STRAIN FOR BUTANOL PRODUCTION
Using screening of transposon random insertion mutants, genes involved in a complex that is a three-component proton motive force-dependent multidrug efflux system were found to be involved in E. coli cell response to butanol. Reduced production of the AcrA and/or AcrB proteins of the complex confers increased butanol tolerance. E. coli strains with reduced AcrA or AcrB production and having a butanol or 2-butanone biosynthetic pathway are useful for production of butanol or 2-butanone.
1 . A recombinant Escherichia coli cell producing butanol or 2-butanone said E. coli cell comprising at least one genetic modification which reduces production of a protein selected from the group consisting of AcrA and AcrB.
2 . The E. coli 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;
c) an isobutanol biosynthetic pathway; and
d) a 2-butanone biosynthetic pathway.
3 . The E. coli cell of claim 1 , wherein the at least one genetic modification is a disruption in a endogenous gene selected from the group consisting of acrA and acrB gene.
4 . The E. coli cell of claim 1 , additionally comprising at least one genetic modification which reduces accumulation of (p)ppGpp.
5 . The E. coli cell of claim 4 , wherein the at least one genetic modification which reduces accumulation of (p)ppGpp reduces production of SpoT or RelA.
6 . The E. coli cell of claim 5 , 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 re/A or in an operon comprising an open reading frame encoding SpoT or RelA.
7 . The E. coli cell of claim 4 , wherein the genetic modification reduces (p)ppGpp synthetic activity of encoded endogenous SpoT protein.
8 . The E. coli cell of claim 4 , wherein the genetic modification increases (p)ppGpp degradative activity by increasing expression of a SpoT with reduced (p)ppGpp synthetic activity.
9 . The recombinant E. coli cell of claim 2 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.
10 . The recombinant E. coli cell of claim 2 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.
11 . The recombinant E. coli cell of claim 2 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.
12 . The recombinant E. coli cell of claim 2 wherein the 2-butanone 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; and
d) at least one genetic construct encoding butanediol dehydratase.
13 . A process for generating the E. coli host cell of claim 1 comprising:
a) providing a recombinant bacterial host cell producing butanol or 2-butanone; and
b) creating at least one genetic modification which redues production of AcrA or AcrB, or both AcrA and AcrB proteins.
14 . A process for production of butanol or 2-butanone from a recombinant E. coli cell comprising:
(a) providing a recombinant E. coli cell which
1) produces butanol or 2-butanone and
2) comprises at least one genetic modification which reduces production of AcrA or AcrB, or both AcrA and AcrB; and
(b) culturing the strain of (a) under conditions wherein butanol or 2-butanone is produced.
15 . The process according to claim 14 , wherein the recombinant E. coli comprises a biosynthetic pathway selected from the group consisting of:
a) a 1-butanol biosynthetic pathway;
b) a 2-butanol biosynthetic pathway;
c) an isobutanol biosynthetic pathway; and
d) a 2-butanone biosynthetic pathway
16 . The process according to claim 14 , wherein the recombinant E. coli cell additionally comprises at least one genetic modification which reduces accumulation of (p)ppGpp.
17 . The process according to claim 16 , wherein the at least one genetic modification which reduces accumulation of (p)ppGpp reduces production of SpoT or RelA.
18 . The process according to claim 17 , 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 re/A or in an operon comprising an open reading frame encoding SpoT or RelA.
19 . The process according to claim 17 , wherein the genetic modification reduces (p)ppGpp synthetic activity of encoded endogenous SpoT protein.
20 . The process according to claim 17 , wherein the genetic modification increases (p)ppGpp degradative activity by increasing expression of a SpoT with reduced (p)ppGpp synthetic activity.
21 . The process according to claim 15 , 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.
22 . The process according to claim 15 , 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.
23 . The process according to claim 15 , 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.
24 . The process according to claim 15 , wherein the 2-butanone 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; and
d) at least one genetic construct encoding butanediol dehydratase.