# Patent

## Title
Strain comprising increased expression of a CFA coding region for butanol production

## Abstract

Screening of fatty acid fed bacteria which are not natural butanol producers identified increased membrane cyclopropane fatty acid as providing improved butanol tolerance. Increasing expression of cyclopropane fatty acid synthase in the presence of the enzyme substrate that is either endogenous to the cell or fed to the cell, increased butanol tolerance. Bacterial strains with increased cyclopropane fatty acid synthase and having a butanol biosynthetic pathway are useful for production of butanol.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Applications 61/015,728 and 61/015,732, both filed Dec. 21, 2007.


FIELD OF INVENTION
The invention relates to the fields of microbiology and genetic engineering. More specifically, altered membrane cyclopropane fatty acid composition was found to play a role in butanol tolerance in bacteria which are not natural butanol producers.
BACKGROUND OF 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.
Methods for the chemical synthesis of butanols are known. For example, 1-butanol may be produced using the Oxo process, the Reppe process, or the hydrogenation of crotonaldehyde (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). 2-Butanol may be produced using n-butene hydration (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). Additionally, isobutanol may be produced using Oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) or Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A: Chem. 220:215-220 (2004)). These processes use starting materials derived from petrochemicals, are generally expensive, and are not environmentally friendly.
Methods of producing butanol by fermentation are also known, where the most popular 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 (Donaldson et al., copending and commonly owned U.S. Patent Application Publication No. US20080182308A1), a 2-butanol biosynthetic pathway (Donaldson et al., copending and commonly owned U.S. Patent Application Publication Nos. US20070259410A1 and US 20070292927A1), and an isobutanol biosynthetic pathway (Maggio-Hall et al., copending and commonly owned U.S. Patent 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 and have some natural tolerance to butanol. Strains of Clostridium that have increased tolerance to 1-butanol have been isolated by chemical mutagenesis (Jain et al. U.S. Pat. No. 5,192,673; and Blaschek et al. U.S. Pat. No. 6,358,717), overexpression of certain classes of genes such as those that express stress response proteins (Papoutsakis et al. 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)). Overexpression in Clostridium of the endogenous gene encoding cyclopropane fatty acid synthase increased the cyclopropane fatty acid content of early log phase cells and initial butanol resistance (Zhao et al. (2003) Appl. and Environ. Microbiology 69:2831-2841).
Desmond et al. (Appl. Environ. Microbiol. 70(10):5929-5936 (2004)) report that overexpression of GroESL, two stress responsive proteins, in Lactococcus lactis and Lactobacillus paracasei produced strains that were able to grow in the presence of 0.5% volume/volume (v/v) [0.4% weight/volume (w/v)] 1-butanol. 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. However, for most bacteria described in the art, particularly those that do not naturally produce butanol, growth is highly inhibited at low concentrations of 1-butanol.
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
The invention provides a recombinant bacterial cell which does not naturally produce butanol, but which is engineered to express a butanol biosynthetic pathway, that comprises a genetic modification that affects an enzyme activity that increases the concentration of cyclopropane fatty acid in the cell membrane fatty acid composition as compared with a wildtype bacterial cell lacking the genetic modification. Such cells have an increased tolerance to butanol as compared with cells that lack the genetic modification. Increased membrane cyclopropane fatty acid composition may be accomplished via increased expression of a gene encoding a cyclopropane fatty acid synthase. The cyclopropane fatty acid synthase substrate is present in the cells either naturally as an endogenous component, or is provided exogenously to the cells.
Accordingly, the invention provides a recombinant bacterial cell which is engineered to produce butanol, and comprises at least one genetic modification affecting an enzyme activity that increases the concentration of cyclopropane fatty acid in the cell membrane fatty acid composition as compared with a wildtype bacterial cell lacking said genetic modification;
wherein the cell contains the substrate for said enzyme; and
wherein the cell does not naturally produce butanol.
In one embodiment the invention provides a recombinant host cell comprising a recombinant biosynthetic pathway selected from the group consisting of:

    
    
        a) a 1-butanol biosynthetic pathway;
        b) a 2-butanol biosynthetic pathway; and
        c) an isobutanol biosynthetic pathway.
    
    


In an alternate embodiment the invention comprises a host cell wherein the at least one genetic modification increases cyclopropane fatty acid synthase activity. In another embodiment the invention provides an additional genetic modification which reduces accumulation of (p)ppGpp as defined here.
In another embodiment the invention provides a process for generating the recombinant cell of the invention comprising:

    
    
        a) providing a recombinant bacterial host cell comprising an engineered butanol biosynthetic pathway that is producing butanol; and
        b) creating at least one genetic modification which increases cyclopropane fatty acid in the cell membrane fatty acid composition above natural levels, provided that the bacterial cell does not naturally produce butanol.
    
    


In another embodiment the invention provides a process for production of butanol from a recombinant bacterial cell comprising:

    
    
        (a) providing a recombinant bacterial host cell that does not naturally produce butanol which
        
            1) is engineered with a butanol biosynthetic pathway and produces butanol; and
            2) comprises at least one genetic modification which affects an enzyme activity that increases the concentration of cyclopropane fatty acid in the cell membrane fatty acid composition as compared with a wildtype bacterial cell lacking said genetic modification; and
        
        
        (b) culturing the strain of (a) under conditions wherein butanol is produced.
    
    





BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS
The various embodiments of the invention can be more fully understood from the following detailed description, the figures, and the accompanying sequence descriptions, which form a part of this application.
FIG. 1A shows a graph of the growth of L. plantarum PN2001, with cfa1 multicopy expression, in the presence of various concentrations of isobutanol. FIG. 1B shows a graph of the growth of L. plantarum PN2014, with control plasmid pFP996, in the presence of various concentrations of isobutanol.
FIG. 2 shows a diagram of domains of the SpoT protein, and the internal fragment used in the insertional disruption plasmid pMPE69 (in A), and a diagram of the structure of the chromosomal spoT locus after insertion of plasmid pMPE69, with positions of the primers used for confirmation (in B).



Table 6 is a table of the Profile HMM for the RelA/SpoT domain. Table 6 is submitted herewith electronically and is incorporated herein by reference.
Table 7 is a table of the Profile HMM for the TGS domain. Table 7 is submitted herewith electronically and is incorporated herein by reference.
Table 8 is a table of the Profile HMM for the HD domain. Table 8 is submitted herewith electronically and is incorporated herein by reference.
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







Summary of Gene and Protein SEQ ID Numbers


for 1-Butanol Biosynthetic Pathway










SEQ ID
SEQ ID



NO:
NO:


Description
Nucleic aid
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
5
6


from Clostridium acetobutylicum ATCC 82




Crotonase from Clostridium acetobutylicum
7
8


ATCC 824




Putative trans-enoyl CoA reductase from
9
10


Clostridium acetobutylicum ATCC 824




Euglena gracilis butyryl-CoA
110
114


dehydrogenase/trans-2-enoyl-CoA




reductase codon optimized




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







Summary of Gene and Protein SEQ ID Numbers


for 2-Butanol Biosynthetic Pathway










SEQ ID
SEQ ID



NO:
NO:


Description
Nucleic aid
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
27
28


subunit from Klebsiella oxytoca ATCC 8724




sadH, 2-butanol dehydrogenase from
29
30


Rhodococcus ruber 219
















TABLE 3







Summary of Gene and Protein SEQ ID Numbers


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
105
113


reductoisomerase)




E. coli ilvD (acetohydroxy acid dehydratase)
33
34


Lactococcus lactis kivD (branched-chain
35
36


α-keto acid decarboxylase), codon optimize




E. coli yqhD (branched-chain alcohol
37
38


dehydrogenase)
















TABLE 4







Representative spoT and relA modification target


genes and encoded proteins













SEQ



Gene
SEQ ID NO:
ID NO:


Organism
name
Nucleic acid
Peptide





E. coli
spoT
39
40


E. coli
relA
41
42


Lactobacillus plantarum WCFS1
spoT
43
44


Bacillus licheniformis ATCC 14580
spoT
45
46


Bacillus subtilis subsp. subtilis str. 168
spoT
47
48


Pseudomonas putida KT2440
relA
49
50


Pseudomonas putida KT2440
spoT
51
52


Enterococcus faecium
spoT-1
53
54


Enterococcus faecium
spoT-2
55
56


Enterococcus faecalis
spoT
57
58


Rhodococcus erythropolis
spoT
59
60
















TABLE 5







Representative cfa genes and encoded proteins












SEQ ID NO:
SEQ ID NO:


Organism
Gene name
Nucleic acid
Peptide





Lactobacillus plantarum
cfa1
61
62


Lactobacillus plantarum
cfa2
63
64


E. coli
cfa
65
66


Pseudomonas putida
cfa
67
68


Enterococcus faecalis
cfa
69
70









SEQ ID NO:71 is the nucleotide sequence of pFP996.
SEQ ID NOs:72 and 73 are primers for PCR amplification of the L. Plantarum fba promoter.
SEQ ID NO:74 is the nucleotide sequence of the L. Plantarum atpB promoter.
SEQ ID NOs:75 and 76 are primers for PCR amplification of the L. Plantarum atpB promoter.
SEQ ID NOs:77 and 78 are primers for PCR amplification of the L. Plantarum bdhB coding region.
SEQ ID NO:79 is the nucleotide sequence of a synthetic DNA fragment containing a 5′ Shine-Delgarno sequence and bdhB coding region.
SEQ ID NOs:80 and 81 are primers for PCR amplification of the L. Plantarum cfa1 coding region.
SEQ ID NOs:82 and 83 are primers for PCR amplification of the L. Plantarum cfa2 coding region.
SEQ ID NOs:84 and 85 are primers for PCR amplification of the E. coli cfa coding region.
SEQ ID NOs:86 and 87 are primers for PCR amplification of an internal fragment of the L. plantarum spoT coding region.
SEQ ID NOs:88 and 89 are primers for PCR amplification of portion of the Bacillus shuttle vector pMK4.
SEQ ID NOs:90 and 91 are primers for PCR amplification to confirm pMPE69 insertion into the spoT gene.
SEQ ID NOs:92 and 93 are primers for PCR amplification of the region from plasmid pDEW849 with the trc promoter and the E. coli cfa coding region.
SEQ ID NOs:94 and 95 are primers for PCR amplification of a DNA fragment from Lactobacillus plantarum (Genbank NC—004567) with homology to ldhL.
SEQ ID NO:96 is the integration vector pFP988.
SEQ ID NOs:97 and 98 are primers for PCR amplification of the Cm resistance gene with its promoter from pC194 (GenBank NC—002013).
SEQ ID NOs:99 and 100 are oligonucleotides for constructing the P11 promoter.
SEQ ID NOs:101 and 102 are primers for PCR amplification of the L. plantarum ldhL promoter.
SEQ ID NOs:103 and 104 are primers for PCR amplification of the L. plantarum cfa1 coding region.
SEQ ID NO:105 is the Bacillus subtilis ilvC coding region.
SEQ ID NOs:106 and 107 are oligonucleotides for constructing the P11 promoter.
SEQ ID NOs:108 and 109 are primers for PCR amplification of the L. plantarum ldhL promoter.
SEQ ID NO:110 is the sequence of a DNA fragment containing a codon optimized Euglena gracilis butyryl-CoA dehydrogenase lacking the normal mitochondrial presequence.
SEQ ID NOs:111 and 112 are primers for PCR amplification of the atpB promoter and cfa1 gene.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a recombinant bacterial cell which does not naturally produce butanol, but which is engineered to express a butanol biosynthetic pathway, that comprises a genetic modification that affects an enzyme activity that increases the concentration of cyclopropane fatty acid in the cell membrane fatty acid composition as compared with a wildtype bacterial cell lacking the genetic modification. Such cells have an increased tolerance to butanol as compared with cells that lack the genetic modification. A tolerant bacterial strain of the invention has at least one genetic modification that causes increased concentration of cyclopropane fatty acid in the cell membrane. Increase in membrane cyclopropane fatty acid may be accomplished via increased expression of a cyclopropane fatty acid synthase.
Host cells of the invention are engineered to produce butanol.
Butanol produced using the present strains may be used as an alternative energy source to fossil fuels. Fermentive production of butanol results in less pollutants than typical petrochemical synthesis.
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 “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 (TER), 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 (GenBank 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—417484, 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 (GenBank 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—417484 (SEQ ID NO:38), NC—000913 (SEQ ID NO:37)), and C. acetobutylicum (GenBank Nos: NP—349892, NC—003030).
The term “dksA” refers to a gene that encodes the DksA protein, which binds directly to RNA polymerase affecting transcript elongation and augmenting the effect of the alarmone ppGpp on transcription initiation.
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.
The term “(p)ppGpp” refers to either ppGpp or pppGpp, or a combination of both compounds.
The term “relA” refers to a gene that encodes a RelA protein which is a mono-functional enzyme with GTP pyrophosphokinase activity (EC 2.7.6.5), for synthesis of (p)ppGpp. Although in the literature some genes encoding enzymes with (p)ppGpp synthesis and degradation activities are called relA, herein these will be referred to as spoT instead of relA.
The term “spoT” refers to a gene that encodes a SpoT protein, which is a bi-functional enzyme with both GTP pyrophosphokinase, (EC 2.7.6.5) activity for synthesis of (p)ppGpp, and ppGpp pyrophosphohydrolase (EC3.1.7.2) activity for degradation of (p)ppGpp. The related RelA and SpoT proteins and their encoding genes are distinguished by both enzyme activities and domain architectures as described below.
The term “RelA/SpoT” domain will refer to a portion of the SpoT or RelA proteins that may be used to identity SpoT or RelA homologs.
As used herein “TGS domain” will refer to a portion of the SpoT or RelA protein that may be used to identity SpoT and RelA homologs. The TGS domain is named after ThrRS, GTPase, and SpoT and has been detected at the amino terminus of the uridine kinase from the spirochaete Treponema pallidum. TGS is a small domain that consists of  ̃50 amino acid residues and is predicted to possess a predominantly beta-sheet structure. Its presence in two types of regulatory proteins (the GTPases and guanosine polyphosphate phosphohydrolases/synthetases) suggests that it has a nucleotide binding regulatory role. The TGS domain is not unique to the SpoT or RelA protein, however, in combination with the presense of the HD domain and the SpoT/RelA domain it is diagnostic for a protein having SpoT function. In combination with the SpoT/RelA domain, the TGS domain is diagnostic for a protein having RelA function.
The term “HD domain” refers to an amino acid motif that is associated with a superfamily of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins and domains associated with nucleotidyltransferases and helicases from bacteria, archaea, and eukaryotes (Yakunin et al., J. Biol. Chem., Vol. 279, Issue 35, 36819-36827, Aug. 27, 2004). The HD domain is not unique to the SpoT protein, however in combination with the SpoT/RelA domain and the TGS domain, it may be used to identify SpoT proteins according to the methods described herein.
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 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 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 events that increase the cyclopropane 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 screening for butanol tolerance. Among the about ten different fatty acids tested, it was found that feeding specifically with the cyclopropane fatty acid dihydrosterculic acid provided increased butanol tolerance.
Increasing Membrane Cyclopropane Fatty Acids
In the bacterial cells of the present invention, at least one genetic modification is made that affects an enzyme activity that increases the concentration of cyclopropane fatty acid in the cell membrane fatty acid composition as compared with a wildtype bacterial cell lacking the genetic modification. The genetic modification may be made in any bacterial cell that does not naturally make butanol. Examples include, but are not limited to, bacterial cells of Escherichia, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, and Enterococcus, Specifically, a genetic modification is made that increases cyclopropane fatty acid synthase activity in the bacterial cell. The activity level may be increased by mutation of the coding region to provide an enzyme with higher levels of activity, or by increased expression of a gene encoding the enzyme. The activity level of the enzyme may be increased by mutagenesis and selection or screening using methods well known to one skilled in the art. Methods for increasing expression of a gene in a bacterial cell are common and well known in the art and may be applied to the exercise of increasing cyclopropane fatty acid synthase.
One method is to increase the level of expression of an endogenous gene encoding cyclopropane fatty acid synthase (of a cfa gene). Increased expression of an endogenous gene may be achieved by introducing multiple copies on a plasmid, introducing multiple copies into the genome, and/or expressing the coding region from a stronger promoter than the natural promoter (either from a plasmid or in the genome). For example, the natural promoter may be replaced with a stronger promoter using promoter replacement methods for exchanging the endogenous transcriptional control elements such as described in Yuan et al. (Metab Eng. (2006) 8:79-90). Increased expression of either the endogenous cfa1 gene (coding region SEQ ID NO: 61; encoded protein SEQ ID NO: 62) or cfa2 gene (coding region SEQ ID NO: 63; encoded protein SEQ ID NO: 64) of Lactobacillus plantarum is described in Examples 1 and 2 herein. Also described herein in Example 3 is increased expression of the endogenous cfa gene (coding region SEQ ID NO: 65; encoded protein SEQ ID NO: 66) in E. coli. Any bacterial cell having at least one endogenous cfa gene may be modified for increased expression of endogenous cyclopropane fatty acid synthase. Some representative cfa genes, including coding region sequences and amino acid sequences of the encoded proteins of various bacteria are given in Table 5 as SEQ ID NOs:61-70. Additional cfa genes may be identified and isolated using methods including bioinformatics, sequence comparison, hybridization, and PCR amplification as described below.
Accordingly the invention provides a recombinant cell wherein the cfa coding region is an isolated nucleic acid molecule selected from the group consisting of:

    
    
        a) an isolated nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NO:61, 63, 65, 67 and 69;
        b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; and
        c) an isolated nucleic acid molecule that encodes a polypeptide having 95% identity based on the Clustal method of alignment when compared to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:61, 63, 65, 67, and 69.
    
    


Alternatively a heterologous cfa gene may be expressed in a bacterial cell to produce an increased level of cyclopropane fatty acid synthase activity. A heterologous cfa gene may be expressed from a plasmid, or introduced into the genome. The natural promoter may be used if it is active in the heterologous cell. 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 heterologous cfa coding region for expression. Examples of promoters and plasmids (vectors) that may be used for transfer and expression of cfa 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.
Cfa Genes Used for Increased Cyclopropane Fatty Acid Synthase Expression
Examples of cfa genes that may be used to increase expression of cyclopropane fatty acid synthase in the present invention, in addition to those listed in Table 5, are identified in the literature and in bioinformatics databases well known to the skilled person. Additionally, the sequences described herein or those recited in the art may be used to identify other homologs in nature. For example each of the cfa nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins from the same or other microbial species. 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 cfa 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 bacteria 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 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 cfa 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.
Bioinformatics approaches include sequence comparisons, either nucleic acid or amino acid, using sequences described herein and sequences in databases available to the public including general sequence databases and specific genome sequence databases. In addition, databases may be used such as Pfam (Pfam: clans, web tools and services: R. D. Finn, J. Mistry, B. Schuster-Böckler, S. Griffiths-Jones, V. Hollich, T. Lassmann, S. Moxon, M. Marshall, A. Khanna, R. Durbin, S. R. Eddy, E. L. L. Sonnhammer and A. Bateman, Nucleic Acids Research (2006) Database Issue 34:D247-D251) which organizes proteins into families of structurally and functionally related members.
Substrate of Cyclopropane Fatty Acid Synthase
In the bacterial cell of the present invention, the substrate for cyclopropane fatty acid synthase is present in the cell such that the genetic modification that increases cyclopropane fatty acid synthase activity leads to increased concentration of cyclopropane fatty acid in the cell membrane. The substrate, which is a cis unsaturated moiety in a fatty acid of a membrane phospholipid, is either endogenous to the cell or is derived from unsaturated fatty acids provided exogenously to the cell. The fatty acid substrates that may be present in the cell or provided to the cell, such as in the growth medium, include but are not limited to oleic acid (C18:1 cis-9), cis-vaccenic acid (C18:1-11) and palmitoleic acid (C16:1). Cyclopropane fatty acid synthase enzymes endogenous to different bacterial cells may prefer different substrates and produce different cyclopropane fatty acids. For example, the cfa encoded enzyme of L. plantarum converts the endogenous substrate cis-vaccenic acid to the cyclopropane fatty acid lactobacillic acid (cis-11,12 methylene-octadecanoic acid cis-11,12-methylene-octadecanoic acid). The cfa encoded enzyme of E. coli converts endogenous cis-vaccenic acid (C18:1-11) and palmitoleic acid (C16:1-9) substrates to the corresponding 19cyclo and 17cyclopropane fatty acids. The L. plantarum cfa2 encoded enzyme does not convert an endogenous (in L. plantarum) substrate to cyclopropane fatty acid, but does convert oleic acid to the cyclopropane fatty acid dihydrosterculic acid when this substrate is fed to the cells in the growth medium. One skilled in the art can readily without undue experimentation determine a substrate for a particular cyclopropane fatty acid synthase and assess that it is present in the cell or if not, provide it in the growth medium.
Butanol Tolerance of Increased Cyclopropane Fatty Acid Synthase Strain
A bacterial strain of the present invention genetically modified for increased cyclopropane fatty acid synthase activity has improved tolerance to butanol. The tolerance of increased cyclopropane fatty acid synthase strains may be assessed by assaying their growth in concentrations of butanol that are detrimental to growth of the parental strains (prior to genetic modification for increased cyclopropane fatty acid synthase activity). 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 genetically modified strain. For example, as shown in Example 2 herein, a cfa1 modified strain of L. plantarum showed improved growth over the parental strain, being inhibited by 62% in 4.5% 2-butanol while the control strain was inhibited by 79%. Also, in the presence of oleic acid a cfa2 modified strain of L. plantarum showed improved growth over the parental strain, being inhibited by 39% in 2.25% isobutanol while control strains were inhibited by 42% and 51%. For example, as shown in Example 3 herein, a cfa modified strain of E. coli showed improved growth over the parental strain, being inhibited by 50% in 0.8% isobutanol while the control strain was inhibited by 42%.
Combined Genetic Modifications for Butanol Tolerance
A separate genetic modification conferring butanol tolerance in bacterial cells is disclosed in commonly owned and co-pending U.S. Ser. No. 61/015,689 which is herein incorporated by reference. The additional modification is one that reduces accumulation of (p)ppGpp. Any genetic modification that reduces (p)ppGpp accumulation in a bacterial cell may be combined with a genetic modification that increases cyclopropane fatty acid synthase activity to confer butanol tolerance. Specifically, modifications that reduce expression of spoT and/or relA genes, or increase degradative activity relative to synthetic activity of SpoT, can reduce accumulation of (p)ppGpp. As summarized in Gentry and Cashel (Molec. Micro. 19:1373-1384 (1996)), the protein encoded by the spoT gene of E. coli (coding region SEQ ID NO:39; protein SEQ ID NO:40) is an enzyme having both guanosine 3′5′-bis(diphosphate) 3′-pyrophosphohydrolase (ppGppase) and 3′,5′-bis(diphosphate synthetase (PSII) activities. In E. coli there is a closely related gene called relA (coding region SEQ ID NO:41; protein SEQ ID NO:42), which encodes an enzyme with 3′,5′-bis(diphosphate synthetase (PSI) activity. In E. coli, the RelA protein is associated with ribosomes and is activated by binding of uncharged tRNAs to the ribosomes. RelA activation and synthesis of (p)ppGpp results in decreased production of ribosomes, and stimulation of amino acid synthesis. The spoT gene product is responsible for synthesis of (p)ppGpp (Hernandez and Bremer, J. Biol. Chem. (1991) 266:5991-9) during carbon source starvation (Chaloner-Larsson and Yamazaki Can. J. Biochem. (1978) 56:264-72; (Seyfzadeh and Keener, Proc. Natl. Acad. Sci. USA (1993) 90:11004-8) in E. coli. 
Any bacterial gene identified as a spoT or relA gene is a target for modification in the corresponding organism to create a strain of one embodiment of the present invention with combined modifications having reduced (p)ppGpp accumulation and increased cyclopropane fatty acid synthase activity providing increased butanol tolerance. SpoT and/or relA genes and gene products from E. coli, Lactobacillus plantarum, Bacillus licheniformis, Pseudomonas putida, Enterococcus faecium, Rhodococcus erythropolis are specifically described herein (see SEQ ID NOs:39-60, listed in Table 4). Many other examples are identified in the literature and in bioinformatics databases well known the skilled person. Additionally, the sequences described herein or those recited in the art may be used to identify other homologs in nature. For example each of the SpoT/RelA nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins from the same or other microbial species using the same methods described above for isolating cfa genes.
Alternatively, because spoT and relA sequences are so well known and abundant, suitable spoT and relA targets may be identified on the basis of various identifying domains and via Profile Hidden Markov Models (HMM). SpoT and RelA proteins are associated with various identifying domains which can be utilized for the identification of homologs using bioinformatic approaches. One such domain is the TGS domain associated with SpoT and RelA, which senses acylated-Acyl Carrier Protein thereby serving as the switch linking the SpoT-dependent stress response to fatty acid metabolism (Battesti and Bouveret (2006) Molecular Microbiology 62:1048-10630). Another domain is the HD domain which is associated with the SpoT protein and is also associated with a superfamily of metal-dependent phosphohydrolases. In addition to the TGS and HD domains, SpoT and RelA each have a RelA/SpoT domain that is common to both proteins.
It is relevant to note here that within the current art there is some inconsistency with respect to the differentiation between SpoT and RelA proteins. For example, although E. coli has both spoT and relA genes, many bacteria have a gene called spoT, but no gene called relA, or vice versa. The genetic nomenclature is not consistent from one species to another with regard to encoded enzyme activity, since in some species the protein called RelA has both synthesis and degradation activities for (p)ppGpp. In Lactobacillus plantarum there is no gene called spoT, and there is a gene called relA (coding region SEQ ID NO:43) which encodes a protein (SEQ ID NO:44) with both ppGppase and PSII activities. In the nomenclature herein, the Lactobacillus plantarum gene called relA falls under the definition of a spoT gene, and is considered to be a spoT gene encoding a SpoT protein.
For the purposes of this invention it will be understood that a SpoT protein is one that structurally contains all of the RelA/SpoT, TGS and HD domains as described below, encodes a SpoT protein which is a bi-functional enzyme with both guanosine 3′5′-bis(diphosphate) 3′-pyrophosphohydrolase (ppGppase) and 3′,5′-bis(diphosphate synthetase (PSII) activities, and whose disruption affects the levels of (p)ppGpp accumulation in the cell. Similarly a RelA protein is one that contains both the RelA/SpoT and TGS domains, encodes a RelA protein which is a mono-functional enzyme with 3′,5′-bis(diphosphate synthetase (PSI) activity, and whose disruption affects the levels of (p)ppGpp accumulation in the cell.
Accordingly, proteins with RelA or SpoT activities have been characterized as containing the RelA/SpoT domain and the TGS domain. These domains were identified by Pfam (Pfam: clans, web tools and services: R. D. Finn, J. Mistry, B. Schuster-Böckler, S. Griffiths-Jones, V. Hollich, T. Lassmann, S. Moxon, M. Marshall, A. Khanna, R. Durbin, S. R. Eddy, E. L. L. Sonnhammer and A. Bateman, Nucleic Acids Research (2006) Database Issue 34:D247-D251), and each is characterized by a Profile Hidden Markov Model (HMM). The Profile HMM is prepared using the hmmsearch algorithm of the HMMER software package (Janelia Farm Research Campus, Ashburn, Va.). The theory behind Profile HMMs is described in Durbin et al. ((1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press) and Krogh et al. ((1994) J. Mol. Biol. 235:1501-1531), which characterizes a set of proteins based on the probability of each amino acid occurring at each position in the alignment of the proteins of the set. The Profile HMM for the RelA/SpoT domain is in Table 5 and the Profile HMM for the TGS domain is in Table 6. In addition to the RelA/SpoT and TGS domains common to RelA and SpoT proteins, SpoT proteins have an HD domain, also identified by Pfam as above. The Profile HMM for the HD domain is given in Table 7. Tables 5, 6 and 7 are submitted herewith electronically and are incorporated herein by reference. Any protein which has an E-value parameter of 0.01 or less when queried using the Profile HMM for the RelA/SpoT domain and Profile HMM for the TGS domain and that lacks an HD domain can be identified as a RelA protein. Any protein which has an E-value parameter of 0.01 or less when queried using the profiles for these two domains and the Profile HMM for the HD domain can be identified as a SpoT protein. A tree of all known sequences fitting these profiles is shown in FIG. 9. Also marked on the tree are a set of RelA-SpoT related proteins that do not fit the Profile HMMs for RelA and SpoT proteins.
Accordingly in one embodiment the invention provides recombinant bacterial cells wherein an additional genetic modification down regulates a genetic construct encoding, or causes reduced production of, a protein having a) an E-value parameter of 0.01 or less when queried using the Pfam Profile HMM for the RelA/SpoT domain; and b) an E-value parameter of 0.01 or less when queried using the Pfam Profile HMM for the TGS domain; and c) an E-value parameter of 0.01 or less when queried using the Pfam Profile HMM for the HD domain. In another embodiment the invention provides a recombinant bacterial cell wherein the genetic modification down regulates a genetic construct encoding, or causes reduced production of, a protein having; a) an E-value parameter of 0.01 or less when queried using the Pfam Profile HMM for the TGS domain; and an E-value parameter of 0.01 or less when queried using the Pfam Profile HMM for the RelA/SpoT domain. Sequences encoding RelA and SpoT proteins identify relA and spoT genes that may be modified to create bacterial strains of the present invention. In the present bacterial strains, a modification is engineered that results in reduced (p)ppGpp accumulation. This may be accomplished by reduction or elimination of expression of an endogenous spoT and/or relA gene in several ways. Synthesis of ppGpp can be blocked by insuring that the pools of charged tRNA are balanced. The genetic modification may down regulate a genetic construct encoding a SpoT or RelA protein. If the bacterial host has only a relA or a spoT gene, then reduced (p)ppGpp accumulation is obtained by modification of the endogenous relA or spoT gene, causing reduced expression to confer butanol tolerance. If the bacterial host has both genes, then both relA and spoT genes are modified, causing reduced expression of both genes, to confer butanol tolerance. The spoT gene may be modified so that there is no expression, if expression of the relA gene is reduced. Alternatively, with relA unmodified, the expression of spoT may be lowered to provide increased tolerance. In addition, when the bacterial host has both genes, then modification for reduced expression of relA is sufficient to confer butanol tolerance under conditions where an aminoacyl-tRNA species is low and RelA production of (p)ppGpp would be high. Thus effects of the relA mutation in limited aminoacyl-tRNA species conditions better exemplifies the impact on butanol tolerance of RelA-dependent (p)ppGpp synthesis. For example, E. coli has both relA and spoT genes as described above. Elimination of spoT expression in a strain where relA expression is reduced, (as demonstrated in Example 3 of commonly owned and co-pending U.S. Ser. No. 61/015,689 which is herein incorporated by reference) confers butanol tolerance. Reduced expression of spoT in a strain where relA expression is unmodified, (as demonstrated in Example 4 of commonly owned and co-pending U.S. Ser. No. 61/015,689 which is herein incorporated by reference) confers butanol tolerance.
Pseudomonas putida also has a relA (coding region of SEQ ID NO:49; encoded protein of SEQ ID NO:50) and a spoT gene (coding region of SEQ ID NO:51; encoded protein of SEQ ID NO:52) which can be modified as described for E. coli to confer butanol tolerance.
Lactobacillus plantarum has only a spoT gene (which is called relA in the literature), and this gene (coding region has SEQ ID NO:43; encoded protein of SEQ ID NO:44) may be modified to reduce expression and confer butanol tolerance. Bacillus licheniformis Bacillus subtilis, Enterococcus faecalis, and Rhodococcus erythropolis each have a SpoT protein (SEQ ID NOs: 46, 48, 58, and 60, respectively). In each bacterial host the encoding spoT gene (coding regions of SEQ ID NOs:45, 47, 57, and 59, respectively) can be modified to reduce expression, reduce (p)ppGpp synthesis, and increase tolerance. Enterococcus faecium has two SpoT proteins (SEQ ID NOs: 54 and 56) encoded by genes with coding regions (SEQ ID NOs: 53 and 55) that may be modified to reduce expression, reduce (p)ppGpp synthesis, and increase tolerance.
Any genetic modification method known by one skilled in the art for reducing the presence of a functional enzyme may be used to alter spoT or relA gene expression to reduce (p)ppGpp accumulation. Methods include, but are not limited to, deletion of the entire gene or a portion of the gene encoding SpoT or RelA, inserting a DNA fragment into the spoT or relA gene (in either the promoter or coding region) so that the protein is not expressed or expressed at lower levels, introducing a mutation into the spoT or relA coding region which adds a stop codon or frame shift such that a functional protein is not expressed, and introducing one or more mutations into the spoT or relA coding region to alter amino acids so that a non-functional or a less enzymatically active protein is expressed. In addition, spoT or relA expression may be blocked by expression of an antisense RNA or an interfering RNA, and constructs may be introduced that result in cosuppression. Moreover, a spoT or relA gene may be synthesized whose expression is low because rare codons are substituted for plentiful ones, and this gene substituted for the endogenous corresponding spoT or relA gene. Such a gene will produce the same polypeptide but at a lower rate. In addition, the synthesis or stability of the transcript may be lessened by mutation. Similarly the efficiency by which a protein is translated from mRNA may be modulated by mutation. All of these methods may be readily practiced by one skilled in the art making use of the known sequences encoding SpoT or RelA enzymes. Hundreds of spoT and relA sequences are publicly available, and representative sequences are listed in Table 4. One skilled in the art may choose specific modification strategies to eliminate or lower the expression of the relA or spoT gene as desired in the situations described above.
Alternatively, to reduce (p)ppGpp accumulation, a genetic modification may be made that increases the (p)ppGpp degradation activity present in a bacterial cell. The endogenous spoT gene may be modified to reduce the (p)ppGpp synthetic function of the encoded protein. Alternatively, a modified spoT gene encoding a protein with only degradative activity may be introduced. Regions of the SpoT protein that are responsible for the synthetic and degradative activities have been mapped (Gentry and Cashel Mol Microbiol. (1996) 19:1373-1384). The RelA/SpoT and TGS domains (described above) function in ppGpp synthesis while the HD domain is responsible for ppGpp hydrolysis. Gentry and Cashel showed that destruction of the HD domain eliminated the hydrolytic activity without loss of biosynthetic capacity while elimination of either of the other 2 domains resulted in loss of the synthetic capacity without loss of the hydrolytic activity. Thus the sequences encoding the RelA/SpoT and/or TGS domains in the endogenous spoT gene may be mutated to reduce (p)ppGpp synthetic activity. For example, in frame deletions eliminating the various dolmans can be readily synthesized in vitro and recombined into the chromosome by standard methods of allelic replacement. Examples of such deletions are readily found in the literature for both RelA (Fujita et al. Biosci. Biotechnol. Biochem. (2002) 66:1515-1523; Mechold et al J. Bacteriol. (2002) 84:2878-88) and SpoT (Battesti and Bouveret (2006) Molecular Microbiology 62:1048-10630). Furthermore, residual degradative capacity can be enhanced by increasing expression of the modified endogenous gene via chromosomal promoter replacements using methods such as described by Yuan et al (Metab. Eng. (2006) 8:79-90), and White et al. (Can. J. Microbiol. (2007) 53:56-62). Alternatively, a mutation affecting the function of either the RelA/SpoT domain or the TGS domain may be made in a spoT gene, and this gene introduced into a bacterial cell to increase (p)ppGpp degradation activity with no increase in synthesis.
DNA sequences surrounding the spoT or relA coding sequence are also useful in some modification procedures and are available for numerous bacteria such as for E. coli in the complete genome sequence of the K12 strain: GenBank Accession #U00096.2. The genome sequences of L. plantarum, L. salivarius, L sakei, L johnsonii, L. acidophilus and L. delbrueckii are known (National Center for Biotechnology Information (NCBI) database), GenbankTM identification as follows:

    
    
        Lactobacillus plantarum WCFS1, complete genome gi|28376974|ref|NC—004567.1|[28376974]
        Lactobacillus salivarius subsp. salivarius UCC118, complete genome gi|90960990|ref|NC—007929.1|[90960990]
        Lactobacillus sakei strain 23K complete genome gi|78609255|emb|CR936503.1|[78609255]
        Lactobacillus johnsonii NCC 533, complete genome gi|42518084|ref|NC—005362.1|[42518084]
        Lactobacillus acidophilus NCFM, complete genome gi|58336354|ref|NC—006814.1|[58336354]
        Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, complete genomegi|104773257|ref|NC—008054.1|[104773257]
    
    


Additional bacterial genome sequences are available from the E. coli Genome Project (Madison, Wis.) and other genome sequencing projects. A listing of microbial genome sequences compiled by the National Library of Medicine includes 567 completed efforts (41 archael and the rest bacterial) with another 841 in progress. In all, 1408 genomes have been or are under investigation, and information on relA and spoT genes or surrounding DNA within these sequences may be used in making relA and/or spoT modifications.
In particular, DNA sequences surrounding the spoT or relA coding sequence are useful for modification methods using homologous recombination. An example of this method is using spoT gene flanking sequences bounding a selectable marker gene to mediate homologous recombination whereby the marker gene replaces the spoT gene. Also partial spoT gene sequences and spoT flanking sequences bounding a selectable marker gene may be used to mediate homologous recombination whereby the marker gene replaces a portion of the spoT gene. In addition, the selectable marker may be bounded by site-specific recombination sites, so that following expression of the corresponding site-specific recombinase, the resistance gene is excised from the spoT gene without reactivating the latter. The site-specific recombination leaves behind a recombination site which disrupts expression of the SpoT enzyme. The homologous recombination vector may be constructed to also leave a deletion in the spoT gene following excision of the selectable marker, as is well known to one skilled in the art. Moreover, promoter replacement methods may be used to exchange the endogenous transcriptional control elements allowing another means to modulate expression (Yuan et al. ibid).
The spoT gene of E. coli is within a demonstrated operon while the spoT gene of Lactobacillus plantarum is within a gene cluster whose structure is consistent with it being an operon. When part of an operon, expression of spoT or relA may also be reduced by genetic modification of a coding region that is upstream of the spoT or relA coding region in the operon. For example in the spoT-containing operon in E. coli, upstream of the spoT coding region are coding regions for gmk (guanosine monophosphate kinase) and rpoZ (DNA-directed RNA polymerase subunit omega). A modification of the gmk or rpoZ coding region which produces a polar effect will reduce or eliminate spoT expression. Polar mutations are typically nonsense, frameshift or insertion mutations. With these types of mutations, transcription may be truncated, translational coupling is prevented, and hence both interrupted and downstream genes are not expressed. This type of modification is described in Example 2 of commonly owned and co-pending U.S. Ser. No. 61/015,689 (which is herein incorporated by reference) where a transposon insertion in rpoZ affects spoT expression and butanol tolerance. In addition, in Examples 3 and 4 of commonly owned and co-pending U.S. Ser. No. 61/015,689 (which is herein incorporated by reference) a polar modification in rpoZ was constructed resulting in butanol tolerance. In addition intergenic regions could be modified to prevent translational coupling when it is found.
In the same manner, the yrvE coding region that is upstream of the spoT coding region in an operon in the Lactobacillus plantarum genome (GenBank Accession #AL935263) may be modified to reduce spoT expression. Using gene organization information from genome sequencing of other bacterial strains allows targeted modification of coding regions in operons upstream of spoT or relA coding regions in those strains to reduce (p)ppGpp synthesis and confer butanol tolerance.
Reduced Response to (p)ppGpp
The effect of reducing accumulation of (p)ppGpp may also be obtained in the present strains by reducing responsiveness to (p)ppGpp. Any modification that affects an enzyme activity that increases the concentration of cyclopropane fatty acid in the cell membrane fatty acid composition may be combined with a modification reducing responsiveness to (p)ppGpp. Mutants with reduced response to (p)ppGpp were found in the RNA polymerase core subunit encoding genes and the RNA polymerase binding protein DksA (Potrykus and Cashel (2008) Ann. Rev. Microbiol. 62:35-51). Reduced expression of any of these proteins may be engineered to reduce the response to (p)ppGpp. In particular, reducing expression of DksA may be engineered in the present strains to confer increased tolerance to butanol and 2-butanone. Expression of the endogenous dksA gene in a target bacterial cell may be reduced using any genetic modification method such as described above for spoT or relA. The dksA gene of a target host cell may be readily recognized by one skilled in the art through bioinformatics analysis, or experimental methods as described for spoT.
Butanol Biosynthetic Pathway
In the present invention, a genetic modification conferring increased cyclopropane fatty acid in the membrane is engineered in a bacterial cell that does not naturally produce butanol, but that is engineered to express a butanol biosynthetic pathway. Either genetic 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 biosynthetic pathway for the production of 1-butanol is described by Donaldson et al. in co-pending and commonly owned U.S. patent application Ser. No. 11/527,995, published as US20080182308A1, which is 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 encoded 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 encoded by the gene given as SEQ ID NO:5;
        c) 3-hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed for example by crotonase encoded by the gene given as SEQ ID NO:7;
        d) crotonyl-CoA to butyryl-CoA, as catalyzed for example by butyryl-CoA dehydrogenase encoded by the gene given as SEQ ID NO:9;
        e) butyryl-CoA to butyraldehyde, as catalyzed for example by butyraldehyde dehydrogenase encoded by the gene given as SEQ ID NO:11; and
        f) butyraldehyde to 1-butanol, as catalyzed for example by 1-butanol dehydrogenase encoded 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.
2-Butanol Biosynthetic Pathway
Biosynthetic pathways for the production of 2-butanol are described by Donaldson et al. in co-pending and commonly owned U.S. Patent Application Publication Nos. US20070259410A1 and US 20070292927A1 which are 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 encoded by the gene given as SEQ ID NO:19;
        b) alpha-acetolactate to acetoin, as catalyzed for example by acetolactate decarboxylase encoded by the gene given as SEQ ID NO:17;
        c) acetoin to 2,3-butanediol, as catalyzed for example by butanediol dehydrogenase encoded by the gene given as SEQ ID NO:21;
        d) 2,3-butanediol to 2-butanone, catalyzed for example by butanediol dehydratase encoded 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 encoded by the gene given as SEQ ID NO:29.

Isobutanol Biosynthetic Pathway

    
    


Biosynthetic pathways for the production of isobutanol are described by Maggio-Hall et al. in copending and commonly owned U.S. patent application Ser. No. 11/586,315, published as US20070092957 A1, which is 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 encoded by the gene given as SEQ ID NO:19;
        b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed for example by acetohydroxy acid isomeroreductase encoded by the gene given as SEQ ID NO:31;
        c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed for example by acetohydroxy acid dehydratase encoded 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 encoded by the gene given as SEQ ID NO:35; and
        e) isobutyraldehyde to isobutanol, as catalyzed for example by a branched-chain alcohol dehydrogenase encoded by the gene given as SEQ ID NO:37.

Construction of Bacterial Strains for Butanol Production

    
    


Any bacterial strain that is genetically 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. Methods described in co-pending and commonly owned U.S. Patent Publication Nos. US20080182308A1, US20070259410A1, US 20070292927A1, and US20070092957 A1 may be used.
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)). Additionally, in vitro transposomes are available to create random mutations in a variety of genomes from commercial sources such as EPICENTRE® (Madison, Wis.).
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, as described in Examples herein. 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 pAMβ1 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 plantarum 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 strains with increased cyclopropane fatty acid synthase activity 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. Glucose and dextrose may be obtained through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, and oats.
In addition, fermentable sugars may be obtained from cellulosic and lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in commonly owned and co-pending 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.
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 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.
For 1-butanol synthesis the first three genes of the pathway are thl, hbd, and crt, encoding the enzymes acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, and crotonase, respectively. The last three genes of the pathway are EgTER, ald, and bdhB, encoding the enzymes butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase and butanol dehydrogenase, respectively.
General Methods:
Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984, and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987. Additional methods used in the Examples are described in manuals including Advanced Bacterial Genetics (Davis, Roth and Botstein, Cold Spring Harbor Laboratory, 1980), Experiments with Gene Fusions (Silhavy, Berman and Enquist, Cold Spring Harbor Laboratory, 1984), Experiments in Molecular Genetics (Miller, Cold Spring Harbor Laboratory, 1972) Experimental Techniques in Bacterial Genetics (Maloy, in Jones and Bartlett, 1990), and A Short Course in Bacterial Genetics (Miller, Cold Spring Harbor Laboratory 1992).
These references include descriptions of the media and buffers used including TE, M9, MacConkey and LB.
All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.
Media and Culture Conditions:
Materials and methods suitable for the maintenance and growth of bacterial cultures were found in Experiments in Molecular Genetics (Jeffrey H. Miller), Cold Spring Harbor Laboratory Press (1972), Manual of Methods for General Bacteriology (Phillip Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), pp. 210-213, American Society for Microbiology, Washington, D.C. or Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland Mass. All reagents and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), Gibco/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified.
LB medium contains following per liter of medium: Bacto-tryptone (10 g), Bacto-yeast extract (5 g), and NaCl (10 g).
Supplements were added as mentioned in the examples. All additions were pre-sterilized before they were added to the media.
Additional growth media used were MRS medium (Acumedia Manufacturers, Inc. Lansing, Mich. or Difco Laboratories, Detroit, Mich.) or semi-synthetic LAB medium, pH7. The composition of the latter medium was as follows:
0.01M Ammonium Sulfate
0.005M Potassium Phosphate, pH 7.0
0.05M MOPS, pH 7.0
1% S10 Metal Mix
0.01 M Glucose
0.2% Yeast Extract
0.01% Casamino Acids
2.5 μg/ml hematin
The composition of S10 Metal Mix is:
200 mM MgCl2 
70 mM Ca Cl2 
5 mM Mn Cl2 
0.1 mM FeCl3 
0.1 mM Zn Cl2 
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.). Isobutanol and 2-butanol were purchased from Sigma Chemical Company (St. Louis, Mo.).

General Molecular Biology Techniques:

Unless otherwise stated, restriction enzyme digestions, ligations, transformations, and methods for agarose gel electrophoresis were performed as described in Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). Polymerase Chain Reactions (PCR) techniques were found in White, B., PCR Protocols: Current Methods and Applications, Volume 15 (1993) Humana Press Inc.
Genetic Transformation of Lactobacillus plantarum 
A loop of Lactobacillus plantarum cells was inoculated into 5 ml of MRS broth from a fresh MRS plate and incubated overnight. The overnight culture was diluted to an OD600 of 0.25 in 25 ml MRS broth and incubated in a shaker for 2-4 hours until the OD600 reached between 0.8 and 1.0. The cells were harvested by centrifugation for 5 min at 4° C. at 4000 rpm (Beckman table top centrifuge, with rotor CO650). The cell pellet was washed twice with 50 ml of SM (326 g sucrose (952 mM), 0.7 mg of MgCl2.6H2O (3.5 mM) in 1 L of water) by spinning at the same speed for 15 minutes. After two washes, the pellet was re-suspended in 400 μl of SM. An aliquot 100 μl of cells was used for each electroporation. 500 ng of plasmid DNA was added to the cell suspension and the mixture was allowed to incubate on ice for 5 minutes before electroporation. Electroporation was carried out with a BioRad Gene Pulser with a setting of 1.5 kV, 25 μF and 800Ω. Immediately after the electric discharge, 500 μl of MRSSM (MRS with 0.5M sucrose and 0.1M MgCl2) was added and the cell suspension was transferred to a 15 ml culture tube. The cells were allowed to recover by incubating at 37° C. for two hours in a shaker. From each transformation mixture, 50-100 μl of mixture was spread onto selection plates with appropriate antibiotic supplement and the plates were incubated at 30° C. The colonies of transformants are usually visible after 2-5 days.
L. plantarum Genomic DNA Purification
L. plantarum genomic DNA was prepared using MasterPure DNA purification kit (Epicentre) as follows. A single colony was grown over night in MRS medium. A culture sample (0.5-1 ml) was centrifuged, the supernatant discarded, and the pellet washed three times with STE [6.7% sucrose, 50 mM Tris-cl, pH8, 1 mM EDTA (filter sterilized with 0.22 μm membrane)]. Add 300 μl of Tissue and cell lysis solution containing Proteinase K, diluted 1 μl of 50 μg/ul Proteinase K into 300 μl of Tissue and cell lysis solution, for each sample and mix thoroughly. The sample was incubated at 65° C. for 5 min, then cooled to 37° C. for 5-10 min. Next 1 μl of RNase (5 μg/μl) was added, the sample mixed thoroughly, incubated at 37° C. for 30 min, and placed on ice for 5 min. 150 μl of MCP protein precipitation reagent (Epicentre, Master pure DNA purification kit) was added to 300 μl of lysed sample. The debris was pelleted by centrifugation in a micro-centrifuge for 3-5 min, and the supernatant transferred to a clean micro-centrifuge tube. 500 μl of iso-propanol was added to the recovered supernatant and the samples inverted 30-40 times. The DNA was pelleted by centrifugation at 4° C. for 10 minutes in a micro-centrifuge, the pellet rinsed twice with 755 ethanol, air-dried, and resuspenced in 35 μl of TE buffer. 5 μl) was run on a gel to assess the concentration.
PCR Amplification
High fidelity amplification of DNA fragments was carried out with Pfx enzyme from Invitrogen (Carlsbad, Calif.). The extension time depends on the length of the fragment to be amplified. As a general rule, 1 min was used per kb of DNA. For example, the PCR conditions normally included 3 minutes initial denaturing at 94° C., 25 cycle of amplification (94° C. 30 sec, 54° C. 30 sec, 68° C. 1 min) and a final 3 min of extension at 68° C. for fragments of 1 kb or less.
Plasmid DNA Purification from L. plantarum 
L. plantarum plasmid-containing strains were grown in 20 ml of MRS medium (with antibiotic if required) without shaking for 16-20 hours at 30° C. The cells were washed one time with sterile deionized water and resuspended in 1 ml of 5 M LiCl. This suspension was incubated for 1 hour at room temperature with shaking. Subsequently, the cells were washed one time with 1 ml of sterile deionized water and resuspended in 1 ml of protoplasting buffer (25 mM sucrose, 50 mM Tris-HCl, pH 8.0, 10 mM EDTA) with 10 mg/ml of lysozyme and 100 μg/ml of RNase, and incubated for 1 hour at 37° C. The protoplasted cells were centrifuged and resuspend in 500 μl of Qiagen buffer P1, then divided in half. Each portion was used separately to extract plasmid DNA according to the Qiagen directions and then combined on one column.
Fatty Acid Methyl Ester (FAME) Analysis
For preparation of samples for fatty acid methyl ester analysis (FAME), the cultures were grown to late log phase in semi-synthetic LAB medium, pH7. The cell pellet was harvested by centrifugation and was washed twice with phosphate buffered saline (PBS, Bio-Rad Laboratories, Hercules, Calif.). Cell pellets were stored at −80° C. until analyzed by FAME. For preparation of samples grown in the presence of oleic acid for FAME, the cultures were grown to late-log phase in semi-synthetic LAB medium, pH7, with additional 5 g/l BSA and 50 mg/l oleic acid (Sigma Chemical Company, St. Louis, Mo.). These cultures were washed and stored as above, except that an additional wash with PBS containing 5 g/l BSA was done prior to the two PBS washes.
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.
Fatty Acid Analysis: Formation of Fatty Acid Methyl Esters by the H2SO4/CH3OH Method
This method forms methyl esters of all the fatty acids in the sample. Both free fatty acids and fatty acids present in ester linkages in the lipids in the sample are derivatized to the methyl esters by this procedure.
2 ml 5% H2SO4 in CH3OH was added to each of the tubes containing sample. The caps were placed on tubes, screwed on about 3⁄4 of the way (i.e., not tight), then the tubes were heated at 80° C. for 1 hr. The tubes were cooled, and then 1 ml H2O and 0.5 ml hexane were added to each. The resulting 2 phase mixture was vortexed; then the two phases were allowed to separate. The top hexane layer, which contained the methylated fatty acids was removed and placed in a separate container. 2 μl of this 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 μm film thickness; catalog #28045-U) was used with an FID detector (GC/FID). For peak identification sample column was used with an Agilent MSD detector was used (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 μm film thickness; catalog #24317) was used.
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 as described previously.
Growth Analysis
For shake flask experiments, the cultures were grown overnight at 30° C. in semi-synthetic LAB medium, pH7 containing 1 μg/ml erythromycin. The next day, the cultures were diluted to an initial OD600 of 0.1 in the same medium, but without erythromycin, and allowed to grow at 30° C., 80 rpm until doubled. The cultures were then diluted 1:1 into the same medium containing various concentrations of isobutanol or 2-butanol and grown at 30° C., 80 rpm. Samples were taken approximately hourly and OD600 was measured. The growth rates, μ(hr−1) were calculated from an exponential curve fit of the OD600 vs. time data. For growth rates for which the curve fit value was negative, a value of 0.00 was recorded. The % growth rate inhibition was calculated as follows: 100-100[μ(isobutanol)/μ(no isobutanol)]. All solvent concentrations are reported as % (w/v).
Bioscreen-C Growth Analysis
For Bioscreen-C (instrument purchased from Growth Curves USA, Piscataway N.J.) growth curves, the cultures were grown overnight in MRS medium containing 1 μg/ml erythromycin at 30° C. The next day, these were diluted to OD600 of 0.1 in the wells of the Bioscreen plate with MRS medium containing 1 μg/ml erythromycin and either 2.25% (w/v) isobutanol or no isobutanol at 30° C. OD was monitored every 15 minutes and growth rates (hr−1) were calculated from an exponential curve fit of the OD600 vs. time data for data in the exponential phase of growth. The % growth rate inhibition was calculated as follows: 100-100[μ(2.25% isobutanol)/μ(no isobutanol)].
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
Constructions for Overexpresssion of cfa1 and cfa2 Genes in L. plantarum 
To express genes in L. plantarum, a series of shuttle vectors derived from pFP996 were constructed. pFP996 (SEQ ID NO: 71) is a shuttle vector for gram-positive bacteria. It can replicate in both E. coli and gram-positive bacteria. It contains the origins of replication from pBR322 (nucleotides #2628 to 5323) and pE194 (nucleotides #43 to 2627). pE194 is a small plasmid isolated originally from a gram positive bacterium, Staphylococcus aureus (Horinouchi and Weisblum J. Bacteriol. (1982) 150(2):804-814). In pFP996, the multiple cloning sites (nucleotides #1 to 50) contain restriction sites for EcoRI, BglII, XhoI, SmaI, ClaI, KpnI, and HindIII. There are two antibiotic resistance markers; one is for resistance to ampicillin and the other for resistance to erythromycin. For selection purposes, ampicillin was used for transformation in E. coli and erythromycin was used for selection in L. plantarum. 
First a vector called pFP996-fba was constructed by inserting the fba promoter region of L. plantarum into the multiple cloning site of pFP996. The fba promoter region was amplified with the Pfx enzyme as described in General Methods using L. plantarum PN0512 strain (ATC #PTA-7727) genomic DNA, as a template with the primer set fba-F and fba-R (SEQ ID NOs:72, 73). The genomic DNA was prepared according to the procedure described in General Methods. The resulting fragment was digested with EcoRI and Xho I and cloned into the corresponding sites in pFP996 to create pFP996-fba.
In order to express the cfa1 and cfa2 genes at higher than normal levels, the corresponding coding regions were expressed from the atpB promoter (SEQ ID NO:74). This promoter was originally cloned along with the bdhB coding region in the vector pFP996-fba. The atpB promoter was amplified using the same L. plantarum genomic DNA as a template using the primer set atpB-F and atpB-R (SEQ ID NOs:75, 76). The promoter was obtained by PCR with a high fidelity enzyme Pfx (Invitrogen). The bdhB coding region with attached 5′ Shine-Delgarno sequence was amplified from a synthetic construct with primer set bdhB-F and bdhb-R (SEQ ID NOs 77, 78) with Pfx enzyme. The synthetic the bdhB coding region and attached 5′ Shine-Delgarno sequence (SEQ ID NO; 79) was obtained from Genscript Corporation (Piscataway, N.J.). The promoter fragment was digested with MluI and Spe I, while the bdhB fragment was digested with SpeI and BamHI. Both PCR products were used in a single ligation reaction with vector pFP996-fba which was linearized with restriction enzymes MluI and BglII. The new vector was designated as pFP996-atpB-bdhB. In this vector the fba promoter was replaced by the atpB promoter and bdhB coding region.
The cfa1 coding region from L. plantarum PNO512 was obtained by amplification of the same genomic DNA with the primer set Cfa1-F and Cfa-1R (SEQ ID NOs:80, 81). The restriction site SpeI and a Shine-Delgarno (SD) sequence for efficient translation was introduced in the forward primer. In the reverse primer, a BglII site was introduced. The DNA fragment obtained by PCR with high fidelity enzyme Pfx was digested with SpeI and Bgl II. The digested product was ligated into the corresponding restriction sites down stream of the atpB promoter in vector pFP996-atpB-bdhB. In the resulting construct, the bdhB coding region was replaced by the cfa1 coding region and this new vector was designated as pFP996-atpB-cfa1. For cloning of the cfa2 gene, its cfa2 coding region was amplified from the same genomic DNA with primer set Cfa2-F and Cfa2-R (SEQ ID NOs:82, 83). The same SD sequence and restriction sites as in the primer set for the cfa1 coding region were used for these two primers. The PCR product from the cfa2 coding region was cloned into the pFP996-atpB-bdhB vector using the same method as described for the cfa1 gene. The vector containing the cfa2 coding region was named pFP996-atpB-cfa2.
To express the cfa1 and cfa2 chimeric genes in L. plantarum, the pFP996-atpB-cfa1 and pFP996-atpB-cfa2 plasmids were each introduced into the host by electroporation as described in General Methods. After electroporation, the cells were spread onto MRS plates supplemented with erythromycin (3 μg/L). Plates were incubated at 30° C. Colonies with the plasmid were initially screened with the primer for the atpB promoter, atpB-F (SEQ ID NO:75), and the reverse primers for either the cfa1 and cfa2 coding regions (SEQ ID NOs:81 and 83, respectively). PCR products of the expected sizes were obtained in each case. Furthermore, plasmid DNA was isolated from each transformant and used to transform E. coli TOP10 (Invitrogen). Plasmid DNA subsequently isolated from the E. coli transformants and digested with restriction enzymes Spe I and Bgl II had the expected restriction digestion patterns, thus further verifying the presence of the cloned genes in the plasmids of the transformed L. plantarum PN0512 strains. The following strain names were given to transformants and controls:













Strain name
Plasmid








PN2001
pFP996-atpB-cfa1-8a



PN2003
pFP996-atpB-cfa1-9a



PN2005
pFP996-atpB-cfa2-a



PN2010
pFP996-atpB-cfa2-4



PN2012
pFP996



PN2014
pFP996









Example 2
Overexpression of cfa1 or cfa2 Overexpression in L. plantarum and Effect Butanol Tolerance
FAME Analysis of L. plantarum PN0512 Transformant Strains
Lactobacillus plantarum strains PN2001, PN2003, PN2005, PN2010, PN2012, and PN2014 were grown and prepared for FAME as described in the General Methods section. The results for cultures grown in semi-synthetic medium lacking added fatty acids are shown in Table 10.







TABLE 10







Membrane composition of PN0512 transformant strains grown


without added fatty acids.









Strain














PN2001
PN2003
PN2005
PN2010
PN2012
PN2014





Gene on
cfa1
cfa1
cfa2
cfa2
none
none


plasmid








membrane








fatty acids








C14:0
<0.1
0.5
<0.1
<0.1
0.3
0.4


C16:0
34.6
31.9
34.9
33.2
30.8
33.4


C16:1
6.0
4.5
6.2
4.9
5.3
5.1


C18:0
13.8
11.6
9.3
9.7
8.6
7.6


C18:1
5.8
7.1
40.8
42.9
47.2
45.9


cyc-C19:0
28.0
32.2
6.4
6.4
5.0
5.6









It is clear from this data that the strains with the cloned cfa1 gene converted a much higher percentage of the C18:1 fatty acid, cis-vaccenic acid, to the cyclopropane fatty acid (cyc-C19:0) derived from it, lactobacillic acid. Thus, strains PN2001 and PN2003 represent strains that have been genetically modified to have increased levels of cyclopropane fatty acids in the membrane lipids.

The same six strains were grown in semi-synthetic medium with added 50 μg/ml oleic acid and 5 g/l BSA as a carrier. FAME was performed with transesterification using CH3ONa in CH3OH as described in General Methods, and results are shown in Table 11. For this analysis a longer 100 meter column was used so that the isomers of monounsaturated 18 carbon fatty acids and the cyclopropane fatty acids derived from them could be distinguished. The only C18:1 fatty acid that L. plantarum synthesized was cis-vaccenic (C18:1 11-cis). This was converted to lactobacillic acid (cyc-C19:0 11-). Oleic acid (C18:1 9-cis) was taken up from the medium and incorporated into the membrane lipids and converted to dihydrosterculic acid (cyc-C19:0 9-).







TABLE 11







Membrane composition of PN0512 transformant strains grown with


added oleic acid.









Strain














PN2001
PN2003
PN2005
PN2010
PN2012
PN2014





Gene on
cfa1
cfa1
cfa2
cfa2
none
none


plasmid








membrane








fatty acids








C16:0
17.3
18.6
19.9
20.0
17.6
16.0


C16:1
4.7
Trace
 2.8
4.5
5.2
4.9


C18:0
1.4
trace
trace
1.4
1.3
1.2


C18:1, 9-cis
22.7
10.6
27.1
24.4
40.7
44.8


C18:1, 11-cis
1.6
trace
17.9
19.1
17.7
12.5


cyc-C19:0, 9-
24.5
43.4
21.9
20.4
9.5
9.8


cyc-C19:0,
27.9
27.4
10.3
10.2
9.4
9.1


11-









The strains with either cfa1 or cfa2 on the multicopy plasmid had increased levels of dihydrosterculic acid (cyc-C19:0 9-) in the membrane lipids as compared with the control strains. Thus, strains PN2001, PN2003, PN2005, and PN2010 represent strains that have been genetically modified to have increased levels of cyclopropane fatty acids in the membrane lipids when oleic acid is present in the growth medium.

Improved Tolerance of the Strains with cfa1 Overexpression to Isobutanol

Lactobacillus plantarum strains PN2001, which overexpresses cfa1, and PN2014, a control strain with the vector alone, were grown in shake flasks in semi-synthetic LAB medium, pH7 with various concentrations of isobutanol as described in General Methods. The growth vs. time for each strain is plotted in FIG. 1 (A:PN2001; B:PN2014). It is clear that the strain PN2001 grew in the presence of 3% isobutanol, while strain PN2014 did not grow in the presence of 3% isobutanol. Furthermore, the growth in 2.75% isobutanol was faster for PN2001 than for PN2014. Growth rates and % growth inhibition were calculated from these growth curves above and are shown in Table 12.







TABLE 12







PN2001 and PN2014 growth rates and % growth


inhibition in the presence of isobutanol













PN2014





PN2001
(control
PN2001, %
PN2014, %


[Isobutanol]
(cfa1 plasmid)
plasmid)
Growth
Growth


% w/v
μ, hr−1
μ, hr−1
inhibition
inhibition














0
0.347
0.343
0
0


2.25
0.187
0.170
46
50


2.5
0.119
0.118
66
66


2.75
0.104
0.037
70
89


3.0
0.029
0.00
92
100


3.2
0.00
0.00
100
100









At 2.75% and 3% isobutanol, the growth of PN2001 overexpressing cfa1 was less inhibited than was the growth of the control strain PN2014. Thus, elevated expression of cfa1 and the resultant increase in membrane cyclopropane fatty acid levels produced improved tolerance to isobutanol.

Improved Tolerance of the Strains with cfa1 Overexpression to 2-butanol

Lactobacillus plantarum strains PN2001, which overexpresses cfa1, and PN2014, a control strain with the vector alone, were grown in shake flasks in semi-synthetic LAB medium, pH7 with various concentrations of 2-butanol as described in General Methods. Growth rates and % growth inhibition calculated from the growth curves are shown in Table 13.







TABLE 13







PN2001 and PN2014 growth rates and % growth


inhibition in the presence of 2-butanol













PN2014





PN2001
(control
PN2001, %
PN2014, %


[2-Butanol]
(cfa1 plasmid)
plasmid)
Growth
Growth


% w/v
μ, hr−1
μ, hr−1
inhibition
inhibition














0
0.284
0.297
0
0


3.5
0.132
0.153
54
48


4.0
0.125
0.123
56
59


4.25
0.111
0.158
61
47


4.5
0.108
0.061
62
79


4.75
0.068
0.034
76
89


5.0
0.078
0.00
73
100









At 4.5, 4.75 and 5.0% 2-butanol, the growth of PN2001 overexpressing cfa1 was less inhibited than was the growth of the control strain PN2014. Thus, elevated expression of cfa1 and the resultant increase in membrane cyclopropane fatty acid levels yielded improved tolerance to 2-butanol.

Improved Tolerance of the Strains with cfa1 or cfa2 Overexpression to Isobutanol when Grown in Medium Containing a Source of Oleic Acid

L. plantarum strains PN2001, PN2003, PN2005, PN2010, PN2012, and PN2014 were tested for growth in a Bioscreen-C growth curve machine in the presence of isobutanol as described in General Methods. These growth assays were conducted in MRS medium, which contains 1 g/L polysorbate 80 (commercially also known as Tween® 80) that serves as a source of oleic acid. Erythromycin at 1 μg/ml was added to the medium to maintain the plasmids. It is well known that L. plantarum grown in MRS medium incorporates oleic acid into membrane lipids (Johnsson et al. (1995) Appl. Environ. Microbiol. 61:4497-4499). Table 14 shows the growth rates in the presence or absence of 2.25% isobutanol and the % growth inhibition.







TABLE 14







PN2001, PN2003, PN2005, PN2010, PN2012 and PN2014


growth rates in MRS medium and % growth inhibition


in the presence of isobutanol












Gene on
μ (no isobutanol),
μ (2.25% isobutanol),
% Growth


Strain
plasmid
hr−1
hr−1
inhibition





PN2001
cfa1
0.47
0.32
32


PN2003
cfa1
0.50
0.33
34


PN2005
cfa2
0.51
0.31
39


PN2010
cfa2
0.54
0.33
39


PN2012
none
0.41
0.20
51


PN2014
none
0.38
0.22
42









The growth of strains PN2001 and PN2003 with the cfa1 overexpression plasmid was less inhibited by 2.25% isobutanol than was the growth of the control strains, PN2012 and PN2014. Likewise, the growth of PN2005 and PN2010 with the cfa2 overexpression plasmid was less inhibited by 2.25% isobutanol than was the growth of the control strains, PN2012 and PN2014, According, in medium containing a source of oleic acid, elevated expression of either cfa1 or cfa2 and the resultant increase in membrane cyclopropane fatty acid levels resulted in improved tolerance to isobutanol.

Example 3
Overexpression of cfa in E. coli and Effect on Butanol Tolerance
Plasmid pDEW849 contains the E. coli cfa coding region under control of the trc promoter in a multicopy plasmid. To construct this plasmid, the cfa coding region was obtained by PCR amplification using chromosomal DNA from E. coli strain MG1655 as template and the primers cfa_sense and cfa_antisense (SEQ ID NOs:84, 85). The cfa_sense primer was designed so that when the amplified DNA is cloned into the pTrcHis2TOPO® vector (Invitrogen, Carlsbad, Calif.), an N-terminal fusion protein would not be formed and thus the native Cfa protein is expressed. The cfa_sense primer also has an EcoRI site that was used to determine orientation of the inserted DNA. The cfa_antisense primer was designed to contain the termination codon of cfa and thus the native Cfa protein is expressed, rather than a C-terminal fusion protein.
A 1165 bp PCR product was obtained from amplification reactions using ExTaqTM (TaKaRa) and the following conditions: 94° C. for 5 minutes, 35 cycles of (94° C. for 1 minute, 60° C. for 2 minutes, 72° C. for 3 minutes), and 72° C. for 15 minutes. The product of the PCR reaction was purified using a Qiaquick PCR clean-up kit (Qiagen) following the manufacturer's instructions and was then ligated into pTrcHis2TOPO® (Invitrogen) following the protocol supplied by the vendor. After transformation of E. coli strain TOP10 (Invitrogen) and selection for ampicillin resistance, plasmid DNA from individual transformants was digested with EcoRI. One plasmid, for which two fragments of sizes 4.4 KB (vector) and 1.2 KB (insert) resulted, was named pDEW849. The presence of the cfa coding region in the correct orientation was confirmed by DNA sequence analysis. Plasmid pDEW849 and a control plasmid, pTrcHis2TOPO®/lacZ (Invitrogen), were moved by transformation to E. coli strain BW25113 (Datsenko and Wanner (2000) Proc. Nat. Acad. Sci. USA 97:6640-6645) selecting for ampicillin resistance, to generate strains DPD4655 and DPD4658, respectively.
E. coli strains DPD4655 and DPD4658 were grown overnight in LB medium containing 100 μg/ml ampicillin at 37° C. The next day, these were diluted to OD600 of 0.1 in the wells of a Bioscreen-C plate (instrument and plates purchased from Growth Curves USA, Piscataway N.J.) with LB medium and either 0.4%, 0.8% (w/v) isobutanol or no isobutanol at 37° C. in triplicate. OD was monitored and the OD at the 3 hour and 15 minute time point was used to calculate the % of growth in the presence of isobutanol as compared with its absence, given in Table 15.







TABLE 15







% of growth vs control in cfa overexpressing E. coli strains










DPD4655
DPD4658



(cfa overexpression)
(control lacZ overexpression)











[Isobutanol] 

% OD

% OD


%
OD600
+isobutanol/
OD600
+isobutanol/


(w/v)
(+/− st dev)
−isobutanol
(+/− st dev)
−isobutanol














0
0.442 +/− 0.006
100
0.413 +/− 0.016
100


0.4
0.318 +/− 0.005
72
0.259 +/− 0.004
63


0.8
0.220 +/− 0.006
50
0.174 +/− 0.011
42









At both 0.4% and 0.8% isobutanol, there was an increased amount of growth for the DPD4655 cultures overexpressing cfa as compared with the control DPD4658 cultures. Thus, overexpression of cfa in E. coli confers tolerance to isobutanol.

Example 4
Construction of Insertional Mutants in Lactobacillus spoT
An internal fragment amplified from the L. plantarum (ATCC #BAA-793; also called NCIMB 8826 and WCFS1) spoT gene (SEQ ID NO:43) was chosen to disrupt both the ppGpp synthase and hydrolase domains. The 601 bp internal fragment, from nucleotide position 235 to 835, counting from the A of the initiator ATG, was chosen as it is predicted to disrupt the function of both domains, removing the first 234 bp of the hydrolase domain, and the last 191 bp of the synthesis domain as shown in FIG. 2, A. The internal fragment was PCR amplified using the primers #5807 and #5808 (SEQ ID NOs:86 and 87). These primers bound a portion of the spoT gene at nucleotide positions 235-259 and 816-835, respectively. Lactobacillus plantarum BAA-793 genomic DNA was used as the template in a PCR reaction using HI-FI Taq polymerase. An annealing temperature of 60° C. and an elongation time of 1 minute were used for 30 rounds of amplification. The amplified PCR product was inserted into the TOPO cloning vector pCR2.1-TOPO as per the manufacturer's (Invitrogen) instructions. The insertion of the PCR product was confirmed by restriction digestion. This construct was designated pTOPO-relAint.
A plasmid pMPE3 was constructed by amplifying a 2.45 kb fragment of the Bacillus shuttle vector pMK4 (purchased from Bacillus Genetics Stock center) using the primers pUCCMNotIR (SEQ ID NO:88) and pUCCMNotIF (SEQ ID NO:89). The amplified fragment contained the multiple cloning site, lacZ-alpha, pMB1 origin of replication and chloramphenicol resistance gene from pMK4. The amplified fragment was digested with NotI and then circularized by ligation, generating pMPE3 which can replicate in E. coli but not in Lactobacillus plantarum. 
The spot fragment was excised from the pTOPO-relAint plasmid using BamHI and PstI sites that flank the inserted PCR product. The resulting  ̃650 bp band was gel purified and ligated into similarly digested plasmid pMPE3. The resulting plasmid was again confirmed by restriction digestion and designated pMPE69.
pMPE69 was transformed into L. plantarum PNO512 (ATCC #PTA-7727), using 5 μg of DNA and 60 μl of competent cells prepared using standard procedures. Aliquots of the transformation were plated on MRS with 10 ug/ml chloramphenicol and incubated at 30° C. overnight. 10 putative single crossover integrants were transferred into MRS with 10 ug/ml of chloramphenicol and again grown up overnight at 30° C. Aliquots of the 10 integrant cultures were used for Instagene [Bio-Rad Laboratories, Hercules, Calif.] preparations of genomic DNA. PCR confirmation of pMPE69 insertion into the spoT gene was performed using primers #5785 and #5798 (SEQ ID NOs:90 and 91). Primer #5785 binds upstream of the spoT gene, while #5798 binds within the pMPE3 backbone. The PCR reactions were predicted to produce a  ̃1400 bp product. This product was observed in 8 of the 10 putative integrants. The first two integrants were saved and designated MS0280 and MS0281. The structure of the chromosomal spoT locus after insertion of plasmid pMPE69, and positions of the primers used for confirmation, are shown in FIG. 2, B.
Example 5
Improved Tolerance with Combination cfa1 Overexpression and spoT Mutation
The purpose of this Example was to test the effect of a combination of cfa1 overexpression and spoT mutation on the isobutanol tolerance of Lactobacillus plantarum. To create a strain with both gene modifications, pFP996-atpB-cfa1 (described in Example 1; also called pFP996-1)) was transformed into the MS0280 strain (described in Example 4; also called PN1300) creating strain PN1312. For a control strain, pFP996 (described in Example 1) was transformed into MS0280 creating PN1310. Strain PN1312, which contained a multicopy expression plasmid with cfa1, was compared to strain PN1310, which contained the vector control.
The growth of the strains over time was followed, and tolerance compared as follows. The strains were cultured in S36 medium (contains 10 mM ammonium sulfate, 5 mM potassium phosphate buffer, pH 6.0, 50 mM MES, pH 6.0, 2 mM MgCl2, 0.7 mM CaCl2, 50 μM MnCl2, 1 μM FeCl3, 1 μM ZnCl2, 1.72 μM CuCl2, 2.53 μM CoCl2, 2.42 μM Na2MoO4, 2 μM thiamine hydrochloride, 0.01 M glucose, 0.2% yeast extract and 0.01% casamino acids) overnight at 30° C. under anaerobic conditions. The next day, cultures were started from the overnight cultures in fresh medium, with initial OD600 values of 0.18. These cultures were incubated at 30° C. under anaerobic conditions for 4 hours. During this time, PN1310 reached an OD600 of 0.83, and PN1312 reach an OD600 of 0.61. This incubation period allowed the cyclopropane fatty acid to increase in the membrane. Following the 4 hour incubation period, 6 ml of the PN1310 culture was dispensed into 4 tubes, while 5 ml of the PN1312 culture was dispensed into 4 tubes. The cells were collected by centrifugation and resuspended as follows: 2 tubes each of 5 ml S36 medium and 2 tubes each of 5 ml S36 medium+3% isobutanol. After obtaining Initial OD600 values, the tubes were incubated at 30° C. under anaerobic conditions. OD600 values were obtained at 2 hours. Duplicate samples were averaged to calculate the change in OD600 values. The results are summarized in Table 16.







TABLE 16







The change in OD600 for both strains in the


presence and absence of isobutanol.












PN1310 (vector)
PN1312 (cfa1)














0%
3%
0%
3%







2 h Δ OD
1.2
0.3
1.2
0.5










The vector alone was inhibited 75% for growth at 3% iso-butanol and the strain with cfa1 overexpression was inhibited 59% for growth at 3% isobutanol. Thus, improvements in tolerance for strain PN1312 (spoT) were obtained at a high concentration of isobutanol.

Example 6
Prophetic
Producing Isobutanol Using E. coli Strain with Increased Expression of cfa
E. coli strains engineered to express an isobutanol biosynthetic pathway are described in commonly owned and co-pending US patent application publication #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 (encoding1,3-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 overexpression of the cfa gene. This plasmid is constructed by amplifying the region from plasmid pDEW849 (described in Example 3) with the trc promoter and the E. coli cfa gene. Both of the primers for amplification (SEQ ID NOs:92 and 93) also have a BsrD I restriction site. The PCR product is digested with BsrD I and 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 E. coli cfa gene are verified by DNA sequence analysis. This cfa plasmid 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), MnSO4.H2O (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. Significantly higher titers of isobutanol are obtained in the cultures of the strains with the cfa plasmid than in the parental strains.
Example 7
Prophetic
Producing 2-Butanol Using E. coli Strain with Increased Expression of cfa
The engineering of E. coli for expression of a 2-butanol biosynthetic pathway is described in commonly owned and co-pending US Patent Application Publication 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 cfa overexpression plasmid (described in Example 6). E. coli NM522/p2BOH with and without the cfa 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. Higher titers are obtained from the strain with the cfa plasmid.
Example 8
Prophetic
Producing 1-Butanol Using E. coli Strain with Increased Expression of cfa
E. coli strains engineered to express a 1-butanol biosynthetic pathway are described in commonly owned and co-pending US Patent Application Publication 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 cfa overexpression plasmid (described in Example 6).
The parental strain and the transformant with the cfa overexpression 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), MnSO4.H2O (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. Titers of 1-butanol are found to be higher in the strain harboring the cfa expression plasmid.
Example 9
Prophetic
Expression of an Isobutanol Biosynthetic Pathway in Lactobacillus plantarum with Increased Expression of cfa1
The purpose of this prophetic Example is to describe how to express an isobutanol biosynthetic pathway in a Lactobacillus plantarum strain that overexpresses cfa1. 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 cfa1 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 ldhL1 locus by homologous recombination. To build the ldhL integration targeting vector, a DNA fragment from Lactobacillus plantarum (Genbank NC—004567) with homology to ldhL is PCR amplified with primers LDH EcoRV F (SEQ ID NO:94) and LDH AatIIR (SEQ ID NO:95). The 1986 bp PCR fragment is cloned into pCR4Blunt-TOPO and sequenced. The pCR4Blunt-TOPO-ldhL1 clone is digested with EcoRV and AatII releasing a 1982 bp ldhL1 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:96) 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 ldhL1 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-ldhL.
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:97) and Cm R (SEQ ID NO:98), 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 ldhL-homology containing integration vector pFP988-ldhL is digested with MluI and SwaI and the 4740 bp vector fragment is gel purified. The Cm cassette fragment is ligated with the pFP988-ldhL 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:99) and P11 R-SpeI (SEQ ID NO:100). 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 ΔldhL1::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 cfa1 Genes.
The remaining two isobutanol genes and cfa1 under the control of the L. plantarum ldhL 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 ldhL promoter is PCR amplified from the genome of L. plantarum ATCC BAA-793 using primers PldhL F-HindIII (SEQ ID NO:101) and PldhL R-BamHI (SEQ ID NO:102). 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 cfa1 coding region is PCR amplified from L. plantarum PN0512 genomic DNA using primers F-SphI-cfa1 and R-SphI-cfa1 (SEQ ID NOs:103 and 104). The 1182 bp PCR product is cloned into pCR4Blunt-TOPO and sequenced. The resulting plasmid, pCR4Blunt-TOPO-cfa1, is digested with SphI releasing the fragment with the cfa1 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:105) 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 cfa1 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 cfa1 gene in the same orientation as ilvC and bdhB is retained and named pTRKH3-ilvC(B.s.)-bdhB-cfa1. 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 ΔldhL1::budB-ilvD-kivD::Cm containing pTRKH3-ilvC(B.s.)-bdhB-cfa1 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. Higher titers of isobutanol are obtained from the strain with the cfa1 gene on the plasmid.
Example 10
Prophetic
Expression of the 1-Butanol Biosynthetic Pathway in Lactobacillus plantarum with Increased Expression of cfa1
The purpose of this prophetic Example is to describe how to express the 1-butanol biosynthetic pathway in a Lactobacillus plantarum strain that overexpresses cfa1. 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 cfa1 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 ldhL1 locus by homologous recombination. To build the ldhL integration targeting vector, a DNA fragment from Lactobacillus plantarum (Genbank NC—004567) with homology to ldhL is PCR amplified with primers LDH EcoRV F (SEQ ID NO:94) and LDH AatIIR (SEQ ID NO:95). The 1986 bp PCR fragment is cloned into pCR4Blunt-TOPO and sequenced. The pCR4Blunt-TOPO-ldhL1 clone is digested with EcoRV and AatII releasing a 1982 bp ldhL1 fragment that is gel-purified. The integration vector pFP988, described in Example 9, 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 ldhL1 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-ldhL.
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:97) and Cm R (SEQ ID NO:98), 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 ldhL-homology containing integration vector pFP988-ldhL is digested with MluI and SwaI and the 4740 bp vector fragment is gel-purified. The Cm cassette fragment is ligated with the pFP988-ldhL 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:106) and P11 R (SEQ ID NO:107). 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 ldhL promoter (Ferain et al., J. Bacteriol. 176:596-601 (1994)). and cfa1 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 ldhL promoter is PCR amplified from the genome of L. plantarum ATCC BAA-793 with primers PldhL F (SEQ ID NO:108) and PldhL R (SEQ ID NO:109). 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 110 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 ldhL 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 atpB promoter and cfa1 gene are amplified using plasmid pFP996-atpB-cfa1 (described in Example 1) as a template and the primers F-NruI-atpB and R-XhoI-cfa1 SEQ ID NOs: 111 and 112). The 1496 bp PCR product is cloned into pCR4Blunt-TOPO and sequenced. The resulting plasmid, pCR4Blunt-TOPO-PatpB-cfa1, is digested with NruI and XhoI releasing the fragment with the atpB promoter and cfa1 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 PatpB-cfa1 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-PatpB-cfa1.
Plasmids pTRKH3-ald-E-B and pTRKH3-ald-E-B-PatpB-cfa1 are transformed into L. plantarum ΔldhL1::T-H-C::Cm by electroporation, as described above.
L. plantarum ΔldhL1::T-H-C::Cm containing pTRKH3-ald-E-B or containing pTRKH3-ald-E-B-PatpB-cfa1 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. Higher titers of 1-butanol are obtained from the strain with the cfa1 gene on the plasmid.








TABLE 6







HMMER2.0 [2.3.2]
Program name and version


NAME RelA_SpoT
Name of input sequence alighment file


DESC Region found in RelA/SpoT proteins
Domain description


LENG 131
Length of alignment


ALPH Amino
Type of residues


MAP yes
Map of the match states to the columns of the alignment


COM hmmbuild -F --wme HMM_Is.ann SEED.ann
Commands used to generate the file: this one means that hmmbuild (default parameters) was 



applied to the alignment file


COM hmmcalibrate --seed 0 HMM_Is.ann
Commands used to generate the file: this one means that hmmcalibrate (default parameters) was 



applied to the hmm profile


NSEQ 105
Number of sequences in the alignment file


DATE Sun Apr 29 16:27:35 2007
When file was generated


XT -8455 -4 -1000 -1000 -8455 -4 -8455 -4



NULT -4 -8455
The transition probability distribution for the null model (single G state).


NULE 595 -1558 85 338 -294 453 -1158 197 249
The symbol emission probability distribution for the null model (G state)


902 -1085 -142 -21 -313 45 531 201 384 -1998 -644



EVD -69.960602 0.225039
The extreme value distribution parameters μ and lambda respectively


The highest probability is highlighted for each position














 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 























TABLE 7







HMMER2.0 [2.3.2]
Program name and version


NAME TGS
Name of input sequence alighment file


DESC TGS domain
Domain description


LENG 75
Length of alignment


ALPH Amino
Type of residues


MAP yes
Map of the match states to the columns of the alignment


COM hmmbuild -F HMM_Is.ann SEED.ann
Commands used to generate the file: this one means that hmmbuild (default patrameters)



was applied to the alignment file


COM hmmcalibrate --seed 0 HMM_Is.ann
Commands used to generate the file: this one means that hmmcalibrate (default parametrs)



was applied to the hmm profile


NSEQ 62
Number of sequences in the alignment file


DATE Fri Apr 27 19:07:53 2007
When file was generated


XT -8455 -4 -1000 -1000 -8455 -4 -8455 -4



NULT -4 -8455
The transition probability distribution for the null model (single G state).


NULE 595 -1558 85 338 -294 453 -1158 197 249 902
The symbol emission probability distribution for the null model (G state)


-1085 -142 -21 -313 45 531 201 384 -1998 -644



EVD -50.809875 0.221806
The extreme value distribution parameters μ and lambda respectively


The highest probability is highlighted for each position

























































































TABLE 8







HMMER2.0 [2.3.2]
Program name and version


NAME HD
Name of input sequence alighment file


DESC HD domain
Domain description


LENG 154
Length of alignment


ALPH Amino
Type of residues


MAP yes
Map of the match states to the columns of the alignment


COM hmmbuild -F HMM_Is.ann SEED.ann
Commands used to generate the file: this one means that hmmbuild (default patrameters)



was applied to the alignment file


COM hmmcalibrate-seed 0 HMM_Is.ann
Commands used to generate the file: this one means that hmmcalibrate (default parametrs)



was applied to the hmm profile


NSEQ 160
Number of sequences in the alignment file


DATE Sun Apr 29 14:25:51 2007
When file was generated


XT -8455 -4 -1000 -1000 -8455 -4 -8455 -4



NULT -4 -8455
The transition probability distribution for the null model (single G state).


NULE 595 -1558 85 338 -294 453 -1158 197 249 902
The symbol emission probability distribution for the null model (G state)


-1085 -142 -21 -313 45 531 201 384 -1998 -644



EVD -43.966183 0.276183
The extreme value distribution parameters μ and lambda respectively


The highest probability is highlighted for each position

## Claims

1. A recombinant bacterial cell which is engineered to produce butanol, and comprises at least one genetic modification affecting an enzyme activity that increases the concentration of cyclopropane fatty acid in the cell membrane fatty acid composition as compared with a wildtype bacterial cell lacking said genetic modification;
wherein the cell contains the substrate for said enzyme; and
wherein the cell does not naturally produce butanol; and
wherein the at least one genetic modification increases expression of a cfa coding region wherein the cfa coding region is selected from the group consisting of:
a) a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NO:61, 63, 65, 67 and 69;
b) a nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; and
c) a nucleic acid molecule that encodes a polypeptide having 95% identity based on the Clustal method of alignment when compared to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:61, 63, 65, 67, and 69; and
wherein the cell is a member of the genera selected from the group consisting of Escherichia and Lactobacillus.

2. The recombinant cell of claim 1 wherein the enzyme substrate is endogenous to said cell.

3. The recombinant cell of claim 1 wherein the enzyme substrate is provided exogenously to said cell.

4. The recombinant cell of claim 1 comprising a recombinant biosynthetic pathway selected from the group consisting of:
a) a 1-butanol biosynthetic pathway;
b) a 2-butanol biosynthetic pathway; and
c) an isobutanol biosynthetic pathway.

5. The recombinant cell of claim 1, additionally comprising at least one genetic modification which reduces accumulation of (p)ppGpp.

6. The recombinant cell of claim 5, wherein the at least one genetic modification which reduces accumulation of (p)ppGpp reduces production of SpoT or RelA.

7. The recombinant cell of claim 6, wherein the at least one genetic modification which reduces accumulation of (p)ppGpp is a disruption in an endogenous gene selected from the group consisting of spoT and relA or in an operon comprising an open reading frame encoding SpoT or RelA.

8. The recombinant cell of claim 5, wherein the genetic modification reduces (p)ppGpp synthetic activity of encoded endogenous SpoT protein.

9. The recombinant cell of claim 5, wherein the genetic modification increases (p)ppGpp degradative activity by increasing expression of a SpoT with reduced (p)ppGpp synthetic activity.

10. The bacterial cell of claim 1 wherein the cell is of the genus Lactobacillus and the genetic modification is overexpression of the Lactobacillus cfa1 coding region of SEQ ID NO:61.

11. The recombinant bacterial cell of claim 4 wherein the 1-butanol biosynthetic pathway comprises:
a) at least one genetic construct encoding an acetyl-CoA acetyltransferase;
b) at least one genetic construct encoding 3-hydroxybutyryl-CoA dehydrogenase;
c) at least one genetic construct encoding crotonase;
d) at least one genetic construct encoding butyryl-CoA dehydrogenase;
e) at least one genetic construct encoding butyraldehyde dehydrogenase; and
f) at least one genetic construct encoding 1-butanol dehydrogenase.

12. The recombinant bacterial cell of claim 4 wherein the 2-butanol biosynthetic pathway comprises:
a) at least one genetic construct encoding an acetolactate synthase;
b) at least one genetic construct encoding acetolactate decarboxylase;
c) at least one genetic construct encoding butanediol dehydrogenase;
d) at least one genetic construct encoding butanediol dehydratase; and
e) at least one genetic construct encoding 2-butanol dehydrogenase.

13. The recombinant bacterial cell of claim 4 wherein the isobutanol biosynthetic pathway comprises:
a) at least one genetic construct encoding an acetolactate synthase;
b) at least one genetic construct encoding acetohydroxy acid isomeroreductase;
c) at least one genetic construct encoding acetohydroxy acid dehydratase;
d) at least one genetic construct encoding branched-chain keto acid decarboxylase; and
e) at least one genetic construct encoding branched-chain alcohol dehydrogenase.

14. A process for generating the recombinant cell of claim 1 comprising:
a) providing a recombinant bacterial host cell comprising an engineered butanol biosynthetic pathway that is producing butanol; and
b) creating at least one genetic modification which increases cyclopropane fatty acid in the cell membrane fatty acid composition above natural levels, provided that the bacterial cell does not naturally produce butanol.

15. A process for production of butanol from a recombinant bacterial cell comprising:
(a) providing the recombinant bacterial host cell of claim 1; and
(b) culturing the host cell of (a) under conditions wherein butanol is produced.

16. The process of claim 15, wherein the bacterial host cell is a member of a genus selected from the group consisting of Escherichia and Lactobacillus.

17. The process of claim 15 wherein the recombinant bacterial host comprises a recombinant biosynthetic pathway selected from the group consisting of:
a) a 1-butanol biosynthetic pathway;
b) a 2-butanol biosynthetic pathway; and
c) an isobutanol biosynthetic pathway.

18. The process of claim 17 wherein the 1-butanol biosynthetic pathway comprises:
a) at least one genetic construct encoding an acetyl-CoA acetyltransferase;
b) at least one genetic construct encoding 3-hydroxybutyryl-CoA dehydrogenase;
c) at least one genetic construct encoding crotonase;
d) at least one genetic construct encoding butyryl-CoA dehydrogenase;
e) at least one genetic construct encoding butyraldehyde dehydrogenase; and
f) at least one genetic construct encoding 1-butanol dehydrogenase.

19. The process of claim 17 wherein the 2-butanol biosynthetic pathway comprises:
a) at least one genetic construct encoding an acetolactate synthase;
b) at least one genetic construct encoding acetolactate decarboxylase;
c) at least one genetic construct encoding butanediol dehydrogenase;
d) at least one genetic construct encoding butanediol dehydratase; and
e) at least one genetic construct encoding 2-butanol dehydrogenase.

20. The process of claim 17 wherein the isobutanol biosynthetic pathway comprises:
a) at least one genetic construct encoding an acetolactate synthase;
b) at least one genetic construct encoding acetohydroxy acid isomeroreductase;
c) at least one genetic construct encoding acetohydroxy acid dehydratase;
d) at least one genetic construct encoding branched-chain keto acid decarboxylase; and
e) at least one genetic construct encoding branched-chain alcohol dehydrogenase.

21. The process of claim 15 wherein the enzyme substrate is endogenous to the cell or provided exogenously to said cell.

22. The process of claim 15 wherein the cell is of the genus Lactobacillus and the genetic modification is overexpression of the Lactobacillus cfa1 coding region of SEQ ID NO:61.

23. The process of claim 15 additionally comprising an additional genetic modification which reduces accumulation of (p)ppGpp reduces production of SpoT or RelA.

24. The process of claim 23, wherein the at least one genetic modification which reduces accumulation of (p)ppGpp is a disruption in an endogenous gene selected from the group consisting of spoT and relA or in an operon comprising an open reading frame encoding SpoT or RelA.

25. The recombinant host cell of claim 1 wherein the host cell is Escherichia coli or Lactobacillus plantarum.

26. The recombinant host cell of claim 4 wherein the host cell is Escherichia coli or Lactobacillus plantarum.