrogenase (which may be encoded, for example, by the gene given as SEQ ID NO:29).
Other suitable biosynthetic pathways for the production of 2-butanol will be apparent to those of skill in the art.
Isobutanol Biosynthetic Pathway
Suitable biosynthetic pathways for the production of isobutanol are described by Maggio-Hall et al. in U.S. Patent Application Publication No. US20070092957 A1, incorporated herein by reference. One isobutanol biosynthetic pathway comprises the following substrate to product conversions:
a) pyruvate to acetolactate, as catalyzed for example by acetolactate synthase (which may be encoded, for example, by the gene given as SEQ ID NO:19);
b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed for example by acetohydroxy acid isomeroreductase (which may be encoded, for example, by the gene given as SEQ ID NO:31);
c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed for example by acetohydroxy acid dehydratase (which may be encoded, for example, by the gene given as SEQ ID NO:33);
d) α-ketoisovalerate to isobutyraldehyde, as catalyzed for example by a branched-chain keto acid decarboxylase (which may be encoded, for example, by the gene given as SEQ ID NO:35); and
e) isobutyraldehyde to isobutanol, as catalyzed for example by a branched-chain alcohol dehydrogenase (which may be encoded, for example, by the gene given as SEQ ID NO:37).
Other suitable biosynthetic pathways for the production of isobutanol will be apparent to those of skill in the art.
Construction of Bacterial Strains for Butanol Production
Any bacterial strain that is modified for butanol tolerance as described herein is additionally genetically modified (before or after modification to tolerance) to incorporate a butanol biosynthetic pathway by methods well known to one skilled in the art. Genes encoding the enzyme activities described above, or homologs that may be identified and obtained by commonly used methods well known to one skilled in the art, are introduced into a bacterial host. Representative coding and amino acid sequences for pathway enzymes that may be used are given in Tables 1, 2, and 3, with SEQ ID NOs:1-38, and 39-42. Typically BLAST (described above) searching of publicly available databases with the provided amino acid sequences is used to identify homologs and their encoding sequences that may be used in butanol biosynthetic pathways in the present cells. For example, proteins having amino acid sequence identities of at least about 70-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or 98% sequence identity to any of the proteins in Tables 1, 2, or 3 and having the noted activities may be identified. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. In addition to using protein or coding region sequence and bioinformatics methods to identify additional homologs, the sequences described herein or those recited in the art may be used to experimentally identify other homologs in nature as described above for fatty acid cistrans isomerase.
Methods described in U.S. Patent Application Publication Nos. US20080182308A1, US20070259410A1, US 20070292927A1, and US20070092957 A1 (all incorporated herein by reference) may be used to engineer bacteria for expression of a butanol biosynthetic pathway. Vectors or plasmids useful for the transformation of a variety of host cells are common and commercially available from companies such as EPICENTRE® (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.), Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly, Mass.). Typically, the vector or plasmid contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the specific species chosen as a production host.
Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements is suitable for the present invention including, but not limited to, lac, ara, tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli and Pseudomonas); the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus subtilis, and Bacillus licheniformis; nisA (useful for expression Gram-positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol. 64(8):2763-2769 (1998)); and the synthetic P11 promoter (useful for expression in Lactobacillus plantarum, Rud et al., Microbiology 152:1011-1019 (2006)). Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included.
Certain vectors are capable of replicating in a broad range of host bacteria and can be transferred by conjugation. The complete and annotated sequence of pRK404 and three related vectors-pRK437, pRK442, and pRK442(H) are available. These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria (Scott et al., Plasmid 50(1):74-79 (2003)). Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 are also available with promoters that can function in a range of Gram-negative bacteria. Plasmid pAYC36 and pAYC37, have active promoters along with multiple cloning sites to allow for the heterologous gene expression in Gram-negative bacteria.
Chromosomal gene replacement tools are also widely available. For example, a thermosensitive variant of the broad-host-range replicon pWV101 has been modified to construct a plasmid pVE6002 which can be used to create gene replacement in a range of Gram-positive bacteria (Maguin et al., J. Bacteriol. 174(17):5633-5638 (1992)).
Expression of a Butanol Biosynthetic Pathway in E. Coli 
Vectors useful for the transformation of E. coli are common and commercially available from the companies listed above. For example, the genes of an isobutanol, 1-butanol, or 2-butanol biosynthetic pathway may be isolated from various sources, as described above, cloned onto a modified pUC19 vector and transformed into E. coli host cells. Alternatively, the genes encoding a butanol biosynthetic pathway may be divided into multiple operons, cloned onto expression vectors, and transformed into various E. coli strains.
Construction of Lactobacillus Strains for Butanol Production
The Lactobacillus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Bacillus subtilis and Streptococcus may be used for Lactobacillus. Non-limiting examples of suitable vectors include pAM61 and derivatives thereof (Renault et al., Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231 (1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al. Appl. Environ. Microbiol. 62:1481-1486 (1996)); pMG1, a conjugative plasmid (Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903 (1994)). Several plasmids from Lactobacillus plantarum have also been reported (van Kranenburg R, Golic N, Bongers R, Leer R J, de Vos W M, Siezen R J, Kleerebezem M. Appl. Environ. Microbiol. 2005 March; 71(3): 1223-1230), which may be used for transformation.
Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired Lactobacillus host cell, may be obtained from Lactobacillus or other lactic acid bacteria, or other Gram-positive organisms. A non-limiting example is the nisA promoter from Lactococcus. Termination control regions may also be derived from various genes native to the preferred hosts or related bacteria.
The various genes for a butanol biosynthetic pathway may be assembled into any suitable vector, such as those described above. The codons can be optimized for expression based on the codon index deduced from the genome sequences of the host strain, such as for Lactobacillus plantarum or Lactobacillus arizonensis. The plasmids may be introduced into the host cell using methods known in the art, such as electroporation, as described in any one of the following references: Cruz-Rodz et al. (Molecular Genetics and Genomics 224:1252-154 (1990)), Bringel and Hubert (Appl. Microbiol. Biotechnol. 33: 664-670 (1990)), and Teresa Alegre, Rodriguez and Mesas (FEMS Microbiology letters 241:73-77 (2004)). Plasmids can also be introduced to Lactobacillus plantatrum by conjugation (Shrago, Chassy and Dobrogosz Appl. Environ. Micro. 52: 574-576 (1986)). The butanol biosynthetic pathway genes can also be integrated into the chromosome of Lactobacillus using integration vectors (Hols et al. Appl. Environ. Micro. 60:1401-1403 (1990); Jang et al. Micro. Lett. 24:191-195 (2003)).
Fermentation of Butanol Tolerant Bacteria for Butanol Production
The present cells with increased membrane unsaturated trans fatty acid composition and having a butanol biosynthesis pathway may be used for fermentation production of butanol.
Fermentation media for the production of butanol must contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Sucrose may be obtained from feedstocks such as sugar cane, sugar beets, cassava, and sweet sorghum, and mixtures thereof. Glucose and dextrose may be obtained through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, and oats, and mixtures thereof. Other fermentable sugars from algae (macroalgae or microalgae).
In addition, fermentable sugars may be obtained from cellulosic and lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in US Patent Application Publication US20070031918A1, which is herein incorporated by reference. Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.
Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose or mixtures of these with C5 sugars such as xylose and/or arabinose.
In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for butanol production.
Typically cells are grown at a temperature in the range of about 25° C. to about 40° C. in an appropriate medium. Suitable growth media are common commercially prepared media such as Bacto Lactobacilli MRS broth or Agar (Difco), Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular bacterial strain will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′:3′-monophosphate, may also be incorporated into the fermentation medium.
Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition.
Fermentations may be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.
Butanol may be produced using a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Batch and fed-batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated by reference.
Butanol may also be produced using continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
It is contemplated that the production of butanol may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for butanol production.
Methods for Butanol Isolation from the Fermentation Medium
Bioproduced butanol may be isolated from the fermentation medium using methods known in the art, such as the methods for ABE fermentations (see for example, Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the butanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.
EXAMPLES
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
The meaning of abbreviations used is as follows: “kb” means kilobase(s), “min” means minute(s), “h” or “hr” means hour(s), “sec’ means second(s), “d” means day(s), “nl” means nanoliter(s), “μl” means microliter(s), “ml” means milliliter(s), “L” means liter(s), “nm” means nanometer(s), “mm” means millimeter(s), “cm” means centimeter(s), “μm” means micrometer(s), “μM” means micromolar, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” means micromole(s), “g” means gram(s), “ng” means nanogram(s), “μg” means microgram(s), “mg” means milligram(s), “rpm” means revolutions per minute, “w/v” means weight/volume, “Cm” means chloramphenicol, “OD” means optical density, and “OD600” means optical density measured at a wavelength of 600 nm.
General Methods
Growth medium was semi-synthetic LAB medium, pH7, with bovine serum albumin (BSA) used as a carrier. In general, the presence of BSA resulted in a medium with little to no cloudiness when fatty acids were added. The composition of this medium is as follows:
0.01 M Ammonium Sulfate
0.005 M Potassium Phosphate, pH 7.0
0.05 M MOPS, pH 7.0
1% S10 Metal Mix
0.01 M Glucose
0.2% Yeast Extract
0.01% Casamino Acids
5 g/l BSA
The composition of S10 Metal Mix is:
200 mM MgCl2 
70 mM CaCl2 
5 mM MnCl2 
0.1 mM FeCl3 
0.1 mM ZnCl2 
0.2 mM Thiamine Hydrochloride
172 μM CuSO4 
253 μM CoCl2 
242 μM Na2MoO4 
All medium ingredients were purchased from Sigma Chemical Company (St. Louis, Mo.) except yeast extract and casamino acids, which were purchased from Beckton, Dickinson and Co (Sparks, Md.). Free fatty acids, added to a final concentration of 50 mg/ml from 1% ethanol stock solutions (stored at −20° C.), were purchased from Sigma Chemical Company (St Louis, Mo.), Isobutanol, 1-butanol, 2-butanol, and methyl ethyl ketone (MEK) were purchased from Sigma Chemical Company (St. Louis, Mo.).
A working stock of Lactobacillus plantarum PN0512 (ATCC # PTA-7727) was prepared to use as a consistent source of inoculum. Cultures were grown in MRS medium (Acumedia Manufacturers, Inc. Lansing, Mich.) at 30° C. overnight. Glycerol was added to a final concentration of 12.5% and aliquots were frozen at −80° C. One aliquot was thawed at room temperature and used to inoculate all tubes in an experiment and then discarded.
For preparation of samples for fatty acid methyl ester analysis (FAME), the working stock was used to inoculate 40 ml of medium containing free fatty acids and the cultures were grown overnight. The cell pellet was harvested by centrifugation and was washed twice with phosphate buffered saline (PBS, Bio-Rad Laboratories, Hercules, Calif.) and 5 g/l BSA, then two more times with PBS. Cell pellets were stored at −80° C. until analyzed by FAME using a transesterification protocol, which quantifies fatty acids that have been incorporated in membrane lipids, but not free fatty acids associated with the cell membrane.
Lipid Extraction
The membrane lipids were extracted by modified Bligh and Dyer protocol (Can. J. Biochem. Physiol. (1959) 37:911-17). The cell pellet prepared as described above was suspended in a mixture of 0.5 ml CHCl3 and 1 ml CH3OH, and transferred to a 13×100 mm tube with a screw top cap. The cap was screwed on about 3⁄4 of the way (i.e., not tight), and the tube was incubated at 40° C. for 30 min. The tube was cooled and an additional 0.5 ml CHCl3 and 1 ml H2O were added the mixture. This results in the formation of two phases. The two phases were equilibrated by vortexing. The two phases were allowed to separate; then the lower CHCl3 layer was removed and transferred to another 13×100 mm tube with a screw top cap. With the cap removed, the CHCl3 was evaporated under a stream of N2. Methyl esters of the fatty acids in the residue were then formed using one of the following procedures.
Formation of Fatty Acid Methyl Esters by Transesterification Using CH3ONa in CH3OH
1 ml freshly made 1.0 M CH3ONa in CH3OH was added to the tubes containing lipid samples extracted by the Bligh and Dyer method as described above. The caps were placed on tubes, screwed on about 3⁄4 of the way (i.e., not tight), then the tubes were heated at 60° C. for 30 minutes. The mixture was chilled in ice bath and 1 ml of 1.0 N HCl was added to the solution in the tubes. The pH of the resulting solution was checked with pH paper to make sure a pH of 7 or lower had been reached. 0.5 ml hexane was added into the test tube and mixed well by vortexing. The tubes were allowed to sit for a few minutes until two phases formed. The top hexane layer was removed and placed in a separate tube for storage until analysis, which was done by GC/FID and/or GC/MS. 2 μl of the hexane layer was injected into an Agilent GC (model 6890)/MS (model 5973). For routine samples a Supelco Equity-1 column (15 m×0.25 mm×0.25 um film thickness; catalog #28045-U) was used with an FID detector (GC/FID). When an unknown peak needed to be identified, the same column was used with an Agilent MSD detector (GC/MS). When samples requiring difficult separations that were impossible to achieve on a 15 m column were analyzed (e.g., the separation of oleic from elaidic acid), a Supelco S-2380 column (100 m×0.25 mm×0.25 um film thickness; catalog #24317) was used.
Growth Analysis
For growth yield experiments, 5 ml of medium with fatty acids and several concentrations of 1-butanol, isobutanol, 2-butanol, or MEK in 15 ml screw cap tubes was inoculated with 12.5 μl of the working stock giving an initial OD600 of 0.012. The caps were tightly sealed and incubated at 30° C. on a roller drum for 20 to 26 hours, at which time 1.0 ml was removed and OD600 was measured with a blank of medium amended with the fatty acid. All solvent concentrations are reported as % (w/v).
Methods for Determining Isobutanol Concentration in Culture Media
The concentration of isobutanol in the culture media can be determined by a number of methods known in the art. For example, a specific high performance liquid chromatography (HPLC) method utilized a Shodex SH-1011 column with a Shodex SH-G guard column, both purchased from Waters Corporation (Milford, Mass.), with refractive index (RI) detection. Chromatographic separation was achieved using 0.01 M H2SO4 as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C. Isobutanol had a retention time of 46.6 min under the conditions used. Alternatively, gas chromatography (GC) methods are available. For example, a specific GC method utilized an HP-INNOWax column (30 m×0.53 mm id, 1 μm film thickness, Agilent Technologies, Wilmington, Del.), with a flame ionization detector (FID). The carrier gas was helium at a flow rate of 4.5 mL/min, measured at 150° C. with constant head pressure; injector split was 1:25 at 200° C.; oven temperature was 45° C. for 1 min, 45 to 220° C. at 10° C./min, and 220° C. for 5 min; and FID detection was employed at 240° C. with 26 mL/min helium makeup gas. The retention time of isobutanol was 4.5 min.
Methods for Determining 2-Butanol Concentration in Culture Media
The concentration of 2-butanol in the culture media can be determined by a number of methods known in the art. For example, a specific high performance liquid chromatography (HPLC) method utilized a Shodex SH-1011 column with a Shodex SH-G guard column, both purchased from Waters Corporation (Milford, Mass.), with refractive index (RI) detection. Chromatographic separation was achieved using 0.01 M H2SO4 as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C. Under the conditions used, 2-butanol had a retention time of 44.3 min. Alternatively, gas chromatography (GC) methods are available. For example, a specific GC method utilized an HP-INNOWax column (30 m×0.53 mm id, 1 μm film thickness, Agilent Technologies, Wilmington, Del.), with a flame ionization detector (FID). The carrier gas was helium at a flow rate of 4.5 mL/min, measured at 150° C. with constant head pressure; injector split was 1:25 at 200° C.; oven temperature was 45° C. for 1 min, 45 to 220° C. at 10° C./min, and 220° C. for 5 min; and FID detection was employed at 240° C. with 26 mL/min helium makeup gas. The retention time of 2-butanol was 5.03 min.
Methods for Determining 1-Butanol Concentration in Culture Media
The concentration of 1-butanol in the culture media can be determined by a number of methods known in the art. For example, a specific high performance liquid chromatography (HPLC) method utilized a Shodex SH-1011 column with a Shodex SH-G guard column, both purchased from Waters Corporation (Milford, Mass.), with refractive index (RI) detection. Chromatographic separation was achieved using 0.01 M H2SO4 as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C. 1-Butanol had a retention time of 52.8 min under the conditions used. Alternatively, gas chromatography (GC) methods are available. For example, a specific GC method utilized an HP-INNOWax column (30 m×0.53 mm id, 1 μm film thickness, Agilent Technologies, Wilmington, Del.), with a flame ionization detector (FID). The carrier gas was helium at a flow rate of 4.5 mL/min, measured at 150° C. with constant head pressure; injector split was 1:25 at 200° C.; oven temperature was 45° C. for 1 min, 45 to 220° C. at 10° C./min, and 220° C. for 5 min; and FID detection was employed at 240° C. with 26 mL/min helium makeup gas. The retention time of 1-butanol was 5.4 min. A similar GC method using a Varian CP-WAX 58(FFAP) CB column (25 m×0.25 mm id×0.2 μm film thickness, Varian, Inc., Palo Alto, Calif.) was also used.
Example 1
Incorporation of Fed Fatty Acids into Membrane Lipids of L. plantarum PN0512
Cultures of Lactobacillus plantarum PN0512 were grown in media containing either oleic acid (cis) or elaidic acid (trans), or no added fatty acid, as described in General Methods, and membrane composition was analyzed also as described in General Methods. The results of FAME analyses shown in Table 1 indicate that when elaidic acid or oleic acid was added to the growth medium of PN0512 these were incorporated into the cell membrane so that the amount of the fed fatty acid was substantially increased in the cell membrane.







TABLE 1







Effect of feeding free fatty acids on membrane composition of


L. plantarum PN0512; amounts are in weight %.









fatty acid in growth medium












Oleic (C18:1,
Elaidic (C18:1,


membrane fatty acid
None
9-cis)
9-trans)













C14:0
<0.1
<0.1
1.8


C16:0
27.1
19.1
16.4


C16:1
 5.8
2
5.6


C18:0
 4.1
1.5
1.5


C18:1, 9-cis
nd*
42.7
nd


C18:1, 9-trans
nd
nd
44


C18:1, 11-cis
42.4
14.4
18.3


cyc-C19:0-9-(cyclopropane
nd
13.3
nd


derived from 9-cis)





cyc-C19:0-11-(cyclopropane
16.4
7.2
12.3


derived from 11-cis)





*nd means not detected






Oleic, elaidic, and dihydrosterculic (cyc-C19:0, 9-) acids are not normally found in the cell membrane of L. plantarum. When elaidic or oleic acids were fed, each increased from 0% to high levels in the cell membrane of strain PN0512. Dihydrosterculic is present when PN0512 is fed oleic acid because cyclopropane fatty acid synthase in PN0512 converts the cis double bond in oleic acid to cyclopropane. Thus these growth conditions yield cell cultures with substantially different cell membranes that were used to determine the effect of elevated trans fatty acid in the membrane lipids on butanol tolerance.

Example 2
Improved Tolerance to Isobutanol with Increased Trans Unsaturated Fatty Acids in the Cell Membrane
Oleic acid (cis) and elaidic acid (trans) differ only in the conformation of the double bond. As shown in Example 1, feeding L. plantarum cells either oleic or elaidic acid resulted in membranes containing an increased amount of the fed fatty acid. Growth in the presence of these fatty acids and various concentrations of isobutanol was tested. Cultures were prepared as described in General Methods. Table 6 displays the average of two independent experiments comparing the growth yield of elaidic acid and oleic acid fed cultures of PN0512 after 20 hours of incubation at 30° C. in different amounts of isobutanol.







TABLE 2







Growth yield of oleic acid and elaidic acid fed L. plantarum


PN0512 in the presence of isobutanol.









[Isobutanol] %
OD600 Oleic
OD600 Elaidic


w/v
fed
fed












0
1.340
1.280


1.0
1.190
1.210


1.5
1.110
1.145


2.0
1.130
1.100


2.5
0.519
0.922


2.7
0.387
0.606


2.9
0.095
0.281


3.1
0.063
0.122


3.3
0.035
0.072


3.5
0.015
0.042









These results show that at concentrations greater than 2% isobutanol, the growth yield of the elaidic acid fed cultures was greater than the growth yield of the oleic acid fed cultures. For example, for cultures grown in 2.5% w/v isobutanol, the growth yield was 78% higher in the elaidic acid fed cultures than in the oleic acid fed cultures. These results are consistent with greater isobutanol tolerance of the culture with a high trans unsaturated fatty acid in the membrane as compared with the culture with high cis unsaturated fatty acid.

Example 3
Improved Tolerance to 1-Butanol with Increased Trans Unsaturated Fatty Acids in the Cell Membrane
Growth of PN0512 in the presence of oleic acid or elaidic acid and various concentrations of 1-butanol was tested. Cultures were prepared as described in General Methods. Table 7 shows the results, giving the average of the OD600 of biological replicates for each culture after overnight growth at 30° C. in different amounts of 1-butanol.







TABLE 7







Growth yield of oleic acid and elaidic acid fed L. plantarum


PN0512 in the presence of 1-butanol.









[1-Butanol]
OD600
OD600


% (w/v)
oleic fed
elaidic fed












0
1.509
1.508


2.0
1.026
1.039


2.25
0.611
1.003


2.5
0.161
0.559


2.75
0.061
0.095


3.0
0.003
0.009









These results show that at concentrations greater than 2% 1-butanol, the growth yield of the elaidic acid fed cultures was greater than the growth yield of the oleic acid fed cultures. For example, for cultures grown in 2.5% w/v 1-butanol, the growth yield was greater than 3 fold higher in the elaidic acid fed cultures than in the oleic acid fed cultures. These results are consistent with greater 1-butanol tolerance of the culture with a high trans unsaturated fatty acid in the membrane as compared with the culture with high cis unsaturated fatty acid.

Example 4
Improved Tolerance to 2-Butanol with Increased Trans Unsaturated Fatty Acids in the Cell Membrane
Growth of PN0512 in the presence of oleic acid or elaidic acid and various concentrations of 2-butanol was tested. Cultures were prepared as described in General Methods. Table 8 shows the results giving the average of the OD600 of biological replicates for each culture after overnight growth at 30° C. in different amounts of 2-butanol.







TABLE 8







Growth yield of oleic acid and elaidic acid fed L. plantarum


PN0512 in the presence of 2-butanol.









[2-butanol]
OD600
OD600


% w/v
Oleic fed
Elaidic fed












0
1.480
1.490


2.0
1.410
1.430


3.0
1.130
1.270


4.0
0.431
1.030


4.5
0.100
0.400


4.7
0.067
0.186


4.9
0.040
0.088


5.1
0.030
0.038


5.3
0.004
0.030


5.5
0.004
0.008









As was observed with isobutanol and 1-butanol, the elaidic acid fed culture demonstrated improved tolerance to 2-butanol when compared to the oleic acid fed culture. For example, for cultures grown in 4.5% w/v 2-butanol, the growth yield was 4 fold higher in the elaidic acid fed cultures than in the oleic acid fed cultures.

Example 5
Specificity of Tolerance Improvements with Increased Trans Unsaturated Fatty Acids in the Cell Membrane
Growth of PN0512 in the presence of oleic acid or elaidic acid and various concentrations of methyl ethyl ketone (MEK) was tested. Cultures were prepared as described in General Methods. Table 9 shows the results, giving the average of the OD600 of biological replicates after overnight growth at 30° C. in different amounts of MEK.







TABLE 9







Growth yield of oleic and elaidic fed L. plantarum


PN0512 in the presence of MEK









[MEK], 
OD600
OD600


% (w/v)
oleic fed
elaidic fed












0
1.53895
1.548


3.5
1.00155
1.0041


4.0
0.9446
0.91055


4.5
0.942
0.88705


5.0
0.6828
0.32345


5.5
0.50045
0.14765









In contrast to the results with isobutanol, 2-butanol, and 1-butanol, elaidic acid fed cultures of PN0512 did not have improved tolerance to MEK as compared with oleic acid fed cultures. Thus, there was specificity in that elevated trans fatty acids improved tolerance to 4 carbon alcohols, but not to a 4 carbon ketone.

Example 6
Genetic Implementation of Elevated Trans Fatty Acids in Cell Membrane (Prophetic)
It may not be desirable for a biological process of butanol production to rely on exogenously added fatty acids to alter membrane properties of a production organism. Thus, genetic changes to the production organism resulting in altered membrane composition can be made. Expression of an enzyme that converts cis unsaturated fatty acids to the trans conformation will increase the levels of trans fatty acids in bacterial cells that do not normally have trans fatty acids. Such an enzyme is the esterified fatty acid cistrans isomerase (cti) of Pseudomonas putida KT2440, encoded by cti (PP—2376; protein with SEQ ID NO:136, coding region with SEQ ID NO:135).
For expression, the coding region of the cti gene is amplified by PCR and cloned into an expression vector. For example, expression in Escherichia coli is accomplished using the pTrcHis2-TOPO vector (Invitrogen Inc., Carlsbad, Calif.). The cti coding region is obtained by PCR amplification using genomic DNA from P. putida KT2440 (ATCC#47054D-5) as a template and the following sense and antisense primers, respectively:







(SEQ ID NO: 153









5′ ACAGGAGAATGAATTCATGGTGCATCGTATCCTTGCC 3′



 







(SEQ ID NO: 154)









5′ TCAGAGGTTCTCGTAGCGGT 3′






The sense primer includes an extension that provides a ribosome binding site and eliminates the short N-terminal fusion in the pTrcHis2-TOPO vector by generating an in-frame termination codon in the primer. The antisense primer includes the stop codon for the coding region, so that the native protein will be expressed in E. coli. Cloning of this fragment into the pTrcHis2-TOPO vector is done following the manufacturer's protocol. Orientation of the cloned insert and verification of the cloned sequence is done by DNA sequence analysis. A plasmid with the cti coding region in the correct orientation for expression controlled by the trc promoter is saved and is named pTrcCti. Transformed E. coli MG1655 (ATCC#700926) cells carrying this plasmid are grown in LB medium (Teknova, Inc. Half Moon Bay, Calif.) at 30° C. or 37° C. Cells harvested and analyzed by FAME are expected to show the presence of trans monounsaturated fatty acids in membrane lipids. Increased tolerance to isobutanol, 1-butanol, and 2-butanol is expected to be evident in growth yield assays of these cells done at 30° C. or 37° C. as described in Examples 2, 3, and 4.
Example 7
Prophetic
Producing Isobutanol Using E. coli Strain with Expression of cti
E. coli strains engineered to express an isobutanol biosynthetic pathway are described in US Patent Application Publication No. US20070092957A1, Examples 9-15, which are herein incorporated by reference. Strain BL21 (DE) 1.5GI yqhD/pTrc99a::budB-ilvC-ilvD-kivD was derived from BL21 (DE3) (Invitrogen) and was engineered to contain an operon expressed from the trc promoter that includes the Klebsiella pneumoniae budB coding region for acetolactate synthase, the E. coli ilvC coding region for acetohydroxy acid reductoisomerase, the E. coli ilvD coding region for acetohydroxy acid dehydratase and the Lactococcus lactis kivD coding region for branched chain α-keto acid decarboxylase. In addition, in this strain the native promoter of the yqhD gene (encoding 1,3-propanediol dehydrogenase) was replaced with the 1.5GI promoter (WO 2003/089621). The same promoter replacement was made in E. coli strain MG1655 to create MG1655 1.5GI-yqhD::Cm, and the same plasmid was introduced resulting in strain MG655 1.5/GI yqhD/pTrc99A::budB-ilvC-ilvD-kivD.
These isobutanol pathway containing strains are engineered for butanol tolerance by introducing a compatible plasmid for expression of a cti gene. Such a compatible plasmid is constructed by amplifying the region from plasmid pTrcCti described in Example 6 with the trc promoter and the E. coli cti gene. Both of the primers for amplification (SEQ ID NOs:155 and 156) also have a BsrD I restriction site. Sense primer: 5′-GCAATGGTTTGACAGCTTATCATCGAC-3′ Antisense primer: 5′-GCAATGGAGGTTCTCGTAGCGGTTCA-3′ The PCR product is partially digested with BsrD I and the largest fragment is ligated into BsrD I digested vector pACYC184 (New England Biolabs, Beverly, Mass.). Transformants of E. coli TOP10 are selected for tetracycline resistance and screened for sensitivity to chloroamphenicol. Plasmid DNA is isolated from tetracycline resistant and chloramphenicol sensitive transformants. The presence of the trc promoter and the cti gene are verified by DNA sequence analysis. This plasmid having the P. putida KT2440 cti coding region expressed from the trc promoter in the pACYC184 vector backbone is named pACYCtrcCti and is used to transform strains BL21 (DE) 1.5GI yqhD/pTrc99a::budB-ilvC-ilvD-kivD and MG655 1.5/GI yqhD/pTrc99A::budB-ilvC-ilvD-kivD selecting for ampicillin resistance and tetracycline resistance.
These strains are analyzed for butanol production. The cells from cultures of each strain are used to inoculate shake flasks (approximately 175 mL total volume) containing 50 or 170 mL of TM3a/glucose medium (with appropriate antibiotics) to represent high and low oxygen conditions, respectively. TM3a/glucose medium contains (per liter): glucose (10 g), KH2PO4 (13.6 g), citric acid monohydrate (2.0 g), (NH4)2SO4(3.0 g), MgSO4.7H2O (2.0 g), CaCl2.2H2O (0.2 g), ferric ammonium citrate (0.33 g), thiamine.HCl (1.0 mg), yeast extract (0.50 g), and 10 mL of trace elements solution. The pH was adjusted to 6.8 with NH4OH. The trace elements solution contains: citric acid.H2O (4.0 g/L), MnSO4H2O (3.0 g/L), NaCl (1.0 g/L), FeSO4.7H2O (0.10 g/L), CoCl2.6H2O (0.10 g/L), ZnSO4.7H2O (0.10 g/L), CuSO4.5H2O (0.010 g/L), H3BO3 (0.010 g/L), and Na2MoO4. 2H2O (0.010 g/L).
The flasks are inoculated at a starting OD600 of ≦0.01 units and incubated at 34° C. with shaking at 300 rpm. The flasks containing 50 mL of medium are closed with 0.2 μm filter caps; the flasks containing 150 mL of medium are closed with sealed caps. IPTG is added to a final concentration of 0.04 mM when the cells reach an OD600 of ≧0.4 units. Approximately 18 h after induction, an aliquot of the broth is analyzed by HPLC (Shodex Sugar SH1011 column (Showa Denko America, Inc. NY) with refractive index (RI) detection) and GC (Varian CP-WAX 58(FFAP) CB, 0.25 mm×0.2 μm×25 m (Varian, Inc., Palo Alto, Calif.) with flame ionization detection (FID)) for isobutanol content, as described in the General Methods section. No isobutanol is detected in control strains. Molar selectivities and titers of isobutanol produced by strains carrying pTrc99A::budB-ilvC-ilvD-kivD are obtained. In preferred embodiments, higher titers of isobutanol are obtained in the cultures of the strains with the cti plasmid than in the parental strains.
Example 8
Prophetic
Producing 2-Butanol Using E. coli Strain with Expression of cti
The engineering of E. coli for expression of a 2-butanol biosynthetic pathway is described in US Patent Application Publication No. US20070259410A1, Examples 6 and 7, which are herein incorporated by reference. Construction is described of two plasmids for upper and lower pathway expression. In pBen-budABC, an NPR promoter (Bacillus amyloliquefaciens neutral protease promoter) directs expression of Klebsiella pneumoniae budABC coding regions for acetolactate decarboxylase, acetolactate synthase, and butanediol dehydrogenase. In pBen-pdd-sadh an NPR promoter directs expression of Klebsiella oxytoca pddABC coding regions for butanediol dehydratase alpha subunit, butanediol dehydratase beta subunit, and butanediol dehydratase gamma subunit, and the Rhodococcus ruber sadh coding region for butanol dehydrogenase. Plasmid p2BOH is described containing both operons, and strain NM522/p2BOH containing this plasmid for 2-butanol pathway expression is described.
The NM522/p2BOH strain is engineered for butanol tolerance by introducing the cti overexpression plasmid pACYCtrcCti (described in Example 7). E. coli NM522/p2BOH with and without the cti plasmid are inoculated into a 250 mL shake flask containing 50 mL of medium and shaken at 250 rpm and 35° C. The medium is composed of: dextrose, 5 g/L; MOPS, 0.05 M; ammonium sulfate, 0.01 M; potassium phosphate, monobasic, 0.005 M; S10 metal mix, 1% (v/v); yeast extract, 0.1% (w/v); casamino acids, 0.1% (w/v); thiamine, 0.1 mg/L; proline, 0.05 mg/L; and biotin 0.002 mg/L, and is titrated to pH 7.0 with KOH. S10 metal mix contains: MgCl2, 200 mM; CaCl2, 70 mM; MnCl2, 5 mM; FeCl3, 0.1 mM; ZnCl2, 0.1 mM; thiamine hydrochloride, 0.2 mM; CuSO4, 172 μM; CoCl2, 253 μM; and Na2MoO4, 242 μM. After 18 h, 2-butanol is detected by HPLC or GC analysis using methods that are well known in the art, for example, as described in the General Methods section above. In preferred embodiments, higher titers are obtained from the strain with the cti plasmid.
Example 9
Prophetic
Producing 1-Butanol Using E. coli Strain with Expression of cti
E. coli strains engineered to express a 1-butanol biosynthetic pathway are described in US Patent Application Publication No. US20080182308A1, Example 13, which is herein incorporated by reference. Two plasmids were constructed that carry genes encoding the 1-butanol pathway. Plasmid pBHR T7-ald contains a gene for expression of butyraldehyde dehydrogenase (ald). Plasmid pTrc99a-E-C-H-T contains a four gene operon comprising the upper pathway, for expression of acetyl-CoA acetyltransferase (thlA), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), and butyryl-CoA dehydrogenase (trans-2-enoyl-CoA reductase, EgTER(opt)) (EgTER(opt), crt, hbd and thlA). In addition, in this strain the native promoter of the yqhD gene (encoding 1,3-propanediol dehydrogenase) was replaced with the 1.5GI promoter (WO 2003/089621).
The 1-butanol producing strain is engineered for butanol tolerance by introducing the cti expression plasmid pACYCtrcCti (described in Example 7).
The parental strain and the transformant with the cti expression plasmid are used to inoculate shake flasks (approximately 175 mL total volume) containing 15, 50 and 150 mL of TM3a/glucose medium (with appropriate antibiotics) to represent high, medium and low oxygen conditions, respectively. TM3a/glucose medium contains (per liter): 10 g glucose, 13.6 g KH2PO4, 2.0 g citric acid monohydrate, 3.0 g (NH4)2SO4, 2.0 g MgSO4.7H2O, 0.2 g CaCl2.2H2O, 0.33 g ferric ammonium citrate, 1.0 mg thiamine.HCl, 0.50 g yeast extract, and 10 mL trace elements solution, adjusted to pH 6.8 with NH4OH. The solution of trace elements contains: citric acid.H2O (4.0 g/L), MnSO4H2O (3.0 g/L), NaCl (1.0 g/L), FeSO4.7H2O (0.10 g/L), CoCl2.6H2O (0.10 g/L), ZnSO4.7H2O (0.10 g/L), CuSO4.5H2O (0.010 g/L), H3BO3 (0.010 g/L), and Na2MoO4.2H2O (0.010 g/L). The flasks are inoculated at a starting OD600 of ≦0.01 units and incubated at 34° C. with shaking at 300 rpm. The flasks containing 15 and 50 mL of medium are capped with vented caps; the flasks containing 150 mL, are capped with non-vented caps to minimize air exchange. IPTG is added to a final concentration of 0.04 mM; the OD600 of the flasks at the time of addition is ≧0.4 units. Approximately 15 h after induction, an aliquot of the broth is analyzed by HPLC (Shodex Sugar SH1011 column) with refractive index (RI) detection and GC (Varian CP-WAX 58(FFAP) CB column, 25 m×0.25 mm id×0.2 μm film thickness) with flame ionization detection (FID) for 1-butanol content, as described in the General Methods section. In preferred embodiments, titers of 1-butanol are found to be higher in the strain harboring the cti expression plasmid.
Example 10
Prophetic
Expression of an Isobutanol Biosynthetic Pathway in Lactobacillus plantarum with Increased Expression of cti
The purpose of this prophetic Example is to describe how to express an isobutanol biosynthetic pathway in a Lactobacillus plantarum strain that expresses cti. The five genes of the isobutanol pathway, encoding five enzyme activities, are divided into two operons for expression. The budB, ilvD and kivD genes, encoding the enzymes acetolactate synthase, acetohydroxy acid dehydratase, and branched-chain α-keto acid decarboxylase, respectively, are integrated into the chromosome of Lactobacillus plantarum by homologous recombination using the method described by Hols et al. (Appl. Environ. Microbiol. 60:1401-1413 (1994)). The remaining two genes of the isobutanol biosynthetic pathway (ilvC and bdhB, encoding the enzymes acetohydroxy acid reductoisomerase and butanol dehydrogenase, respectively) and the cti gene are cloned into an expression plasmid and transformed into the Lactobacillus strain carrying the integrated isobutanol genes. Lactobacillus plantarum is grown in MRS medium (Difco Laboratories, Detroit, Mich.) at 37° C., and chromosomal DNA is isolated as described by Moreira et al. (BMC Microbiol. 5:15 (2005)).
Integration
The budB-ilvD-kivD cassette under the control of the synthetic P11 promoter (Rud et al., Microbiology 152:1011-1019 (2006)) is integrated into the chromosome of Lactobacillus plantarum ATCC BAA-793 (NCIMB 8826) at the IdhL1 locus by homologous recombination. To build the IdhL integration targeting vector, a DNA fragment from Lactobacillus plantarum (Genbank NC—004567) with homology to IdhL is PCR amplified with primers LDH EcoRV F (SEQ ID NO:140) and LDH AatIIR (SEQ ID NO:141). The 1986 bp PCR fragment is cloned into pCR4Blunt-TOPO and sequenced. The pCR4Blunt-TOPO-IdhL1 clone is digested with EcoRV and AatII releasing a 1982 bp IdhL1 fragment that is gel-purified. The integration vector pFP988 (a Bacillus integration vector that contains an E. coli replicon from pBR322, an ampicillin antibiotic marker for selection in E. coli and two sections of homology to the sacB gene in the Bacillus chromosome that directs integration of the vector and intervening sequence by homologous recombination; given as SEQ ID NO:142) is digested with HindIII and treated with Klenow DNA polymerase to blunt the ends. The linearized plasmid is then digested with AatII and the 2931 bp vector fragment is gel purified. The EcoRV/AatII IdhL1 fragment is ligated with the pFP988 vector fragment and transformed into E. coli Top10 cells. Transformants are selected on LB agar plates containing ampicillin (100 μg/mL) and are screened by colony PCR to confirm construction of pFP988-IdhL.
To add a selectable marker to the integrating DNA, the Cm resistance gene with its promoter is PCR amplified from pC194 (GenBank NC—002013) with primers Cm F (SEQ ID NO:143) and Cm R (SEQ ID NO:144), amplifying a 836 bp PCR product. This PCR product is cloned into pCR4Blunt-TOPO and transformed into E. coli Top10 cells, creating pCR4Blunt-TOPO-Cm. After sequencing to confirm that no errors are introduced by PCR, the Cm cassette is digested from pCR4Blunt-TOPO-Cm as an 828 bp MluI/SwaI fragment and is gel purified. The IdhL-homology containing integration vector pFP988-IdhL is digested with MluI and SwaI and the 4740 bp vector fragment is gel purified. The Cm cassette fragment is ligated with the pFP988-IdhL vector creating pFP988-DldhL::Cm.
Finally the budB-ilvD-kivD cassette which includes the Klebsiella pneumoniae budB coding region (SEQ ID NO:19), the E. coli ilvD coding region (SEQ ID NO:33), and the codon optimized Lactococcus lactis kivD coding region (SEQ ID NO:35) from pFP988DssPspac-budB-ilvD-kivD (described in Examples 1, 4, 9, 10, 11, 12, 14, and 20 of US 2007-0092957 A1) is modified to replace the amylase promoter with the synthetic P11 promoter. Then, the whole operon is moved into pFP988-DldhL::Cm. The P11 promoter is built by oligonucleotide annealing with primers P11 F-StuI (SEQ ID NO:145) and P11 R-SpeI (SEQ ID NO:146). The annealed oligonucleotide is gel-purified on a 6% Ultra PAGE gel (Embi Tec, San Diego, Calif.). The plasmid pFP988DssPspac-budB-ilvD-kivD, containing the amylase promoter, is digested with StuI and SpeI and the resulting 10.9 kbp vector fragment is gel-purified. The isolated P11 fragment is ligated with the digested pFP988DssPspac-budB-ilvD-kivD to create pFP988-P11-budB-ilvD-kivD. Plasmid pFP988-P11-budB-ilvD-kivD is then digested with StuI and BamHI and the resulting 5.4 kbp P11-budB-ilvD-kivD fragment is gel-purified. pFP988-DldhL::Cm is digested with HpaI and BamHI and the 5.5 kbp vector fragment isolated. The budB-ilvD-kivD operon is ligated with the integration vector pFP988-DldhL::Cm to create pFP988-DldhL-P11-budB-ilvD-kivD::Cm.
Integration of pFP988-DldhL-P11-budB-ilvD-kivD::Cm into L. plantarum BAA-793 to Form L. plantarum IdhL1::budB-ilvD-kivD::Cm Comprising Exogenous budB, ilvD, and kivD Genes.
Electrocompetent cells of L. plantarum are prepared as described by Aukrust, T. W., et al. (In: Electroporation Protocols for Microorganisms; Nickoloff, J. A., Ed.; Methods in Molecular Biology, Vol. 47; Humana Press, Inc., Totowa, N.J., 1995, pp 201-208). After electroporation, cells are outgrown in MRSSM medium (MRS medium supplemented with 0.5 M sucrose and 0.1 M MgCl2) as described by Aukrust et al. supra for 2 h at 37° C. without shaking. Electroporated cells are plated for selection on MRS plates containing chloramphenicol (10 μg/mL) and incubated at 37° C. Transformants are initially screened by colony PCR amplification to confirm integration, and initial positive clones are then more rigorously screened by PCR amplification with a battery of primers.
Plasmid Expression of ilvC, bdhB and cti1 Genes.
The remaining two isobutanol genes and cti1 under the control of the L. plantarum IdhL promoter (Ferain et al., J. Bacteriol. 176:596-601 (1994)) are expressed from plasmid pTRKH3 (O'Sullivan D J and Klaenhammer T R, Gene 137:227-231 (1993)). The IdhL promoter is PCR amplified from the genome of L. plantarum ATCC BAA-793 using primers PldhL F-HindIII (SEQ ID NO:147) and PldhL R-BamHI (SEQ ID NO:148). The 411 bp PCR product is cloned into pCR4Blunt-TOPO and sequenced. The resulting plasmid, pCR4Blunt-TOPO-PldhL is digested with HindIII and BamHI releasing the PldhL fragment. The cti coding region is PCR amplified from Pseudomonas putida KT240 genomic DNA using primers SEQ ID NOs:153 and 154 from Ex 6). The PCR product is cloned into pCR4Blunt-TOPO and sequenced. The resulting plasmid, pCR4Blunt-TOPO-cti, is digested with SphI releasing the fragment with the cti coding region.
Plasmid pTRKH3 is digested with SphI and partially digested with HindIII. The gel-purified approximately 7 Kb vector fragment is ligated with the PldhL fragment and the gel-purified 2.4 kbp BamHI/SphI fragment containing ilvC(B.s.)-bdhB, which includes the Bacillus subtilis ilvC coding region (SEQ ID NO:41) and the Clostridium acetobutylicum bdhB coding region (SEQ ID NO:13) from a Bacillus expression plasmid pBDPgroE-ilvC(B.s.)-bdhB (described in Example 20 of US 2007-0092957 A1) in a three-way ligation. The ligation mixture is transformed into E. coli Top 10 cells and transformants are grown on Brain Heart Infusion (BHI, Difco Laboratories, Detroit, Mich.) plates containing erythromycin (150 mg/L). Transformants are screened by PCR to confirm construction. The resulting plasmid, pTRKH3-ilvC(B.s.)-bdhB, is digested with SphI, treated with calf intestinal alkaline phosphatase, and ligated with the cti coding region fragment. The ligation mixture is transformed into E. coli Top 10 cells and transformants are grown on Brain Heart Infusion (BHI, Difco Laboratories, Detroit, Mich.) plates containing erythromycin (150 mg/L). The transformants are screened by PCR and one with the cti gene in the same orientation as ilvC and bdhB is retained and named pTRKH3-ilvC(B.s.)-bdhB-cti. This plasmid and plasmid pTRKH3-ilvC(B.s.)-bdhB are transformed into L. plantarum ΔldhL1::budB-ilvD-kivD::Cm by electroporation, as described above.
L. plantarum ΔIdhL1::budB-ilvD-kivD::Cm containing pTRKH3-ilvC(B.s.)-bdhB-cti or containing pTRKH3-ilvC(B.s.)-bdhB are inoculated into a 250 mL shake flask containing 50 mL of MRS medium plus erythromycin (10 μg/mL) and grown at 37° C. for 18 to 24 h without shaking, after which isobutanol is detected by HPLC or GC analysis. In preferred embodiments, higher titers of isobutanol are obtained from the strain with the cti gene on the plasmid.
Example 11
Prophetic
Expression of the 1-Butanol Biosynthetic Pathway in Lactobacillus plantarum with Expression of cti
The purpose of this prophetic Example is to describe how to express the 1-butanol biosynthetic pathway in a Lactobacillus plantarum strain that expresses cti. The six genes of the 1-butanol pathway, encoding six enzyme activities, are divided into two operons for expression. The first three genes of the pathway (thl, hbd, and crt, encoding the enzymes acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, and crotonase, respectively) are integrated into the chromosome of Lactobacillus plantarum by homologous recombination using the method described by Hols et al. (Appl. Environ. Microbiol. 60:1401-1413 (1994)). The last three genes of the 1-butanol pathway (EgTER, ald, and bdhB, encoding the enzymes butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase and butanol dehydrogenase, respectively) and cti are cloned into an expression plasmid and transformed into the Lactobacillus strain carrying the integrated upper pathway 1-butanol genes. Lactobacillus is grown in MRS medium (Difco Laboratories, Detroit, Mich.) at 37° C. Chromosomal DNA is isolated from Lactobacillus plantarum as described by Moreira et al. (BMC Microbiol. 5:15 (2005)).
Integration
The thl-hbd-crt cassette under the control of the synthetic P11 promoter (Rud et al., Microbiology 152:1011-1019 (2006)) is integrated into the chromosome of Lactobacillus plantarum ATCC BAA-793 (NCIMB 8826) at the IdhL1 locus by homologous recombination. To build the IdhL integration targeting vector, a DNA fragment from Lactobacillus plantarum (Genbank NC—004567) with homology to IdhL is PCR amplified with primers LDH EcoRV F (SEQ ID NO:140) and LDH AatIIR (SEQ ID NO:141). The 1986 bp PCR fragment is cloned into pCR4Blunt-TOPO and sequenced. The pCR4Blunt-TOPO-IdhL1 clone is digested with EcoRV and AatII releasing a 1982 bp IdhL1 fragment that is gel-purified. The integration vector pFP988, described in Example 10, is digested with HindIII and treated with Klenow DNA polymerase to blunt the ends. The linearized plasmid is then digested with AatII and the 2931 bp vector fragment is gel-purified. The EcoRV/AatII IdhL1 fragment is ligated with the pFP988 vector fragment and transformed into E. coli Top10 cells. Transformants are selected on LB agar plates containing ampicillin (100 μg/mL) and are screened by colony PCR to confirm construction of pFP988-IdhL.
To add a selectable marker to the integrating DNA, the Cm gene with its promoter is PCR amplified from pC194 (Genbank NC—002013) with primers Cm F (SEQ ID NO:143) and Cm R (SEQ ID NO:144), amplifying a 836 bp PCR product. The amplicon is cloned into pCR4Blunt-TOPO and transformed into E. coli Top10 cells, creating pCR4Blunt-TOPO-Cm. After sequencing to confirm that no errors are introduced by PCR, the Cm cassette is digested from pCR4Blunt-TOPO-Cm as an 828 bp MluI/SwaI fragment and is gel-purified. The IdhL-homology containing integration vector pFP988-IdhL is digested with MluI and SwaI and the 4740 bp vector fragment is gel-purified. The Cm cassette fragment is ligated with the pFP988-IdhL vector creating pFP988-DldhL::Cm.
Finally the thl-hbd-crt cassette from pFP988Dss-T-H-C (described in WO2007041269 Examples 13 and 14, which are herein incorporated by reference) including the Clostridium acetobutylicum thlA, hbd, and crt coding regions (SEQ ID NOs:1, 5, and 7 respectively) is modified to replace the amylase promoter with the synthetic P11 promoter. Then, the whole operon is moved into pFP988-DldhL::Cm. The P11 promoter is built by oligonucleotide annealing with primer P11 F (SEQ ID NO:149) and P11 R (SEQ ID NO:150). The annealed oligonucleotide is gel-purified on a 6% Ultra PAGE gel (Embi Tec, San Diego, Calif.). The plasmid pFP988Dss-T-H-C is digested with XhoI and SmaI and the 9 kbp vector fragment is gel-purified. The isolated P11 fragment is ligated with the digested pFP988Dss-T-H-C to create pFP988-P11-T-H-C. Plasmid pFP988-P11-T-H-C is digested with XhoI and BamHI and the 3034 bp P11-T-H-C fragment is gel-purified. pFP988-DldhL::Cm is digested with XhoI and BamHI and the 5558 bp vector fragment isolated. The upper pathway operon is ligated with the integration vector to create pFP988-DldhL-P11-THC::Cm.
Integration of pFP988-DldhL-P11-THC::Cm into L. plantarum BAA-793 to Form L. plantarum ΔldhL1::T-H-C::Cm Comprising Exogenous thl, hbd, and crt Genes
Electrocompetent cells of L. plantarum are prepared as described by Aukrust, T. W., et al. (In: Electroporation Protocols for Microorganisms; Nickoloff, J. A., Ed.; Methods in Molecular Biology, Vol. 47; Humana Press, Inc., Totowa, N.J., 1995, pp 201-208). After electroporation, cells are outgrown in MRSSM medium (MRS medium supplemented with 0.5 M sucrose and 0.1 M MgCl2) as described by Aukrust et al. supra for 2 h at 37° C. without shaking. Electroporated cells are plated for selection on MRS plates containing chloramphenicol (10 μg/mL) and incubated at 37° C. Transformants are initially screened by colony PCR amplification to confirm integration, and initial positive clones are then more rigorously screened by PCR amplification with a battery of primers.
Plasmid Expression of EgTER, ald, and bdhB Genes.
The three remaining 1-butanol genes under the control of the L. plantarum IdhL promoter (Ferain et al., J. Bacteriol. 176:596-601 (1994)). and cti under control of the atpB promoter are expressed from plasmid pTRKH3 (O'Sullivan D J and Klaenhammer T R, Gene 137:227-231 (1993)). The IdhL promoter is PCR amplified from the genome of L. plantarum ATCC BAA-793 with primers PldhL F (SEQ ID NO:151) and PldhL R (SEQ ID NO:152). The 369 bp PCR product is cloned into pCR4Blunt-TOPO and sequenced. The resulting plasmid, pCR4Blunt-TOPO-PldhL is digested with SacI and BamHI releasing the 359 bp PldhL fragment.
pHT01-ald-EB (described in WO2007041269 Examples 9, 13 and 14) including the Clostridium beijerinckii ald coding region, the Clostridium acetobutylicum bdhB and a codon optimized Euglena gracilis TER fragment (SEQ ID NOs:11, 13, and 39 respectively) is digested with SacI and BamHI and the 10503 bp vector fragment is recovered by gel purification. The PldhL fragment and vector are ligated creating pHT01-Pldhl-ald-EB.
To subclone the IdhL promoter-ald-EgTER-bdh cassette, pHT01-Pldhl-ald-EB is digested with MluI and the ends are treated with Klenow DNA polymerase. The linearized vector is digested with SalI and the 4270 bp fragment containing the PldhL-AEB fragment is gel-purified. Plasmid pTRKH3 is digested with SalI and EcoRV and the gel-purified vector fragment is ligated with the PldhL-AEB fragment. The ligation mixture is transformed into E. coli Top 10 cells and transformants are plated on Brain Heart Infusion (BHI, Difco Laboratories, Detroit, Mich.) plates containing erythromycin (150 mg/L). Transformants are screened by PCR to confirm construction of pTRKH3-ald-E-B.
The cti gene is amplified from Pseudomonas putida KT2440 genomic DNA as described in example 6. The PCR product is cloned into pCR4Blunt-TOPO and sequenced. The resulting plasmid, pCR4Blunt-TOPO-cti, is digested with NruI and XhoI releasing the fragment with the cti coding region.
The plasmid pTRKH3-ald-E-B is digested with NruI and XhoI and the large fragment is gel purified and ligated with the cti fragment. The ligation mixture is transformed into E. coli Top 10 cells and transformants are grown on Brain Heart Infusion (BHI, Difco Laboratories, Detroit, Mich.) plates containing erythromycin (150 mg/L). Transformants are screened by PCR to confirm construction of plasmid pTRKH3-ald-E-B-cti, where cti is expressed from the same promoter as ald-E-b.
Plasmids pTRKH3-ald-E-B and pTRKH3-ald-E-B-cti are transformed into L. plantarum ΔldhL1::T-H-C::Cm by electroporation, as described above.
L. plantarum ΔIdhL1::T-H-C::Cm containing pTRKH3-ald-E-B or containing pTRKH3-ald-E-B-PatpB-cti are inoculated into a 250 mL shake flask containing 50 mL of MRS medium plus erythromycin (10 μg/mL) and grown at 37° C. for 18 to 24 h without shaking. After 18 h to 24, 1-butanol is detected by HPLC or GC analysis. In preferred embodiments, higher titers of 1-butanol are obtained from the strain with the cti gene on the plasmid.

## Claims

1. An isobutanol tolerant Lactobacillus bacterial cell comprising:
(a) an engineered isobutanol biosynthetic pathway, wherein the Lactobacillus bacterial cell comprises heterologous genes encoding an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase or dihydroxyacid dehydratase, a branched-chain keto acid decarboxylase, and a branched-chain alcohol dehydrogenase that perform the following substrate to product conversions:
(i) pyruvate to acetolactate catalyzed by the acetolactate synthase,
(ii) acetolactate to 2,3-dihydroxyisovalerate catalyzed by the acetohydroxy acid isomeroreductase,
(iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate catalyzed by the acetohydroxy acid dehydratase or dihydroxyacid dehydratase,
(iv) α-ketoisovalerate to isobutyraldehyde catalyzed by the branched-chain keto acid decarboxylase, and
(v) isobutyraldehyde to isobutanol catalyzed by the branched-chain alcohol dehydrogenase; and

(b) at least one gene encoding a heterologous fatty acid cistrans isomerase, wherein the isobutanol tolerant Lactobacillus bacterial cell has increased concentration of membrane unsaturated trans fatty acids as compared with that of the corresponding Lactobacillus bacterial cell that lacks the at least one gene encoding a heterologous fatty acid cistrans isomerase.

2. The isobutanol tolerant Lactobacillus bacterial cell of claim 1, wherein the concentration of at least one unsaturated trans fatty acid selected from the group consisting of elaidic acid, vaccenic acid, and C16:1 trans fatty acid is increased as compared with that of the corresponding Lactobacillus bacterial cell that lacks the at least one gene encoding a heterologous fatty acid cistrans isomerase.

3. The isobutanol tolerant Lactobacillus bacterial cell of claim 1, wherein said cell produces isobutanol and the growth yield of the cell is at least 1.6 to about 3.5-fold higher in a culture medium comprising 2.5% isobutanol than the corresponding Lactobacillus bacterial cell that lacks the at least one gene encoding a heterologous fatty acid cistrans isomerase.

4. The isobutanol tolerant Lactobacillus bacterial cell of claim 1, wherein the concentration of at least one membrane unsaturated trans fatty acid is about 44-fold higher than that of the corresponding Lactobacillus bacterial cell that lacks the at least one gene encoding a heterologous fatty acid cistrans isomerase.

5. A method for the production of isobutanol comprising:
(I) providing an isobutanol tolerant Lactobacillus bacterial cell that comprises:
(a) an engineered isobutanol biosynthetic pathway, wherein the Lactobacillus bacterial cell comprises heterologous genes encoding an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase or dihydroxyacid dehydratase, a branched-chain keto acid decarboxylase, and a branched-chain alcohol dehydrogenase that perform the following substrate to product conversions:
(i) pyruvate to acetolactate catalyzed by the acetolactate synthase,
(ii) acetolactate to 2,3-dihydroxyisovalerate catalyzed by the acetohydroxy acid isomeroreductase,
(iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate catalyzed by the acetohydroxy acid dehydratase or dihydroxyacid dehydratase,
(iv) α-ketoisovalerate to isobutyraldehyde catalyzed by the branched-chain keto acid decarboxylase, and
(v) isobutyraldehyde to isobutanol catalyzed by the branched-chain alcohol dehydrogenase; and

(b) at least one gene encoding a heterologous fatty acid cistrans isomerase, wherein the isobutanol tolerant Lactobacillus bacterial cell has increased concentration of membrane unsaturated trans fatty acids as compared with that of the corresponding Lactobacillus bacterial cell that lacks the at least one gene encoding a heterologous fatty acid cistrans isomerase;

(II) feeding the Lactobacillus bacterial cell of step (I) at least one trans fatty acid under conditions wherein the concentration of unsaturated trans fatty acids in the membrane of the cell are increased; and
(III) growing the Lactobacillus bacterial cell of step (II) under conditions wherein isobutanol is produced.

6. A method for the production of isobutanol comprising:
(I) providing an isobutanol tolerant Lactobacillus bacterial cell that comprises:
(a) an engineered isobutanol biosynthetic pathway, wherein the Lactobacillus bacterial cell comprises heterologous genes encoding an acetolactate synthase, an acetohydroxy acid isomeroreductase, an acetohydroxy acid dehydratase or dihydroxyacid dehydratase, a branched-chain keto acid decarboxylase, and a branched-chain alcohol dehydrogenase that perform the following substrate to product conversions:
(i) pyruvate to acetolactate catalyzed by the acetolactate synthase,
(ii) acetolactate to 2,3-dihydroxyisovalerate catalyzed by the acetohydroxy acid isomeroreductase,
(iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate catalyzed by the acetohydroxy acid dehydratase or dihydroxyacid dehydratase,
(iv) α-ketoisovalerate to isobutyraldehyde catalyzed by the branched-chain keto acid decarboxylase, and
(v) isobutyraldehyde to isobutanol catalyzed by the branched-chain alcohol dehydrogenase; and

(b) at least one gene encoding a heterologous fatty acid cistrans isomerase, wherein the isobutanol tolerant Lactobacillus bacterial cell has increased concentration of membrane unsaturated trans fatty acids as compared with that of the corresponding Lactobacillus bacterial cell that lacks the at least one gene encoding a heterologous fatty acid cistrans isomerase; and

(II) growing the Lactobacillus bacterial cell of step (I) under conditions wherein isobutanol is produced.