# Patent

## Title
Biofuel production by recombinant microorganisms

## Abstract

Provided herein are metabolically-modified microorganisms useful for producing biofuels. More specifically, provided herein are methods of producing high alcohols including isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol from a suitable substrate.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part application of U.S. patent application Ser. No. 12/028,727, filed Feb. 8, 2008, now U.S. Pat. No. 8,975,049, which application claims priority to U.S. Provisional Application Ser. No. 60/900,477 filed Feb. 9, 2007, Ser. No. 60/900,546, filed Feb. 9, 2007, Ser. No. 60/921,927, filed Apr. 4, 2007, and Ser. No. 60/956,634, filed Aug. 17, 2007, the disclosures of which are incorporated herein by reference. The present application also claims priority to U.S. Provisional Application No. 61/088,696, filed Aug. 13, 2008, the disclosure of which is incorporated herein by reference.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with Government support of Grant No. DE-FC03-02ER63421 awarded by the Department of Energy. The U.S. government has certain rights in the invention.


TECHNICAL FIELD
Metabolically-modified microorganisms and methods of producing such organisms are provided. Also provided are methods of producing biofuels by contacting a suitable substrate with a metabolically-modified microorganism and enzymatic preparations there from.
BACKGROUND
Demand for biofuels as a substitute for petroleum is expected to increase because of economic and environmental concerns. The common bio-fuel, ethanol, is not ideal because it has a lower energy density than gasoline and must be mixed with gasoline at a limited concentration range in order to serve as a transportation fuel. Ethanol is also hygroscopic and corrosive, which poses a problem for storage and distribution systems.
SUMMARY
The disclosure provides a recombinant microorganism that produces an alcohol selected from the group consisting of: (a) 1-propanol, (b) 1-butanol, (c) 2-methyl 1-butanol (2 MB), (d) 3-methyl 1-butanol, (e) isobutanol, and (f) 2-phenylethanol, wherein the alcohol is produced from a metabolite comprising 2-keto acid. In one embodiment, the organism produces from about 695 to about 2800 mg/L of 1-propanol compared to a WT parental organisms. In one embodiment, the organism comprises a mutant cimA polypeptide. In another embodiment, the organism produces about 22 to about 395 mg/L of 1-butanol compared to a WT parental organism. In another embodiment, the organisms produce about 650 and 750 mg/L 2 MB for organisms comprising a modified threonine pathway, between about 525 and 625 mg/L 2 MB for organisms with a modified citramalate pathway, or between 1300 and 1400 mg/L 2 MB for organisms comprising a modified threonine and citramalate pathway compared to a WT parental organism. In a further embodiment, the wild-type parental organism comprises a WT cimA.
In one embodiment, the disclosure provides a mutant cimA polypeptide comprising at least about 70% identity to a wild-type (WT) cimA from Methanocaldococcus jannaschii (SEQ ID NO:89), wherein the mutant cimA comprises from about 9- to 22-fold higher production levels of 1-propanol and 1-butanol in an engineered pathway for their production when compared to the same pathway comprising a WT cimA. In another embodiment, the mutant cimA comprises from about 1-10 mutated amino acids as compared to the WT cimA from Methanocaldococcus jannaschii. In yet another embodiment, the mutant cimA comprises the sequence of SEQ ID NO:89 having a mutation at one or more of the following positions: Ile47, Lys435, Val441, His126 or Thr204. In yet another embodiment, the mutant cim A comprises a sequence as set forth SEQ ID NO:89 and having one or more of the following mutations: a Ile47Val, a Lys435Asn, a Val441Ala, a His126Gln or a Thr204Ala, an Arg53Ser, a Glu183Lys, a Leu253Ser, Lys265Arg, Phe349Cys, an Asp86Gly, and Asp328Val, a Glu114Val, a Leu238Ser, a Glu111Lys, a Glu121Val, a Met250Val or a Lys32Asn. In a specific embodiment, the cimA mutant comprises SEQ ID NO:89 and has a mutation at Ile47Val and one or more additional mutations as set forth above. For example, the cimA mutant can comprise SEQ ID NO:89 and an Ile47Val, His126Gln, and a Thr204Ala. The cimA mutatant comprising 147V, H126Q and T204A may further comprise one or more mutations selected from R53S, E183K, L253S, K265R, F349C, D141E, I286V, L327H, D86G, D328V, E114V, L238S, E111K, E121V, M250V and K32N. The cimA mutant may further be truncated following residue 373 of the WT cimA. In yet another embodiment, the mutant cimA comprises a sequence as set forth in SEQ ID NO:89 and having 1-50, 1-20 or 1-10 conservative amino acid substitutions and having one or more of a Ile47Val, a His126Gln or a Thr204Ala mutation.
In one embodiment, a recombinant organism expressing the mutant cimA produces less than about 240 mg/L of ethanol at about 112 hrs of culture. In yet a further embodiment, the yield of ethanol per gram of glucose is less than about 0.0037 g/g. In one embodiment, the microorganism comprises reduced ethanol production compared to a parental microorganism. In yet another embodiment, the microorganism comprises a reduction or inhibition in the conversion of acetyl-coA to ethanol. In yet a further embodiment, the recombinant microorganism comprises a reduction of an ethanol dehydrogenase thereby providing a reduced ethanol production capability. In one embodiment, the microorganism comprises expression or elevated expression of an enzyme that converts pyruvate to alpha-keto-isovalerate. In a further embodiment, the enzyme is 2-keto-acid decarboxylase (e.g., Pdc, Pdc1, Pdc5, Pdc6, Aro10, Thi3, Kivd, and KdcA, a homolog or variant of any of the foregoing, and a polypeptide having at least 60% identity to any one of the foregoing and having 2-keto-acid decarboxylase activity). In another embodiment, the 2-keto-acid decarboxylase is encoded by a polynucleotide having at least 60% identity to a nucleic acid selected from the group consisting of pdc, pdc1, pdc5, Pdc6, aro10, thi3, kivd, kdcA, a homolog or variant of any of the foregoing, or a fragment thereof and wherein the polynucleotide encodes a polypeptide having 2-keto acid decarboxylase activity. In a specific embodiment, the 2-keto-acid decarboxylase is encoded by a polynucleotide derived from a kivd gene, or homolog thereof. In one embodiment, the microorganism comprises elevated expression or activity of a 2-keto-acid decarboxylase and an alcohol dehydrogenase, as compared to a parental microorganism. In one embodiment, the alcohol dehydrogenase is selected from the group consisting of Adh1, Adh2, Adh3, Adh4, Adh5, Adh6, Sfa1, a homolog or variant of any of the foregoing, and a polypeptide having at least 60% identity to any one of the foregoing and having alcohol dehydrogenase activity. In yet another embodiment, the alcohol dehydrogenase is encoded by a polynucleotide having at least 60% identity to a nucleic acid selected from the group consisting of an adh1, adh2, adh3, adh4, adh5, adh6, sfa1 gene, and a homolog of any of the foregoing and wherein the polynucleotide encodes a protein having 2-alcohol dehydrogenase activity. In one embodiment, the recombinant microorganism comprises one or more deletions or knockouts in a gene encoding an enzyme that catalyzes the conversion of acetyl-coA to ethanol, catalyzes the conversion of pyruvate to lactate, catalyzes the conversion of fumarate to succinate, catalyzes the conversion of acetyl-coA and phosphate to coA and acetyl phosphate, catalyzes the conversion of acetyl-coA and formate to coA and pyruvate, condensation of the acetyl group of acetyl-CoA with 3-methyl-2-oxobutanoate (2-oxoisovalerate), isomerization between 2-isopropylmalate and 3-isopropylmalate, catalyzes the conversion of alpha-keto acid to branched chain amino acids, synthesis of Phe Tyr Asp or Leu, catalyzes the conversion of pyruvate to acetyl-coA, catalyzes the formation of branched chain amino acids, catalyzes the formation of alpha-ketobutyrate from threonine, catalyzes the first step in methionine biosynthesis, and catalyzes the catabolism of threonine. For example, the microorganism can comprise one or more gene deletions selected from the group consisting of adhE, ldhA, frdBC, fnr, pta, pflB, leuA, leuB, leuC, leuD, ilvE, tyrB, poxB, ilvB, ilvI, ilvA, metA, tdh, homologs of any of the foregoing and naturally occurring variants of any of the foregoing. In a specific embodiment, a genotype of the microorganism is selected from the group consisting of: (a) a deletion or knockout selected from the group consisting of ΔadhE, ΔldhA, Δpta, ΔleuA, ΔleuB, ΔleuC, ΔleuD, ΔpoxB, ΔilvB, ΔilvI, ΔmetA, Δtdh and any combination thereof and comprising an expression or increased expression of kivd, ThrABC and adh2, wherein the microorganism produces 1-propanol; (b) a deletion or knockout selected from the group consisting of ΔadhE, ΔldhA, Δpta, ΔpoxB, ΔilvB, ΔilvI, ΔmetA, Δtdh, and any combination thereof and comprising an expression or increased expression of kivd, ThrABC and adh2 wherein the microorganism produces 1-butanol; (c) a deletion or knockout selected from the group consisting of ΔilvB, ΔilvI, ΔmetA, Δtdh, and any combination thereof and comprising an expression or increased expression of kivd, ThrABC and adh2 wherein the microorganism produces 2-methyl 1-butanol; (d) a deletion or knockout selected from the group consisting of ΔadhE, ΔldhA, ΔfrdB, ΔfrdC, Δfnr, Δpta, ΔpflB, ΔilvE, ΔtyrB, and any combination thereof and comprising an expression or increased expression of kivd, ThrABC and adh2 wherein the microorganism produces 3-methyl 1-butanol; (e) a ΔldhA, ΔfrdB, ΔfrdC, Δfnr, Δpta, ΔpflB, ΔleuA, ΔilvE, ΔpoxB, ΔilvA, and any combination thereof and comprising an expression or increased expression of kivd, ThrABC and adh2 wherein the microorganism produces isobutanol; or (f) a deletion or knockout selected from the group consisting of ΔadhE, ΔldhA, ΔfrdB, ΔfrdC, Δfnr, Δpta, ΔpflB, ΔleuA, ΔilvE, ΔpoxB, ΔilvA, and any combination thereof and comprising an expression or increased expression of kivd, ThrABC and adh2 wherein the microorganism produces 2-phenylethanol. In a further embodiment, the ThrABC comprises a feedback resistant ThrA*. In one embodiment, a recombinant microorganism comprises an alcohol production profile substantially identical or improved compared to the alcohol production profile of the microorganisms identified herein as SA237 and BuOH23.
Provided herein are metabolically-modified microorganisms that include recombinant biochemical pathways useful for producing biofuels such as isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-propanol, 2-phenylehtanol, or 1-butanol via conversion of a suitable substrate by a metabolically engineered microorganism. Also provided are methods of producing biofuels using microorganisms described herein.
In one embodiment, a recombinant microorganism that produces an alcohol is provided. The alcohol can be 1-propanol, 1-butanol, 2-methyl-1-butanol, 2-phenylethanol or 3-methyl-1-butanol. In general, the alcohol may be produced fermentatively or non-fermentatively (i.e., with or without the presence of oxygen) from a metabolite comprising 2-keto acid. In some aspects, the 2-keto acid includes 2-ketobutyrate, 2-ketoisovalerate, 2-ketovalerate, 2-keto-3-methylvalerate, 2-keto-4-methyl-pentanoate, or phenylpyruvate. In other aspects, the recombinant microorganism includes elevated expression or activity of a 2-keto-acid decarboxylase and an alcohol dehydrogenase, as compared to a parental microorganism. The 2-keto-acid decarboxylase may be Pdc6 from Saccharomyces cerevisiae, Aro10 from Saccharomyces cerevisiae, Thi3 from Saccharomyces cerevisiae, Kivd from Lactococcus lactis, or Pdc from Clostridium acetobutylicum, or homologs thereof. The 2-keto-acid decarboxylase can be encoded by a polynucleotide derived from a gene selected from PDC6 from S. cerevisiae, ARO10 from S. cerevisiae, THIS from S. cerevisiae, kivd from L. lactis, or pdc from C. acetobutylicum, or homologs thereof. In some aspects, the alcohol dehydrogenase may be Adh2 from S. cerevisiae, or homologs thereof, encoded by a polynucleotide derived from the ADH2 gene from S. cerevisiae. The microorganism can include elevated expression or activity of acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy-acid dehydratase, 2-keto-acid decarboxylase, and alcohol dehydrogenase, as compared to a parental microorganism. In some aspects, the microorganism can include elevated expression of acetolactate synthase, acetohydroxy acid isomeroreductase, dihydroxy-acid dehydratase, 2-keto-acid decarboxylase, and alcohol dehydrogenase. In some aspects, the recombinant microorganism further includes an elevated level of pyruvate as compared to a parental microorganism. Accordingly, the recombinant microorganism may further include the deletion or inhibition of expression of an adhE, ldh, frd, fnr, pflB, ackA, or pta gene, or any combination thereof. In particular, the recombinant microorganism can include a deletion of adh, ldh, frd alone or in combination with fnr, fnr and pta, or pta and pflB. In some aspects, the recombinant microorganism may further include deletion of a portion of the recombinant microorganism genome, such as nucleotides from about 1,397,551 to about 1,439,877 of the E. coli genome. In one aspect, the acetohydroxy acid synthase may be encoded by a polynucleotide derived from the ilvIH operon, ilvBN operon, ilvGM in E. coli, or the alsS gene from Bacillus subtilis, or homologs thereof. The ilvI gene of the ilvIH operon encodes an acetohydroxyacid synthase large subunit polypeptide and the ilvH gene of the ilvIH operon encodes an acetohydroxyacid synthase small subunit polypeptide. In another aspect, the acetohydroxy acid isomeroreductase may be encoded by a polynucleotide derived from an ilvC gene in E. coli, or homologs thereof. In another aspect, the dihydroxy-acid dehydratase may be encoded by a polynucleotide derived from an ilvD gene, or homologs thereof. In yet another aspect, the 2-keto-acid decarboxylase may be encoded by a polynucleotide derived from a kivd gene from Lactococcus lactis or homologs thereof, or an ARO10 gene from S. cerevisiae, or homologs thereof. In a further aspect, the alcohol dehydrogenase may be encoded by a polynucleotide derived from an ADH2 gene from S. cerevisiae, or homologs thereof.
In general the ilvIH operon of Escherichia coli encodes acetohydroxy acid synthase, the first enzyme in the isoleucine, valine and leucine biosynthetic pathway. The acetohydroxy acid synthase III isozyme, which catalyzes the first common step in the biosynthesis of isoleucine, leucine, and valine in Escherichia coli K-12, is composed of two subunits, the ilvI (acetohydroxyacid synthase III large subunit) and ilvH (acetohydroxyacid synthase small subunit) gene products. The ilvC gene of Escherichia coli encodes acetohydroxy acid isomeroreductase, the second enzyme in the parallel isoleucine-valine biosynthetic pathway. The ilvD gene of Escherichia coli encodes dihydroxy-acid dehydratase, the third enzyme in the isoleucine-valine biosynthetic pathway. In some aspects the recombinant microorganism included an elevated expression of acetolactate synthase. The acetolactate synthase can be AlsS from Bacillus subtilis or ILV2 from Pichia stipitis (including variants of ILV2 such as ILV2Δ126).
In one embodiment, a recombinant microorganism that produces 1-butanol is provided. The microorganism includes a cimA mutant and can comprise elevated expression or activity of 2-isopropylmalate synthase, beta-isopropylmalate dehydrogenase, isopropylmalate isomerase, and threonine dehydratase, as compared to a parental microorganism. In another embodiment, the recombinant microorganism further includes increased levels of 2-ketovalerate, as compared to a parental microorganism. In another embodiment, the recombinant microorganism further includes decreased levels of 2-ketoisovalerate, 2-keto-3-methyl-valerate, or 2-keto-4-methyl-pentanoate, or any combination thereof, as compared to a parental microorganism. Accordingly, the microorganism may further include the deletion or inhibition of expression of an ilvD gene, as compared to a parental microorganism. In one aspect, the 2-isopropylmalate synthase may be encoded by a polynucleotide derived from a leuA gene, or homologs thereof. In another aspect, the beta-isopropylmalate dehydrogenase may be encoded by a polynucleotide derived from a leuB gene, or homologs thereof. In yet another aspect, the isopropylmalate isomerase may be encoded by a polynucleotide derived from a leuCD operon, or homologs thereof. In general the leuC gene of the leuCD operon encodes an isopropylmalate isomerase large subunit polypeptide and the leuD gene of the leuCD operon encodes an isopropylmalate isomerase small subunit polypeptide. In another aspect, the threonine dehydratase may be encoded by a polynucleotide derived from an ilvA gene, or homologs thereof. In yet another aspect, the threonine dehydratase may be encoded by a polynucleotide derived from a tdcB gene, or homologs thereof. In yet another embodiment, the recombinant microorganism may further include elevated expression or activity of phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, homoserine dehydrogenase, aspartate-semialdehyde dehydrogenase, homoserine kinase, threonine synthase, L-serine dehydratase, or threonine dehydratase, or any combination thereof, as compared to a parental microorganism. In some aspects, the phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, homoserine dehydrogenase, aspartate-semialdehyde dehydrogenase, homoserine kinase, threonine synthase, L-serine dehydratase, and threonine dehydratase, are encoded by a polynucleotide derived from the ppc, pyc, aspC, thrA, asd, thrB, thrC, sdaAB, and tdcB genes, respectively, or homologs thereof.
In one embodiment, a recombinant microorganism that produces 1-propanol is provided. The microorganism includes a cimA mutant and can include elevated expression or activity of alpha-isopropylmalate synthase, LeuB of Leptospira interrogans, isopropylmalate isomerase, and threonine dehydratase, as compared to a parental microorganism. In one aspect, the alpha-isopropylmalate synthase may be encoded by a polynucleotide derived from a cimA gene (e.g., a mutant cimA), or homologs thereof. The cimA gene may be a mutant of a Leptospira interrogans cimA gene or Methanocaldococcus jannaschii cimA gene.
In one aspect, the disclosure provides a mutant CimA comprising at least about 70% identity to WT cimA from Methanocaldococcus jannaschii (SEQ ID NO:89), wherein the mutant cimA comprises from about 9- to 22-fold higher production levels of 1-propanol and 1-butanol in an engineered pathway for their production when compared to the same pathway comprising a WT cimA. In another aspect, the mutant cimA comprises from about 1-10 mutated amino acids as compared to the WT cimA from Methanocaldococcus jannaschii. In yet another embodiment, the mutant cimA comprises the sequence of SEQ ID NO:89 having a mutation at one or more of the following positions: Ile47, His126 or Thr204. In yet another embodiment, the mutant cimA comprises the sequence of SEQ ID NO:89 having a mutation at one or more of the following positions: Ile47, Lys435, Val441, His126 or Thr204. In yet another embodiment, the mutant cim A comprises a sequence as set forth SEQ ID NO:89 and having one or more of the following mutations: a Ile47Val, a Lys435Asn, a Val441Ala, a His126Gln or a Thr204Ala, an Arg53Ser, a Glu183Lys, a Leu253Ser, Lys265Arg, Phe349Cys, an Asp86Gly, and Asp328Val, a Glu114Val, a Leu238Ser, a Glu111Lys, a Glu121Val, a Met250Val or a Lys32Asn. In a specific embodiment, the cimA mutant comprises SEQ ID NO:89 and having a mutation at Ile47Val and one or more additional mutations as set forth above. For example, the cimA mutant can comprise SEQ ID NO:89 and an Ile47Val, His126Gln, and a Thr204Ala. The cimA mutatant comprising 147V, H126Q and T204A may further comprise one or more mutations selected from r53S, E183K, L253S, K265R, F349C, D141E, I286V, L327H, D86G, D328V, E114V, L238S, E111K, E121V, M250V and K32N. The cimA mutant may further be truncated following residue 373 of the WT cimA. In yet another embodiment, the mutant cimA comprises a sequence as set forth in SEQ ID NO:89 and having 1-50, 1-20 or 1-10 conservative amino acid substitutions and having one or more of a Ile47Val, a His126Gln or a Thr204Ala mutation.
In another aspect, the beta-isopropylmalate dehydrogenase may be encoded by a polynucleotide derived from a leuB gene, or homologs thereof. In another aspect, the isopropylmalate isomerase may be encoded by a polynucleotide derived from a leuCD operon, or homologs thereof. In yet another embodiment, the recombinant microorganism may further include elevated expression or activity of phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, homoserine dehydrogenase, aspartate-semialdehyde dehydrogenase, homoserine kinase, threonine synthase, L-serine dehydratase, or threonine dehydratase, or any combination thereof, as compared to a parental microorganism. In some aspects, the phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, homoserine dehydrogenase, aspartate-semialdehyde dehydrogenase, homoserine kinase, threonine synthase, L-serine dehydratase, and threonine dehydratase, are encoded by a polynucleotide derived from the ppc, pyc, aspC, thrA, asd, thrB, thrC, sdaAB, and tdcB genes, respectively, or homologs thereof.
In another embodiment, a recombinant microorganism that produces 2-methyl 1-butanol is provided. The microorganism includes elevated expression or activity of threonine dehydratase, acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy-acid dehydratase, 2-keto-acid decarboxylase, and alcohol dehydrogenase, as compared to a parental microorganism, wherein the recombinant microorganism produces 2-methyl 1-butanol. In some aspects, the threonine dehydratase may be encoded by a polynucleotide derived from an ilvA gene, or homologs thereof. In another aspect, the threonine dehydratase may be encoded by a polynucleotide derived from a tdcB gene, or homologs thereof. In another embodiment, the recombinant microorganism further includes increased levels of 2-keto-3-methyl-valerate, as compared to a parental microorganism. In yet another aspect, the 2-keto-acid decarboxylase may be encoded by a polynucleotide derived from a kivd gene, or homologs thereof, or a PDC6 gene, or homologs thereof, or THI3 gene, or homologs thereof.
In another embodiment, a recombinant microorganism that produces phenylethanol is provided. The microorganism includes elevated expression or activity of chorismate mutase P/prephenate dehydratase, chorismate mutase T/prephenate dehydrogenase, 2-keto-acid decarboxylase and alcohol dehydrogenase, as compared to a parental microorganism. In one aspect, the chorismate mutase P/prephenate dehydratase may be encoded by a polynucleotide derived from a pheA gene, or homologs thereof. In another aspect, the chorismate mutase T/prephenate dehydrogenase may be encoded by a polynucleotide derived from a tyrA gene, or homologs thereof. In yet another embodiment, the recombinant microorganism further includes increased levels of phenylpyruvate, as compared to a parental microorganism.
In another embodiment, a recombinant microorganism that produces 3-methyl 1-butanol is provided. The microorganism includes elevated expression or activity of acetohydroxy acid synthase or acetolactate synthase, acetohydroxy acid isomeroreductase, dihydroxy-acid dehydratase, 2-isopropylmalate synthase, isopropylmalate isomerase, beta-isopropylmalate dehydrogenase, 2-keto-acid decarboxylase, and alcohol dehydrogenase, as compared to a parental microorganism. In some aspects, the acetohydroxy acid synthase may be encoded by a polynucleotide derived from an ilvIH operon, or homologs thereof. In another aspect, the acetolactate synthase may be encoded by a polynucleotide derived from an alsS gene, or homologs thereof. In another aspect, the acetolactate synthase may be encoded by a polynucleotide derived from an ilvMG operon, or homologs thereof. In another embodiment, the recombinant microorganism further includes increased levels of 2-ketoisocaproate, as compared to a parental microorganism. In yet another aspect, the acetolactate synthase may be encoded by a polynucleotide derived from an ilvNB operon, or homologs thereof.
In one embodiment, a method of producing a recombinant microorganism that converts a suitable substrate or metabolic intermediate to 1-butanol is provided. The method includes transforming a microorganism with one or more recombinant polynucleotides encoding a citramalate synthase mutant. In another embodiment, a method of producing a recombinant microorganism that converts a suitable substrate or metabolic intermediate to 1-butanol is provided. The method includes transforming a microorganism with one or more recombinant polynucleotides encoding polypeptides comprising 2-isopropylmalate synthase activity, beta-isopropylmalate dehydrogenase activity, isopropylmalate isomerase activity, threonine dehydratase activity, and/or citramalate synthase activity. In another embodiment, a method of producing a recombinant microorganism that converts a suitable substrate or metabolic intermediate to isobutanol, is provided. The method includes transforming a microorganism with one or more recombinant polynucleotides encoding polypeptides comprising acetohydroxy acid synthase activity, acetohydroxy acid isomeroreductase activity, dihydroxy-acid dehydratase activity, 2-keto-acid decarboxylase activity, and alcohol dehydrogenase activity.
In another embodiment, a method of producing a recombinant microorganism that converts a suitable substrate or metabolic intermediate to 1-propanol, is provided. The method includes transforming a microorganism with one or more recombinant polynucleotides encoding a citramalate synthase mutant. The method may further include transforming a microorganism with one or more additional recombinant polynucleotides encoding polypeptides comprising alpha-isopropylmalate synthase activity, beta-isopropylmalate dehydrogenase activity, isopropylmalate isomerase activity, and threonine dehydratase activity
In one embodiment, a method of producing a recombinant microorganism that converts a suitable substrate or metabolic intermediate to 2-methyl 1-butanol, is provided. The method includes transforming a microorganism with one or more recombinant polynucleotides encoding a citramalate synthase mutant. The method may further include transforming a microorganism with one or more additional recombinant polynucleotides encoding polypeptides comprising threonine dehydratase activity, acetohydroxy acid synthase activity, acetohydroxy acid isomeroreductase activity, dihydroxy-acid dehydratase activity, 2-keto-acid decarboxylase activity, and alcohol dehydrogenase activity.
In another embodiment, a method of producing a recombinant microorganism that converts a suitable substrate or metabolic intermediate to 3-methyl 1-butanol, is provided. The method includes transforming a microorganism with one or more recombinant polynucleotides encoding a citramalate synthase mutant. The method can further include transforming a microorganism with one or more additional recombinant polynucleotides encoding polypeptides comprising acetohydroxy acid synthase activity or acetolactate synthase activity, acetohydroxy acid isomeroreductase activity, dihydroxy-acid dehydratase activity, 2-isopropylmalate synthase activity, isopropylmalate isomerase activity, beta-isopropylmalate dehydrogenase activity, 2-keto-acid decarboxylase activity, and alcohol dehydrogenase activity. In another embodiment, a method of producing a recombinant microorganism that converts a suitable substrate or metabolic intermediate to phenylethanol, is provided. The method includes transforming a microorganism with one or more recombinant polynucleotides encoding polypeptides comprising chorismate mutase P/prephenate dehydratase activity, chorismate mutase T/prephenate dehydrogenase activity, 2-keto-acid decarboxylase activity, and alcohol dehydrogenase activity
In another embodiment, a method of producing an alcohol, is provided. The method includes providing a recombinant microorganism provided herein; culturing the microorganism of in the presence of a suitable substrate or metabolic intermediate and under conditions suitable for the conversion of the substrate to an alcohol; and detecting the production of the alcohol. In various aspects, the alcohol is selected from 1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 2-phenylethanol and 3-methyl 1-butanol. In another aspect, the substrate or metabolic intermediate includes a 2-keto acid, such as 2-ketobutyrate, 2-ketoisovalerate, 2-ketovalerate, 2-keto 3-methylvalerate, 2-keto 4-methyl-pentanoate, or phenylpyruvate.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the disclosure and, together with the detailed description, serve to explain the principles and implementations of the invention.
FIG. 1A-C depicts pathways useful in understanding the disclosure. (A) depicts an exemplary synthetic non-fermentative pathway using 2-keto acid metabolism for alcohol production. (B) depicts an exemplary alcohol production pathway in genetically engineered E. coli. Gray arrowheads represent the 2-keto acid degradation pathway. Enzymes, LeuABCD, IlvA, and IlvIHCD represent amino acid biosynthesis pathways. Double lines represent a side-reaction of amino acid biosynthesis pathways. (C) depicts a general pathway of a microorganism and identifies enzymes that can be disrupted or knocked out in the organism to improve flux in a desired direction (e.g., increase flux for the synthesis of pyruvate, and decrease flux of pyruvate to other competitive pathways).
FIG. 2A-C depicts modified amino acid biosynthesis pathways for improved isobutanol and 1-butanol production. Panel A shows isobutanol production with or without the engineered ilvIHCD pathway. Left panel: isobutanol production; Right panel: isobutanol yield per g of glucose. Theoretical maximum yield of isobutanol is 0.41 g/g. Panel B shows 1-butanol production with or without the engineered ilvA-leuABCD pathway from glucose. Left panel: 1-butanol production; Right panel: 1-propanol production in the same strain. Panel C shows 1-butanol production with L-threonine addition. Left panel: 1-butanol production; Right panel: 1-propanol production from the same strain.
FIG. 3 depicts the effect of isobutanol on cell growth. A time course for cell growth of the wild type and the high tolerant mutant, with or without 2% isobutanol is shown. Both strains were grown in LB to exponential phase. At OD600 ̃3.5, 2% isobutanol was added to the medium. Triangles: wild type without isobutanol; diamonds: wild type with isobutanol; squares: the high tolerant mutant without isobutanol; circles: high tolerant mutant with isobutanol.
FIG. 4 depicts an exemplary pathway for the production of isobutanol in E. coli. 
FIG. 5 depicts detection of isobutanol production by mass spectrometry.
FIG. 6 depicts mass spectrometry data.
FIG. 7 depicts an exemplary pathway for the production of 2-keto-isovalerate from pyruvate.
FIG. 8 depicts an exemplary pathway for leucine biosynthesis.
FIG. 9 depicts an exemplary pathway for isoleucine biosynthesis.
FIG. 10 depicts an exemplary pathway for butanol biosynthesis including 2-ketobutyrate as a biosynthetic intermediate.
FIG. 11 depicts an exemplary pathway for butanol biosynthesis from pyruvate.
FIG. 12 depicts an exemplary pathway for butanol biosynthesis including threonine as a biosynthetic intermediate.
FIGS. 13A and B depicts exemplary biosynthetic pathways for the production of (A) isobutanol (e.g., 2-methylpropyl alcohol), 3-methyl 1-butanol, 1-butanol, ethanol, 2-methyl 1-butanol, and 1-propanol. (B) A Schematic representation of the pathway for 1-propanol and 1-butanol production. The engineered citramalate pathway consists of four enzymatic steps from pyruvate to 2-ketobutyrate.
FIG. 14 depicts exemplary biosynthetic pathways for the production of phenylethanol, ethanol, 3-methyl 1-butanol, and isobutanol (e.g., 2-methylpropyl alcohol).
FIG. 15 depicts a nucleic acid sequence (SEQ ID NO:27) derived from a kivd gene encoding a polypeptide having 2-keto-acid decarboxylase activity.
FIG. 16 depicts a nucleic acid sequence (SEQ ID NO:29) derived from a PDC6 gene encoding a polypeptide having 2-keto-acid decarboxylase activity.
FIG. 17 depicts a nucleic acid sequence (SEQ ID NO:31) derived from an ARO10 gene encoding a polypeptide having 2-keto-acid decarboxylase activity.
FIG. 18 depicts a nucleic acid sequence (SEQ ID NO:33) derived from a THI3 gene encoding a polypeptide having 2-keto-acid decarboxylase activity.
FIG. 19 depicts a nucleic acid sequence (SEQ ID NO:35) derived from a pdc gene encoding a polypeptide having 2-keto-acid decarboxylase activity.
FIG. 20 depicts a nucleic acid sequence (SEQ ID NO:37) derived from an ADH2 gene encoding a polypeptide having alcohol dehydrogenase activity.
FIG. 21 depicts a nucleic acid sequence (SEQ ID NO:39) derived from an ilvI gene encoding a polypeptide having acetolactate synthase large subunit activity.
FIG. 22 depicts a nucleic acid sequence (SEQ ID NO:41) derived from an ilvH gene encoding a polypeptide having acetolactate synthase small subunit activity.
FIG. 23 depicts a nucleic acid sequence (SEQ ID NO:43) derived from an ilvC gene encoding a polypeptide having acetohydroxy acid isomeroreductase activity.
FIG. 24 depicts a nucleic acid sequence (SEQ ID NO:45) derived from an ilvD gene encoding a polypeptide having dihydroxy-acid dehydratase activity.
FIG. 25 depicts a nucleic acid sequence (SEQ ID NO:47) derived from an ilvA gene encoding a polypeptide having threonine dehydratase activity.
FIG. 26 depicts a nucleic acid sequence (SEQ ID NO:49) derived from a leuA gene encoding a polypeptide having 2-isopropylmalate synthase activity.
FIG. 27 depicts a nucleic acid sequence (SEQ ID NO:51) derived from a leuB gene encoding a polypeptide having beta-isopropylmalate dehydrogenase activity.
FIG. 28 depicts a nucleic acid sequence (SEQ ID NO:53) derived from a leuC gene encoding a polypeptide having isopropylmalate isomerase large subunit activity.
FIG. 29 depicts a nucleic acid sequence (SEQ ID NO:55) derived from a leuD gene encoding a polypeptide having isopropylmalate isomerase small subunit activity.
FIG. 30 depicts a nucleic acid sequence (SEQ ID NO:57) derived from a cimA gene encoding a polypeptide (SEQ ID NO:89) having alpha-isopropylmalate synthase activity.
FIG. 31 depicts a nucleic acid sequence (SEQ ID NO:59) derived from an ilvM gene encoding a polypeptide having acetolactate synthase large subunit activity.
FIG. 32 depicts a nucleic acid sequence (SEQ ID NO:61) derived from an ilvG gene encoding a polypeptide having acetolactate synthase small subunit activity.
FIG. 33 depicts a nucleic acid sequence (SEQ ID NO:63) derived from an ilvN gene encoding a polypeptide having acetolactate synthase large subunit activity.
FIG. 34 depicts a nucleic acid sequence (SEQ ID NO:65) derived from an ilvB gene encoding a polypeptide having acetolactate synthase small subunit activity.
FIG. 35 depicts a nucleic acid sequence (SEQ ID NO:67) derived from an adhE2 gene encoding a polypeptide having alcohol dehydrogenase activity.
FIG. 36 depicts a nucleic acid sequence (SEQ ID NO:69) derived from a Li-cimA gene encoding a polypeptide having alpha-isopropylmalate synthase activity.
FIG. 37 depicts a nucleic acid sequence (SEQ ID NO:71) derived from a Li-leuC gene encoding a polypeptide having isopropylmalate isomerase large subunit activity.
FIG. 38 depicts a nucleic acid sequence (SEQ ID NO:73) derived from a Li-leuD gene encoding a polypeptide having isopropylmalate isomerase small subunit activity.
FIG. 39 depicts a nucleic acid sequence (SEQ ID NO:75) derived from a Li-leuB gene encoding a polypeptide having beta-isopropylmalate dehydrogenase activity.
FIG. 40 depicts a nucleic acid sequence (SEQ ID NO:77) derived from a pheA gene encoding a polypeptide having chorismate mutase P/prephenate dehydratase activity.
FIG. 41 depicts a nucleic acid sequence (SEQ ID NO:79) derived from a TyrA gene encoding a polypeptide having chorismate mutase T/prephenate dehydratase activity.
FIG. 42 depicts a nucleic acid sequence (SEQ ID NO:81) derived from an alsS gene encoding a polypeptide having acetolactate synthase activity.
FIG. 43 depicts the correlation between knockout mutations and the production of isobutanol for various organisms.
FIG. 44 depicts the comparison of isobutanol production in normal shake flasks and in screw cap flasks.
FIG. 45 depicts the influence of the addition of different complex media components on isobutanol production in a fed batch over 200 hrs.
FIG. 46 shows a metabolic pathway from glucose to 3-methyl-1-butanol. All genes are from E. coli unless otherwise noted. BS=Bacillus subtilis; LL=Lactococcus lactis; SC=Saccharomyces cerevisiae. 
FIG. 47A-B shows an initial production of 3-methyl-1-butanol. Checkered columns indicate isobutanol; solid columns are for 3-methyl-1-butanol. (A) 3-methyl-1-butanol production in JCL16. Strains carrying either ilvIH (EC) or alsS (BS) with chromosomal or plasmid based expression of leuABCD were assayed for alcohol production. (B) 3-methyl-1-butanol production in JCL260. Strains carrying either ilvIH (IAA92) or alsS (IAA85) were tested for alcohol production.
FIG. 48A-B is a graph and a table depicting α-keto acid production. Checkered columns indicate 2-ketoisovalerate; solid columns are for 2-ketoisocaproate. (A) Production of α-keto acids in JCL260 background. Strains were tested for production of 2-ketoisovalerate (isobutanol) and 2-ketoisocaproate (3-methyl-1-butanol). The RBS change is for the leuA gene product only (IPMS). (B) Production of α-keto acids in L-leucine synthesis knockout backgrounds. ‘Δ’ indicates deletion.
FIG. 49 show 3-methyl-1-butanol production with removal of feedback inhibition. JCL260 hosts harboring VVT IPMS (IAA88) and IPMS(G462D) (IAA89) were compared for growth and alcohol production. Growth and alcohol production was quantified in the strain harboring WT IPMS (IAA90) in a JCL260 ΔilvE ΔtyrB (IAA69) background.
FIG. 50 is a schematic illustration of the propanol and butanol production via the threonine biosynthetic pathway in the genetically engineered E. coli. Depicted are disruption of the particular pathways; open rectangular boxes represent the precursors to the alcohol production. Also depicted is the unnatural norvaline pathway. Valine, leucine, isoleucine, proline, and methionine biosynthetic pathways are indicated by their corresponding abbreviations enclosed by gray boxes.
FIG. 51 shows the effect of thrA*BC over-expression on the alcohol and major metabolite productions in BW WT. Time course of propanol, butanol, major by-products, growth and glucose consumption in CRS-BuOH 12 (filled diamond) and CRS-BuOH 31 (open diamond) is shown. Both CRS-BuOH 12 and CRS-BuOH 31 were BW WT strains. CRS-BuOH 31 contained pSA62 and pSA55I while CRS-BuOH 12 contained an additional plasmid pCS49 which carried the thrA*BC behind PLlacO1. Cells were cultured as described in materials and methods.
FIG. 52A-B shows a comparison of alcohol productions in various knock-out strains. A. Strains were numbered as CRS-BuOH 12, 32, 2, 11, 23 from left to right. All strains contained the same sets of plasmids pCS49, pSA62, and pSA55I. Cells were cultured for 72 hours as described herein. The data shown is the 72nd hour time point. B. Time course of growths for each of the strains shown in A.
FIG. 53 shows a comparison of propanol and butanol production using alternative feedback resistant threonine dehydratase and 2-isopropylmalate synthase. BW ΔmetA, Δtdh, ΔilvB, ΔilvI was used as the background strain for the comparison. Strains were numbered as CRS-BuOH 11, 18, 19, 20 and all contained pSA62 and pSA55I in addition to the plasmids indicated below the figure. Gene names of the specific threonine dehydratase and 2-isopropylmalate synthase expressed are listed below the plasmid number. Cells were cultured for 72 hours as described herein. The data shown is the 72nd hour time point.
FIG. 54A-E shows a time course of propanol, butanol and metabolic by-products in CRS-BuOH 23. A. Production of 1-propanol and 1-butanol. Filled diamonds represent butanol and opened diamonds indicate propanol. B. Production of the major by-product acetate. C. Production of the minor by-products pyruvate, lactate and ethanol. Filled diamond represents pyruvate, opened square represents lactate, and cross represents ethanol. D. Consumption of glucose. E. Growth of CRS-BuOH 23 in the 100 hour time period.
FIG. 55 shows the amino acid sequence of cimA from M. jannaschii (SEQ ID NO:89).
FIG. 56A-B shows transfer of the citramalate pathway to E. coli. (A) Schematic representation of the synthetic operons. (B) Time courses for the growth of E. coli strain SA405 (ΔilvA ΔtdcB) and JCL16. OD600, optical density at 600 nM. Cells were incubated in M9 medium containing glucose at 30° C. Diamonds, JCL16; circles, squares, and triangles, SA405 with pSA63 (circles), pCS27 (without cimA-leuABCD) (squares), or L-isoleucine (39.5 μg/ml) (triangles).
FIG. 57A-D shows progress of the evolution of CimA. (A) Amino acid mutations are shown in the schematic representation of CimA. The gray bar indicates the putative regulator domain. The titers of 1-propanol and 1-butanol are shown (mg/L). The relative titers based on the strain with the wild type CimA are in parenthesis. (B) Time courses for the growth of an E. coli strain (SA408 [ΔilvA ΔtdcB ΔilvI]) containing the derivative of pSA63 (cimA-leuABCD). Cells were incubated in M9 medium containing glucose at 30° C. Circles, wild-type (WT) CimA; triangles, CimA2; diamonds, CimA3.7; and squares, control (blank plasmid). (C) Specific activities (M CoA produced/min/M protein) of the wild type (WT) (squares) and CimA3.7 (circles) at various temperatures. (D) Specific activities of the wild type and CimA3.7 at 30° C. in the presence of various concentrations of L-isoleucine.
FIG. 58A-F shows 1-Propanol and 1-butanol production with CimA3.7. Time profiles of cell growth with IPTG (squares) and without IPTG (open circles) (A); 1-propanol (B), 1-butanol (C), and ethanol (D) production; glucose consumption (E); and organic acid production (acetate [diamonds], lactate [circles], formate [triangles]) (F) from KS145/pSA55/pSA142 (containing cimA3.7). Cultures were grown at 30° C. in M9 medium containing 72 g/liter glucose and 5 g/liter yeast extract. OD500, optical density at 600 nm.
FIG. 59A-B shows Sequence Analysis of CimA3.7 A. Structure of Mycobacterium tuberculosis LeuA. The residues in active site and the bound 2-ketoisovalerate are colored blue and orange, respectively. The cartoon image on the left contains the regulator domain while the image on the right does not. The corresponding residues to mutations in CimA3.7 are colored red. B. Amino acid sequence alignment of CimA (M. jannaschii) (SEQ ID NO:89), LeuA (E. Coli) and LeuA (M. tuberculosis). Multiple sequence alignment was carried out using CLUSTAL W•(23). Fully conserved residues are shaded. The residues in the active site are shown with (*). Residue mutations in CimA3.7 are labeled with red. Gaps in the sequence are shown with (−).
FIG. 60 shows 1-propanol and 1-butanol production with the citramalate pathway. (Left panel) 1-Propanol production. (Right panel) 1-Butanol production in the same strain. The host is KS145, and overexpressed genes are indicated below the axis. Cultures were grown at 30° C. in M9 medium containing 5 g/liter yeast extract with or without 72 g/liter glucose for 40 h.
FIG. 61 shows 2-methyl-1-butanol production with a combination of the citramalate and the Threonine pathway. Using this combination of pathways, the production of 2-methyl-1-butanol increases relative to using either the citramalate or the Threonine pathway alone.
FIG. 62 shows production of 2-methyl-1-butanol with a combination of citramalate and threonine pathways.



Like reference symbols in the various drawings indicate like elements.
DETAILED DESCRIPTION
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the microorganism” includes reference to one or more microorganisms, and so forth.
Also, the use of “or” means “and/or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.
It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.
Any publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.
The disclosure provides an isolated polypeptide and polynucleotide useful in the metabolic engineering of microorganisms for the production of 1-butanol and 1-propanol as well as other higher alcohols.
The disclosure provides a mutant citramalate reductase having increased having increased enzymatic activity and the production of ketobutyrate. In one embodiment, the mutant citramalate is evolved from cimA of M. jannaschii. In another embodiment, the cimA is citramalate synthase (Leptospira biflexa serovar Patoc) gi|116664687|gb|ABK13757.1|(116664687); citramalate synthase (Leptospira biflexa serovar Monteralerio) gi|116664685|gb|ABK13756.1|(116664685); citramalate synthase (Leptospira interrogans Serovar Hebdomadis) gi|116664683|gb|ABK13755.1|(116664683); citramalate synthase (Leptospira interrogans Serovar Pomona) gi|1166646811gb|ABK13754.1|(116664681); citramalate synthase (Leptospira interrogans Serovar Australis) gi|116664679|gb|ABK13753.1|(116664679); citramalate synthase (Leptospira interrogans Serovar Autumnalis) gi|116664677|gb|ABK13752.1|(116664677); citramalate synthase (Leptospira interrogans Serovar Pyrogenes) gi|116664675|gb|ABK13751.1|(116664675); citramalate synthase (Leptospira interrogans Serovar Canicola) gi|116664673|gb|ABK13750.1|(116664673); citramalate synthase (Leptospira interrogans Serovar Lai) gi|116664671|gb|ABK13749.1|(116664671); CimA (Leptospira meyeri serovar Semaranga) gi|119720987|gb|ABL98031.1|(119720987); (R)-citramalate synthase gi|2492795|sp|Q58787.1|CIMA_METJA (2492795); (R)-citramalate synthase gi|22095547|sp|P58966.1|CIMA_METMA (22095547); (R)-citramalate synthase gi|22001554|sp|Q8TJJ1.1|CIMA_METAC (22001554); (R)-citramalate synthase gi|22001553|sp|026819.1|CIMA_METTH (22001553); (R)-citramalate synthase gi|22001555|sp|Q8TYB1.1|CIMA_METKA (22001555); (R)-citramalate synthase (Methanococcus maripaludis S2) gi|4535858||ref|NP_988138.1|(45358581); (R)-citramalate synthase (Methanococcus maripaludis S2) gi|44921339|emb|CAF30574.1|(44921339); and similar to (R)-citramalate synthase (Candidatus Kuenenia stuttgartiensis) gi|91203541|emb|CAJ71194.1|(91203541), each sequence associated with the foregoing accession numbers is incorporated herein by reference.
A mutant cimA of the disclosure can be derived from any of the foregoing citramalate synthases. The mutant cimA comprises substitutions of amino acids with smaller sidechains in areas outside the active site. Such substitutions include those with lower residue hydrophobicity, substitution of native residues with uncharged polar residues, and substitutions that resulted in the replacement of a charged residue with a non-charged residue. Such substitutions are consistent with the differences between the thermophilic and mesophilic proteins of the same class.
In one embodiment, the CimA employed in the directed evolution process described herein was isolated from M. jannaschii, an extremely thermophilic archaeon. The acquired mutations are not located near the catalytic center, and comprise mutations that stabilize that active structure in 30° C. It has been shown that the thermophilic proteins have higher residue volume, higher residue hydrophobicity, more charged amino acids, and fewer uncharged polar residues than mesophilic proteins. Twelve out of eighteen substitutions identified in CimA3s resulted in the selection of amino acids with smaller sidechains. Eight substitutions resulted in the selection of lower residue hydrophobicity, while the replacement of native residues with uncharged polar residues was observed six times. Additionally, eight substitutions resulted in the replacement of a charged residue with a non-charged residue. Continued analysis of these mutations should provide further insight into the mechanism which grants higher activity of CimA in E. coli. Most of the substitutions observed were consistent with the differences between the thermophilic and mesophilic proteins.
The disclosure provides (i) a substantially purified polypeptide comprising SEQ ID NO:89 having at least mutations at one or more of the following positions: Ile47, His126, Thr204, and having citramalate synthase activity; (ii) a substantially purified polypeptide having from 1-30, 1-20, 1-10 or 1-5 conservative amino acid substitutions to (i) and having citramalate synthase activity; (iii) a substantially purified polypeptide comprising a sequence that is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO:89 and wherein the polypeptide comprises a residue selected from the group consisting of a Val at position 47, a Gln at position 126, an Ala at position 204, and any combination of the foregoing and having citramalate synthase activity; (iv) a substantially purified polypeptide comprising a sequence as set forth in SEQ ID NO:89 having a mutation selected from the group consisting of a Ile47Val, a His126Gln, a Thr204Ala, and any combination of the foregoing and having citramalate synthase activity; and (v) any of (i)-(iv) wherein the C-terminal domain of the polypeptide comprising SEQ ID NO:89 after residue 373 is absent or truncated.
As described below cimA mutants can be screened for activity by measuring the production of ketobutyrate using techniques known in the art. Furthermore, additional mutations can be made using techniques in the art to obtain substitutions (e.g., conservative substitutions) and/or deletions that provide cimA mutants having increased citramalte synthase activity using the present disclosure and skill in the art.
The disclosure also provides polynucleotides encoding any of the foregoing polypeptides above and as further described herein; SEQ ID NO:57 sets forth a cimA cDNA sequence that can be used to identify and develop the mutant cimA polypeptide of the disclosure based upon the degeneracy of the genetic code. Accordingly, the disclosure provides an isolated polynucleotide comprising a sequence encoding: (i) a substantially purified polypeptide comprising SEQ ID NO:89 having at least mutations at one or more of the following positions: Ile47, His126, Thr204, or any combination thereof and having citramalate synthase activity; (ii) a substantially purified polypeptide having from 1-30, 1-20, 1-10 or 1-5 conservative amino acid substitutions to (i) and having citramalate synthase activity; (iii) a substantially purified polypeptide comprising a sequence that is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO:89 and wherein the polypeptide comprises a residue selected from the group consisting of a Val at position 47, a Gln at position 126, an Ala at position 204, and any combination of the foregoing and having citramalate synthase activity; (iv) a substantially purified polypeptide comprising a sequence as set forth in SEQ ID NO:89 having a mutation selected from the group consisting of a Ile47Val, a His126Gln, a Thr204Ala, and any combination of the foregoing and having citramalate synthase activity; and (v) any of (i)-(iv) wherein the C-terminal domain of a polypeptide comprising SEQ ID NO:89 after residue 373 is absent or truncated. The polynucleotide can be DNA or RNA or a combination thereof.
Butanol is hydrophobic and less volatile than ethanol. 1-Butanol has an energy density closer to gasoline. Butanol at 85 percent strength can be used in cars without any change to the engine (unlike ethanol) and it produces more power than ethanol and almost as much power as gasoline. Butanol is also used as a solvent in chemical and textile processes, organic synthesis and as a chemical intermediate. Butanol also is used as a component of hydraulic and brake fluids and as a base for perfumes.
The native producers of 1-butanol, such as Clostridium acetobutylicum, also produce byproducts such as acetone, ethanol, and butyrate as fermentation products. However, these microorganisms are relatively difficult to manipulate. Genetic manipulation tools for these organisms are not as efficient as those for user-friendly hosts such as E. coli and physiology and their metabolic regulation are much less understood, prohibiting rapid progress towards high-efficiency production. Furthermore, no native microorganisms have been identified to produce from glucose other higher alcohols such as isobutanol, 2-methyl 1-butanol, 3-methyl 1-butanol, and 2-phenylethanol to industrially relevant quantities, despite the small amounts that have been identified as microbial byproducts.
The disclosure provides metabolically engineered microorganisms comprising biochemical pathways for the production of higher alcohols including 1-butanol, 1-propanol, 2-methyl-1-butanol, and 3-methyl-1-butanol from a suitable substrate. A metabolically engineered microorganism of the disclosure comprises one or more recombinant polynucleotides within the genome of the organism or external to the genome within the organism. The microorganism can comprise a reduction, disruption or knockout of a gene found in the wild-type organism and/or introduction of a heterologous polynucleotide including the introduction of an evolved polypeptide having an improved enzymatic activity compared to a wild-type polypeptide.
The disclosure also includes metabolically engineered biosynthetic pathways that utilize an organism's native amino acid pathway. Biofuel production utilizing the organism's native amino acid pathways offers several advantages. Not only does it avoid the difficulty of expressing a large set of foreign genes but it also minimizes the possible accumulation of toxic intermediates. Contrary to the butanol production pathway found in many species of Clostridium, the engineered amino acid biosynthetic routes for biofuel production circumvent the need to involve oxygen-sensitive enzymes and CoA-dependent intermediates. The disclosure provides a much more host-friendly biofuel production system utilizing the organism's native metabolites in the amino acid biosynthetic pathway to produce biofuels.
In one aspect, the disclosure provides a recombinant microorganism comprising elevated expression or an improved activity of at least one target enzyme as compared to a parental microorganism or encodes an enzyme not found in the parental organism that increases flux towards the production of a ketobutyrate. In another or further aspect, the microorganism comprises a reduction, disruption or knockout of at least one gene encoding an enzyme that competes with a metabolite necessary for the production of a desired higher alcohol product. The recombinant microorganism produces at least one metabolite involved in a biosynthetic pathway for the production of, isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol. In general, the recombinant microorganisms comprises at least one recombinant metabolic pathway that comprises a target enzyme and may further include a reduction in activity or expression of an enzyme in a competitive biosynthetic pathway. The pathway acts to modify a substrate or metabolic intermediate in the production of isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol. The target enzyme is encoded by, and expressed from, a polynucleotide derived from a suitable biological source. In some embodiments, the polynucleotide comprises a gene derived from a bacterial or yeast source and recombinantly engineered into the microorganism of the disclosure.
As used herein, the term “metabolically engineered” or “metabolic engineering” involves rational pathway design and assembly of biosynthetic genes, genes associated with operons, and control elements of such polynucleotides, for the production of a desired metabolite, such as a 2-keto acid or higher alcohol, in a microorganism. “Metabolically engineered” can further include optimization of metabolic flux by regulation and optimization of transcription, translation, protein stability and protein functionality using genetic engineering and appropriate culture condition including the reduction of, disruption, or knocking out of, a competing metabolic pathway that competes with an intermediate leading to a desired pathway. A biosynthetic gene can be heterologous to the host microorganism, either by virtue of being foreign to the host, or being modified by mutagenesis, recombination, and/or association with a heterologous expression control sequence in an endogenous host cell. In one aspect, where the polynucleotide is xenogenetic to the host organism, the polynucleotide can be codon optimized.
The term “biosynthetic pathway”, also referred to as “metabolic pathway”, refers to a set of anabolic or catabolic biochemical reactions for converting (transmuting) one chemical species into another. Gene products belong to the same “metabolic pathway” if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.
For example, L-leucine is synthesized through biosynthetic pathway inherent to L-leucine which diverges from the intermediate (2-ketoisovalerate) of L-valine biosynthesis system. In Escherichia, the L-valine biosynthesis and biosynthesis inherent to L-leucine are carried out by a group of enzymes encoded by ilvGMEDA operon and those encoded by leuABCD operon, respectively.
The leuABCD operon includes leuA, leuB, leuC and leuD genes. Among them, leuA encodes α-isopropylmalate synthase, leuB encodes β-isopropylmalate dehydrogenase, leuC and leuD encodes α-isopropylmalate isomerase. Of these enzymes, α-isopropylmalate synthase catalyzes the synthetic reaction from α-ketoisovalerate to α-isopropylmalate, α-isopropylmalate isomerase catalyzes the isomerization reaction from α-isopropylmalate to β-isopropylmalate and β-isopropylmalate dehydrogenase catalyzes the dehydrogenation reaction from β-isopropylmalate to α-ketoisocaproic acid which is the final intermediate of L-leucine biosynthesis. Escherichia possess four kinds of transaminases, namely, transaminase A (aspartate-glutamate aminotransferase) encoded by aspC gene, transaminase B (BCAA aminotransferase) encoded by ilvE gene which is included in ilvGMEDA operon, transaminase C (alanine-valine aminotransferase) encoded by avtA gene and transaminase D (tyrosine aminotransferase) encoded by tyrB gene. These enzymes participate in various amination reactions. Of these enzymes, transaminase B and transaminase D catalyze the above-mentioned amination reaction from α-ketoisocaproic acid to L-leucine. Transaminase C and transaminase D catalyze the final step of L-valine biosynthetic pathway, which includes a common pathway among the L-valine biosynthesis and L-leucine biosynthesis.
Also, the expression of leuABCD operon is repressed by L-leucine. Expression of ilvBN gene encoding acetohydroxy acid synthase I suffers concerted repression by L-valine and L-leucine, expression of ilvGM gene encoding acetohydroxy acid synthase II suffers concerted repression by L-isoleucine, L-valine and L-leucine, and expression of ilvIH gene encoding acetohydroxy acid synthase III suffers repression by L-leucine.
The term “substrate” or “suitable substrate” refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme. The term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate, or derivatives thereof. Further, the term “substrate” encompasses not only compounds that provide a carbon source suitable for use as a starting material, such as any biomass derived sugar, but also intermediate and end product metabolites used in a pathway associated with a metabolically engineered microorganism as described herein. A “biomass derived sugar” includes, but is not limited to, molecules such as glucose, sucrose, mannose, xylose, and arabinose. The term biomass derived sugar encompasses suitable carbon substrates ordinarily used by microorganisms, such as 6 carbon sugars, including but not limited to glucose, lactose, sorbose, fructose, idose, galactose and mannose all in either D or L form, or a combination of 6 carbon sugars, such as glucose and fructose, and/or 6 carbon sugar acids including, but not limited to, 2-keto-L-gulonic acid, idonic acid (IA), gluconic acid (GA), 6-phosphogluconate, 2-keto-D-gluconic acid (2 KDG), 5-keto-D-gluconic acid, 2-ketogluconatephosphate, 2,5-diketo-L-gulonic acid, 2,3-L-diketogulonic acid, dehydroascorbic acid, erythorbic acid (EA) and D-mannonic acid.
The term “alcohol” includes for example 1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol or 2-phenylethanol. The term “1-butanol” or “n-butanol” generally refers to a straight chain isomer with the alcohol functional group at the terminal carbon. The straight chain isomer with the alcohol at an internal carbon is sec-butanol or 2-butanol. The branched isomer with the alcohol at a terminal carbon is isobutanol, and the branched isomer with the alcohol at the internal carbon is tert-butanol.
Recombinant microorganisms provided herein can express a plurality of target enzymes involved in pathways for the production of, for example, isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol, from using a suitable carbon substrate.
Accordingly, metabolically “engineered” or “modified” microorganisms are produced via the introduction of genetic material into a host or parental microorganism of choice thereby modifying or altering the cellular physiology and biochemistry of the microorganism. Through the introduction of genetic material the parental microorganism acquires new properties, e.g. the ability to produce a new, or greater quantities of, an intracellular metabolite. In an illustrative embodiment, the introduction of genetic material into a parental microorganism results in a new or modified ability to produce an alcohol such as isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol. The genetic material introduced into the parental microorganism contains gene(s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of an alcohol and may also include additional elements for the expression and/or regulation of expression of these genes, e.g. promoter sequences.
An engineered or modified microorganism can also include in the alternative or in addition to the introduction of a genetic material into a host or parental micoorganism, the disruption, deletion or knocking out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism. Through the reduction, disruption or knocking out of a gene or polynucleotide the microorganism acquires new or improved properties (e.g., the ability to produced a new or greater quantities of an interacellular metabolite, improve the flux of a metabolite down a desired pathway, and/or reduce the production of undesirable by-products).
The disclosure demonstrates that the expression of one or more heterologous polynucleotide or over-expression of one or more heterologous polynucleotide encoding a polypeptide having ketoacid decarboxylase and a polypeptide having alcohol dehydrogenase in the presence of a polypeptide having α-isopropylmalate synthase, a polypeptide having β-isopropylmalate dehydrogenase, a polypeptide having α-isopropylmalate isomerase, a polypeptide having threonine dehydratease, a polypeptide having homoserine dehydrogenase activity, a polypeptide having homoserine kinase activity, and a polypeptide having threonine synthase activity.
For example, the disclosure demonstrates that with over-expression of the heterologous kivd and adh2 and the E. coli ilvA, leuA, leuB, leuC, leuD (or a Leu operon, e.g., leuABCD), and thrA, thrB, thrC or a Thr operon (e.g., thrABC, the thrA may be a feedback resistive polypeptide such as thrA*) the production of 1-butanol and 1-propanol can be obtained. The production of 1-butanol uses 2-ketovalerate, which involves the intermediate 2-ketobutyrate and the unnatural norvaline biosynthetic pathway. Since Kivd has similar affinity towards both 2-ketoacids and 2-ketobutyrate is a secondary substrate for LeuA, 1-propanol was co-produced with 1-butanol in similar amounts.
Microorganisms provided herein are modified to produce metabolites in quantities not available in the parental microorganism. A “metabolite” refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process. A metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., 2-keto acid) in, or an end product (e.g., isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol) of metabolism. Metabolites can be used to construct more complex molecules, or they can be broken down into simpler ones. Intermediate metabolites may be synthesized from other metabolites, perhaps used to make more complex substances, or broken down into simpler compounds, often with the release of chemical energy.
Exemplary metabolites include glucose, pyruvate, isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol, and 2-keto acids. As depicted in FIG. 1A, exemplary 2-keto acid intermediates include 2-ketobutyrate, 2-ketoisovalerate, 2-ketovalerate, 2-keto 3-methylvalerate, 2-keto 4-methyl-pentanoate, and phenylpyruvate. The exemplary 2-keto acids shown in FIG. 1A may be used as metabolic intermediates in the production of isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol. For example, as shown in FIG. 1B a recombinant microorganism metabolically engineered to provide elevated expression of 2-isopropylmalate synthase, beta-isopropylmalate dehydrogenase and isopropylmalate isomerase enzymes encoded by, for example, a Leu operon (e.g., LeuABCD) produces 2-ketovalerate from 2-ketobutyrate. The 2-ketovalerate metabolite may be used to produce 1-butanol by additional enzymes produced by the metabolically, modified microorganism. Additionally, 1-propanol and 2-methyl 1-butanol can be produced from 2-ketobutyrate and 2-keto-3-methyl-valerate by a recombinant microorganism metabolically engineered to express or over-express acetohydroxy acid synthase, alpha-ketoacid decarboxylase, and alcohol dehydrogenase enzymes encoded by, for example, ilvIHDC, kdc and adh genes. Further, the metabolite 2-ketoisovalerate can be produced by a recombinant microorganism metabolically engineered to express or over-express acetohydroxy acid synthase enzymes encoded by, for example, ilvIHCD genes. This metabolite can then be used in the production of 3-methyl 1-butanol. The metabolites pyruvate and phenylpyruvate can be used to produce 2-phenylethanol by a recombinant microorganism metabolically engineered to express or over-express alpha-ketoacid decarboxylase, and alcohol dehydrogenase enzymes encoded by, for example, kdc and adh. Additional metabolites and genes are shown in FIG. 1B.
Accordingly, provided herein are recombinant microorganisms that produce isobutanol and in some aspects may include the elevated expression of target enzymes such as acetohydroxy acid synthase (e.g., ilvIH operon), acetohydroxy acid isomeroreductase (e.g., ilvC), dihydroxy-acid dehydratase (e.g., ilvD), 2-keto-acid decarboxylase (e.g., PDC6, ARO10, THIS, kivd, or pdc), and alcohol dehydrogenase (e.g., ADH2). The microorganism may further include the deletion or inhibition of expression of an ethanol dehydrogenase (e.g., an adhE), ldh (e.g., an ldhA), frd (e.g., an frdB, an frdC or an frdBC), fnr, leuA, ilvE, poxB, ilvA, pflB, or pta gene, or any combination thereof, to increase the availability of pyruvate or reduce enzymes that compete for a metabolite in a desired biosynthetic pathway. In some aspects the recombinant microorganism may include the elevated expression of acetolactate synthase (e.g., alsS), acteohydroxy acid isomeroreductase (e.g., ilvC), dihydroxy-acid dehydratase (e.g., ilvD), 2-keto acid decarboxylase (e.g., PDC6, ARO10, TH13, kivd, or pdc), and alcohol dehydrogenase (e.g., ADH2). With reference to alcohol dehydrogenases, although ethanol dehydrogenase is an alcohol dehydrogenase, the synthesis of ethanol is undesirable as a by-product in the biosynthetic pathways. Accordingly, reference to an increase in alcohol dehydrogenase activity or expression in a microorganism specifically excludes ethanol dehydrogenase activity.
Also provided are recombinant microorganisms that produce 1-butanol and include the expression of a mutant citramalate synthase (e.g., a mutant cimA) an may further include the elevated expression of target enzymes such as 2-isopropylmalate synthase (e.g., leuA), beta-isopropylmalate dehydrogenase (e.g., leuB), isopropylmalate isomerase (e.g., leuC, leuD, or leuCD operon), threonine dehydratase (e.g., ilvA). The microorganism may further include decreased levels of 2-ketoisovalerate, 2-keto-3-methyl-valerate, or 2-keto-4-methyl-pentanoate, or any combination thereof, as compared to a parental microorganism. In addition, the microorganism may include a disruption, deletion or knockout of expression of a dihydroxy-acid dehydratase (e.g. ilvD gene), as compared to a parental microorganism. A recombinant microorganism that produces 1-butanol may include further elevated expression or activity of phosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartate aminotransferase, homoserine dehydrogenase, aspartate-semialdehyde dehydrogenase, homoserine kinase, threonine synthase, L-serine dehydratase, and/or threonine dehydratase, encoded by a nucleic acid sequences derived from the ppc, pyc, aspC, thrA, asd, thrB, thrC, sdaAB, and tdcB genes, respectively, or homologs thereof.
Also provided are recombinant microorganisms that produce 1-propanol comprising the expression of a mutant citramalate synthase (e.g., a cimA mutant), beta-isopropylmalate dehydrogenase (e.g., leuB), isopropylmalate isomerase (e.g., leuCD operon) and threonine dehydratase.
Also provided are recombinant microorganisms that produce 2-methyl 1-butanol comprising the expression of a mutant citramalate synthase (e.g., a cimA mutant) and may include the elevated expression of target enzymes such as threonine dehydratase (e.g., ilvA, tdcB, ilv1), acetohydroxy acid synthase (e.g., ilvIH operon), acetohydroxy acid isomeroreductase (e.g., ilvC), dihydroxy-acid dehydratase (e.g., ilvD), 2-keto-acid decarboxylase (e.g., PDC6, ARO10, THI3, kivd, and/or pdc, and alcohol dehydrogenase (e.g., ADH2).
Also provided are recombinant microorganisms that produce 3-methyl 1-butanol comprising the expression of a mutant citramalte synthase (e.g., a cimA mutant) and may include the elevated expression of target enzymes such as acetolactate synthase (e.g., alsS), acetohydroxy acid synthase (e.g., ilvIH), acetolactate synthase (e.g., ilvMG) or (e.g., ilvNB), acetohydroxy acid isomeroreductase (e.g., ilvC), dihydroxy-acid dehydratase (e.g., ilvD), 2-isopropylmalate synthase (leuA), isopropylmalate isomerase (e.g., leuC, D or leuCD operon), beta-isopropylmalate dehydrogenase (e.g., leuB), 2-keto-acid decarboxylase (e.g., kivd, PDC6, or THI3), and alcohol dehydrogenase (e.g., ADH2).
Also provided are recombinant microorganisms that produce phenylethanol and may include the elevated expression of target enzymes such as chorismate mutase P/prephenate dehydratase (e.g., pheA), chorismate mutase T/prephenate dehydrogenase (e.g., tyrA), 2-keto-acid decarboxylase (e.g., kivd, PDC6, or THI3), and alcohol dehydrogenase (e.g., ADH2).
As previously noted the target enzymes described throughout this disclosure generally produce metabolites. For example, the enzymes 2-isopropylmalate synthase (leuA), beta-isopropylmalate dehydrogenase (leuB), and isopropylmalate isomerase (leuC, leuD or leuCD operon) may produce 2-ketovalerate from a substrate that includes 2-ketobutyrate. In addition, the target enzymes described throughout this disclosure are encoded by polynucleotides. For example, threonine dehydratase can be encoded by a polynucleotide derived from an ilvA gene or, for example, from the ilv1 gene of Pichia stipitis (or a modified ilv1 or variant thereof). A threonine deaminase may be derived from the genus Saccharomyces, Escherichia or Corynebacterium. 
Acetohydroxy acid synthase can be encoded by a polynucleotide derived from an ilvIH operon. Acetohydroxy acid isomeroreductase can be encoded by a polynucleotide derived from an ilvC gene. Dihydroxy-acid dehydratase can be encoded by a polynucleotide derived from an ilvD gene. 2-keto-acid decarboxylase can be encoded by a polynucleotide derived from a PDC6, ARO10, THI3, kivd, and/or pdc gene. Alcohol dehydrogenase can be encoded by a polynucleotide derived from an ADH2 gene. Additional enzymes and exemplary genes are described throughout this document. Homologs of the various polypeptides and polynucleotides can be derived from any biologic source that provides a suitable polynucleotide encoding a suitable enzyme. Homologs, for example, can be identified by reference to various databases.
The disclosure identifies specific genes useful in the methods, compositions and organisms of the disclosure; however it will be recognized that absolute identity to such genes is not necessary. For example, changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically such changes comprise conservative mutation and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for expression of a function enzyme activity using methods known in the art.
Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptide can also be used to clone and express the polynucleotides encoding such enzymes.
As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, a process sometimes called “codon optimization” or “controlling for species codon bias.”
Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl. Acids Res. 17:477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al. (1996) Nucl. Acids Res. 24: 216-218). Methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Pat. No. 6,015,891, and the references cited therein.
Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure. The native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as they modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
In addition, homologs of enzymes useful for generating metabolites (e.g., keto thiolase, acetyl-CoA acetyltransferase, hydroxybutyryl CoA dehydrogenase, crotonase, crotonyl-CoA reductase, butyryl-coA dehydrogenase, alcohol dehydrogenase (ADH)) are encompassed by the microorganisms and methods provided herein. The term “homologs” used with respect to an original enzyme or gene of a first family or species refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.
A protein has “homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences).
As used herein, two proteins (or a region of the proteins) are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, reference to a kivd gene includes homologs (e.g., pdc6, aro10, thI3, pdc, kdcA, pdc1, pdc5) from other organisms encoding an enzyme having substantially similar enzymatic activity, as well as genes having at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 98, or 99% identity to the referenced gene and which encodes an enzyme having substantially similar enzymatic activity as the referenced gene. For example, pyruvate decarboxylase of Kluyveromyces lactis has 37% identity to Kivd at the amino acids level; kivd and thI3 are 32% identical at the nucleic acid level; Alcohol dehydrogenase of Schizosaccharomyces pombe has 52% identity to ADH2 of Saccharomyces cerevisiae at the amino acid sequence level; S. cerevisiae adh2 and Lactococcus Lactis adh are 49% identical; KIVD (Lactococcus lactis) and PDC6 (Saccharomyces cerevisiae) share 36% identity (Positives=322/562 (57%), Gaps=24/562 (4%)); KIVD (Lactococcus lactis and THIS (Saccharomyces cerevisiae) share 32% identity (Positives=307/571 (53%), Gaps=35/571 (6%)); kivd (Lactococcus lactis) and ARO10 (Saccharomyces cerevisiae) share 30% identikit (Positives=296/598 (49%), Gaps=65/598 (10%)); ARO10 (Saccharomyces cerevisiae) and PDC6 (Saccharomyces cerevisiae) share 34% identity (Positives=320/616 (51%), Gaps=61/616 (9%)); ARO10 (Saccharomyces cerevisiae) and THI3 (Saccharomyces cerevisiae) share 30% identity (Positives=304/599 (50%), Gaps=48/599 (8%)); ARO10 (Saccharomyces cerevisiae) and Pyruvate decarboxylase (Clostridium acetobutylicum ATCC 824) share 30% identity (Positives=291/613 (47%), Gaps=73/613 (11%)); PDC6 ((Saccharomyces cerevisiae) and THI3 (Saccharomyces cerevisiae) share 50% identikit (Positives=402/561 (71%), Gaps=17/561 (3%)); PDC6 (Saccharomyces cerevisiae) and Pyruvate decarboxylase (Clostridium acetobutylicum ATCC 824) share 38% identity (Positives=328/570 (57%), Gaps=30/570 (5%)); and THI3 (Saccharomyces cerevisiae) and Pyruvate decarboxylase (Clostridium acetobutylicum ATCC 824) share 35% identity (Positives=284/521 (54%), Gaps=25/521 (4%)). Sequence for each of the genes and polypeptides/enzymes listed herein can be readily identified using databases available on the World-Wide-Web (see, e.g., http:(//)eecoli.kaist.ac.kr/main.html). In addition, the amino acid sequence and nucleic acid sequence can be readily compared for identity using commonly used algorithms in the art.
When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson et al., 1994, hereby incorporated herein by reference).
The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1.
A typical algorithm used comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996; Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul, 1997). Typical parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, hereby incorporated herein by reference). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereby incorporated herein by reference.
The following table and the disclosure provides non-limiting examples of genes and homologs for each gene having polynucleotide and polypeptide sequences available to the skilled person in the art.







TABLE 1







Depicts recombinant pathways for the production of various


higher alcohols (“+” = expression, increase expression or


activity/“−” = reduced expression or activity or knockout*).























2-M-1-





1-butanol

1-propanol

3-M-1-
butanol



Exemplary

(via L-
1-butanol
(via L-
1-propanol
butanol
(via L-


Enzyme
Gene(s)
isobutanol
threonine)
(via pyruvate)
threonine)
(via pyruvate)
(via pyruvate)
threonine)





Ethanol Dehydrogenase
adhE
−
−
−
−
−
−
−


Lactate Dehydrogenase
ldhA
−
−
−
−
−
−
−


Fumarate reductase
frdBC
−




−
−



fnr
−




−
−


acetate kinase
ackA
−
−
−
−
−
−
−


Phosphate
pta
−
−
−
−
−
−
−


acetyltransferase


Formate acetyltransferase
pflB
−




−
−


α-isopropylmalate
leuA
−
+
+


+


synthase


β-isopropylmalate
leuB

+
+
−
+
+


dehydrogenase,


α-isopropylmalate
leuC

+
+

+
+


isomerase


α-isopropylmalate
leuD

+
+

+


isomerase


BCAA aminotransferase
ilvE
−




−


tyrosine aminotransferase
tyrB, tyrAT





−


pyruvate dehydrogenase
poxB
−
−
−
−
−


acetolactate synthase
ilvB, ilv2,

−
−
−
−



ilvI, alsS,



ilv6


threonine dehydratase
ilvA, tdcB
−
+
+
+
+

+


homoserine
metA

−
−
−
−

−


transsuccinylase


L-threonine 3-
tdh

−
−
−
−

−


dehydrogenase


acetohydroxy acid
ilvHI, ilvNB,
+




+
+


synthase
ilvGM, alsS


acetohydroxy acid
ilvC, ilv5
+




+
+


isomeroredutase


dihydroxy-acid
ilvD, ilv3
+




+
+


dehyratase


2-ketoacid decarboxylase
pdc6, aro10,
+
+
+
+
+
+
+



thI3, kivd,



pdc, kdcA,



pdc1, pdc5


alcohol dehydrogenase
adh1, adh2,
+
+
+
+
+
+
+



adh3, adh4,



adh5, adh6,



sfa1


citramalate synthase
cimA


+

+





*knockout or a reduction in expression are optional in the synthesis of the product, however, such knockouts increase various substrate intermediates and improve yield.






Tables 2-8 set forth reaction pathways for various recombinant microorganism of the disclosure including a list of exemplary genes and homologs and organism source.







TABLE 2





1-butanol production pathway via L-threonine















Reaction 1


L-threonine -> 2-keto-butyrate


Genes


ilvA (E. coli). rdcB (E. coli) or homologs thereof


Reaction 2


2-keto-butyrate -> 2-ethylmalate


Genes


leuA (E. coli) or homologs thereof


Reaction 3


2-ethylmalate -> 3-ethylmalate


Genes


leuCD (E. coli) or homologs thereof


Reaction 4


3-ethylmalate -> 2-ethyl-3-oxosuccinate


Genes


leuB (E. coli) or homologs thereof


Reaction 5


2-ethyl-3-oxosuccinate -> 2-keto-valerate


Gene


n/a (spontaneous reaction)


Reaction 6


2-keto-valerate -> butrylaldehyde


Genes


kivd (Lactococcus lactis), kdcA (Lactococcus lactis), PDC1


(Saccharomyces cerevisiae), PDC5 (Saccharomyces cerevisiae).


PDC6 (Saccharomyces cerevisiae) THI3 (Saccharomyces cerevisiae),


ARO10 (Saccharomyces cerevisiae)or homologs thereof


Reaction 7


butrylaldehyde -> 1-butanol


Genes


ADH1 (Saccharomyces cerevisiae), ADH2 (Saccharomyces cerevisiae),


ADH3 (Saccharomyces cerevisiae), ADH4 (Saccharomyces cerevisiae),


ADH5(Saccharomyces cerevisiae), ADH6 (Saccharomyces cerevisiae),


SFA1 (Saccharomyces cerevisiae) or homologs thereof
















TABLE 3





1-butanol production pathway via pyruvate















Reaction 1


Pyruvate + Acetyl-CoA -> (R)-citramalate


Genes


cimA (Methanocaldococcus jannaschii), cimA (Leptospira interrogans)


or homologs thereof


Reaction 2


(R)-citramalate -> citraconate


Genes


leuCD (Leptospira interrogans), leuCD (E. coli) or homologs thereof


Reaction 3


citraconate -> β-methyl-D-malate


Genes


leuCD (Leptospira interrogans), leuCD (E. coli) or homologs thereof


Reaction 4


β-methyl-D-malate -> 2-keto-butyrate


Genes


leuB (Leptospira interrogans), leuB (E. coli) or homologs thereof


Reaction 5


2-keto-butyrate -> 2-ethylmalate


Genes


leuA (E. coli) or homologs thereof


Reaction 3


2-ethylmalate ->3-ethylmalate


Genes


leuCD (E. coli) or homologs thereof


Reaction 4


3-ethylmalate -> 2-ethyl-3-oxosuccinate


Genes


leuB (E. coli) or homologs thereof


Reaction 5


2-ethyl-3-oxosuccinate -> 2-keto-valerate


Genes


(spontaneous)


Reaction 6


2-keto-valerate -> butrylaldehyde


Genes


kivd (Lactococcus lactis), kdcA (Lactococcus lactis), PDC1


(Saccharomyces cerevisiae), PDC5 (Saccharomyces cerevisiae),


PDC6 (Saccharomyces cerevisiae) THI3 (Saccharomyces cerevisiae),


ARO10 (Saccharomyces cerevisiae) or homologs thereof


Reaction 7


butrylaldehyde -> 1-butanol


Genes


ADH1 (Saccharomyces cerevisiae), ADH2 (Saccharomyces cerevisiae),


ADH3 (Saccharomyces cerevisiae), ADH4 (Saccharomyces cerevisiae),


ADH5 (Saccharomyces cerevisiae), ADH6 (Saccharomyces cerevisiae),


SFA1 (Saccharomyces cerevisiae) or homologs thereof
















TABLE 4





1-propanol production pathway via L-threonine















Reaction 1


L-threonine -> 2-keto-butyrate


Genes


ilvA (E. coli), tdcB (E. coli) or homologs thereof


Reaction 2


2-keto-butyrate -> propyl aldehyde


Genes


kivd (Lactococcus lactis), kdcA (Lactococcus lactis), PDC1


(Saccharomyces cerevisiae), PDC5 (Saccharomyces cerevisiae),


PDC6 (Saccharomyces cerevisiae) THI3 (Saccharomyces cerevisiae),


ARO10 (Saccharomyces cerevisiae)or homologs thereof


Reaction 3


propyl aldehyde -> 1-propanol


Genes


ADH1 (Saccharomyces cerevisiae), ADH2 (Saccharomyces cerevisiae),


ADH3 (Saccharomyces cerevisiae), ADH4 (Saccharomyces cerevisiae),


ADH5 (Saccharomyces cerevisiae), ADH6 (Saccharomyces cerevisiae),


SFA1 (Saccharomyces cerevisiae) or homologs thereof
















TABLE 5





1-propanol production pathway via pyruvate















Reaction 1


Pyruvate + Acetyl-CoA -> (R)-citramalate


Genes


cimA (Methanocaldococcus jannaschii), cimA (Leptospira interrogans)


or homologs thereof


Reaction 2


(R)-citramalate -> citraconate


Genes


leuCD (Leptospira interrogans), leuCD (E. coli) or homologs thereof


Reaction 3


citraconate -> β-methyl-D-malate


Genes


leuCD (Leptospira interrogans), leuCD (E. coli) or homologs thereof


Reaction 4


β-methyl-D-malate -> 2-keto-butyrate


Genes


leuB (Leptospira interrogans), leuB (E. coli) or homologs thereof


Reaction 5


2-keto-butyral -> butrylaldehyde


Genes


kivd (Lactococcus lactis), kdcA (Lactococcus lactis), PDC1


(Saccharomyces cerevisiae), PDC5 (Saccharomyces cerevisiae),


PDC6 (Saccharomyces cerevisiae) THI3 (Saccharomyces cerevisiae),


ARO10 (Saccharomyces cerevisiae)or homologs thereof


Reaction 7


butrylaldehyde -> 1-butanol


Genes


ADH1 (Saccharomyces cerevisiae), ADH2 (Saccharomyces cerevisiae),


ADH3 (Saccharomyces cerevisiae), ADH4 (Saccharomyces cerevisiae),


ADH5 (Saccharomyces cerevisiae), ADH6 (Saccharomyces cerevisiae),


SFA1 (Saccharomyces cerevisiae) or homologs thereof
















TABLE 6





2-methyl-1-butanol production pathway (via L-threonine)
















Reaction 1



L-threonine -> 2-keto-butyrate



Genes



ilvA (E. coli), tdcB (E. coli) or homologs thereof



Reaction 2



2-keto-butyrate -> 2-aceto-2-hydroxy-butyrate



Genes



ilvHI (E. coli), ilvNB (E. coli), ilvGM (E. coli), alsS (Bacillus subtilis)



or homologs thereof



Reaction 3



2-aceto-2-hydroxy-butyrate -> 2,3-dihydroxy-3-methylvalerate



Genes



ilvC (E. coli) or homologs thereof



Reaction 4



2,3-dihydroxy-3-methylvalerate -> 2-keto-3-methyl-valerate



Genes



ilvD (E. coli) or homologs thereof



Reaction 5



2-keto-3-methyl-valerate -> 2-methylbutyraldehyde



Genes



kivd (Lactococcus lactis), kdcA (Lactococcus lactis), PDC1



(Saccharomyces cerevisiae), PDC5 (Saccharomyces cerevisiae),



PDC6 (Saccharomyces cerevisiae) THI3 (Saccharomyces cerevisiae),



ARO10 (Saccharomyces cerevisiae)or homologs thereof



Reaction 6



2-methylbutyraldehyde -> 2-methyl-1-butanol



Genes



ADH1 (Saccharomyces cerevisiae), ADH2 (Saccharomyces cerevisiae),



ADH3 (Saccharomyces cerevisiae), ADH4 (Saccharomyces cerevisiae),



ADH5 (Saccharomyces cerevisiae), ADH6 (Saccharomyces cerevisiae),



SFA1 (Saccharomyces cerevisiae) or homologs thereof
















TABLE 7





3-methyl-1-butanol production pathway (via pyruvate)















Reaction 1


pyruvate -> 2-acetolactate


Gene


ilvHI (E. coli), ilvNB (E. coli), ilvGM (E. coli), alsS (Bacillus subtilis)


or homologs thereof


Reaction 2


2-acetolactate -> 2,3-dihydroxy-isovalerate


Genes


ilvC (E. coli) or homologs thereof


Reaction 3


2,3-dihydroxy-isovalerate -> 2-keto-isovalerate


Genes


ilvD (E. coli) or homologs thereof


Reaction 4


2-keto-isovalerate -> 2-isopropylmalate


Genes


leuA (E. coli) or homologs thereof


Reaction 5


2-isopropylmalate -> 3-isopropylmalate


Genes


leuCD (E. coli) or homologs thereof


Reaction 6


3-isopropylmalate -> 2-isopropyl-3-oxosuccinate


Genes


leuB (E. coli) or homologs thereof


Reaction 7


2-isopropyl-3-oxosuccinate -> 2-ketoisocaproate


Gene


(spontaneous)


Reaction 8


2-ketoisocaproate -> 3-methylbutyraldehyde


Genes


kivd (Lactococcus lactis), kdcA (Lactococcus lactis), PDC1


(Saccharomyces cerevisiae), PDC5 (Saccharomyces cerevisiae),


PDC6 (Saccharomyces cerevisiae) THI3 (Saccharomyces cerevisiae),


ARO10 (Saccharomyces cerevisiae)or homologs thereof


Reaction 9


3-methylbutyraldehyde -> 3-methyl-1-butanol


Genes


ADH1 (Saccharomyces cerevisiae), ADH2 (Saccharomyces cerevisiae),


ADH3 (Saccharomyces cerevisiae), ADH4 (Saccharomyces cerevisiae),


ADH5 (Saccharomyces cerevisiae), ADH6 (Saccharomyces cerevisiae),


SFA1 (Saccharomyces cerevisiae) or homologs thereof
















TABLE 8





phenyl-ethanol production pathway (via chorismate)
















Reaction 1



chorismate -> prephenate



Genes



tyrA (E. coli), pheA (E. coli) or homologs thereof



Reaction 2



prephenate -> phenylpyruvate



Genes



pheA (E. coli) or homologs thereof



Reaction 3



phenylpyruvate -> phenylaldehyde



Genes



kivd (Lactococcus lactis), kdcA (Lactococcus lactis), PDC1



(Saccharomyces cerevisiae), PDC5 (Saccharomyces cerevisiae),



PDC6 (Saccharomyces cerevisiae) THI3 (Saccharomyces cerevisiae),



ARO10 (Saccharomyces cerevisiae)or homologs thereof



Reaction 4



phenylaldehyde -> 2-phenylethanol



Genes



ADH1 (Saccharomyces cerevisiae), ADH2 (Saccharomyces cerevisiae),



ADH3(Saccharomyces cerevisiae), ADH4 (Saccharomyces cerevisiae),



ADH5(Saccharomyces cerevisiae), ADH6 (Saccharomyces cerevisiae),



SFA1 (Saccharomyces cerevisiae) or homologs thereof









The disclosure provides accession numbers for various genes, homologs and variants useful in the generation of recombinant microorganism described herein. It is to be understood that homologs and variants described herein are exemplary and non-limiting. Additional homologs, variants and sequences are available to those of skill in the art using various databases including, for example, the National Center for Biotechnology Information (NCBI) access to which is available on the World-Wide-Web.
Ethanol Dehydrogenase (also referred to as Aldehyde-alcohol dehydrogenase) is encoded in E. coli by adhE. adhE comprises three activities: alcohol dehydrogenase (ADH); acetaldehyde/acetyl-CoA dehydrogenase (ACDH); pyruvate-formate-lyase deactivase (PFL deactivase); PFL deactivase activity catalyzes the quenching of the pyruvate-formate-lyase catalyst in an iron, NAD, and CoA dependent reaction. Homologs are known in the art (see, e.g., aldehyde-alcohol dehydrogenase (Polytomella sp. Pringsheim 198.80) gi|40644910|emb|CAD42653.2|(406-44910); aldehyde-alcohol dehydrogenase (Clostridium botulinum A str. ATCC 3502) gi|148378348|ref|YP_001252889.1|(148378348); aldehyde-alcohol dehydrogenase (Yersinia pestis CO92) gi|16122410|ref|NP_405723.1|(16122410); aldehyde-alcohol dehydrogenase (Yersinia pseudotuberculosis IP 32953) gi|51596429|ref|YP_070620.1|(51596429); aldehyde-alcohol dehydrogenase (Yersinia pestis CO92) gi|115347889|emb|CAL20810.1|(115347889); aldehyde-alcohol dehydrogenase (Yersinia pseudotuberculosis IP 32953) gi|51589711|emb|CAH21341.1|(51589711); Aldehyde-alcohol dehydrogenase (Escherichia coli CFT073) gi|26107972|gb℄AAN80172.1|AE016760_31 (26107972); aldehyde-alcohol dehydrogenase (Yersinia pestis biovar Microtus str. 91001) gi|45441777|ref|NP_993316.1|(45441777); aldehyde-alcohol dehydrogenase (Yersinia pestis biovar Microtus str. 91001) gi|45436639|gb|AAS62193.1|(45436639); aldehyde-alcohol dehydrogenase (Clostridium perfringens ATCC 13124) gi|110798574|ref|YP_697219.1|(110798574); aldehyde-alcohol dehydrogenase (Shewanella oneidensis MR-1)gi|24373696|ref|NP_717739.1|(24373696); aldehyde-alcohol dehydrogenase (Clostridium botulinum A str. ATCC 19397) gi|153932445|ref|YP_001362747.1|(153932445); aldehyde-alcohol dehydrogenase (Yersinia pestis biovar Antigua str. E1979001) gi|165991833|gb|EDR44134.1|(165991833); aldehyde-alcohol dehydrogenase (Clostridium botulinum A str. Hall) gi|153937530|ref|YP_001386298.1|(153937530); aldehyde-alcohol dehydrogenase (Clostridium perfringens ATCC 13124) gi|110673221|gb|ABG82208.1|(110673221); aldehyde-alcohol dehydrogenase (Clostridium botulinum A str. Hall) gi|152933444|gb|ABS38943.1|(152933444); aldehyde-alcohol dehydrogenase (Yersinia pestis biovar Orientalis str. F1991016) gi|165920640|gb|EDR37888.1|(165920640); aldehyde-alcohol dehydrogenase (Yersinia pestis biovar Orientalis str. IP275)gi|165913933|gb|EDR32551.1|(165913933); aldehyde-alcohol dehydrogenase (Yersinia pestis Angola) gi|162419116|ref|YP_001606617.1|(162419116); aldehyde-alcohol dehydrogenase (Clostridium botulinum F str. Langeland) gi|153940830|ref|YP_001389712.1|(153940830); aldehyde-alcohol dehydrogenase (Escherichia coli HS) gi|157160746|ref|YP_001458064.1|(157160746); aldehyde-alcohol dehydrogenase (Escherichia coli E24377A) gi|157155679|ref|YP_001462491.1|(157155679); aldehyde-alcohol dehydrogenase (Yersinia enterocolitica subsp. enterocolitica 8081) gi|123442494|ref|YP_001006472.1|(123442494); aldehyde-alcohol dehydrogenase (Synechococcus sp. JA-3-3Ab) gi|86605191|ref|YP_473954.1|(86605191); aldehyde-alcohol dehydrogenase (Listeria monocytogenes str. 4b F2365) gi|46907864|ref|YP_014253.1|(46907864); aldehyde-alcohol dehydrogenase (Enterococcus faecalis V583) gi|29375484|ref|NP_814638.1|(29375484); aldehyde-alcohol dehydrogenase (Streptococcus agalactiae 2603V/R) gi|22536238|ref|NP_687089.1|(22536238); aldehyde-alcohol dehydrogenase (Clostridium botulinum A str. ATCC 19397) gi|152928489|gb|ABS33989.1|(152928489); aldehyde-alcohol dehydrogenase (Escherichia coli E24377A) gi|157077709|gb|ABV17417.1|(157077709); aldehyde-alcohol dehydrogenase (Escherichia coli HS) gi|157066426|gb|ABV05681.1|(157066426); aldehyde-alcohol dehydrogenase (Clostridium botulinum F str. Langeland) gi|152936726|gb|ABS42224.1|(152936726); aldehyde-alcohol dehydrogenase (Yersinia pestis CA88-4125) gi|149292312|gb|EDM42386.1|(149292312); aldehyde-alcohol dehydrogenase (Yersinia enterocolitica subsp. enterocolitica 8081) gi|122089455|emb|CAL12303.1|(122089455); aldehyde-alcohol dehydrogenase (Chlamydomonas reinhardtii) gi|92084840|emb|CAF04128.1|(92084840); aldehyde-alcohol dehydrogenase (Synechococcus sp. JA-3-3Ab) gi|88553733|gb|ABC98691.1|(86553733); aldehyde-alcohol dehydrogenase (Shewanella oneidensis MR-1) gi|24348056|gb|AAN55183.1|AE015655_9 (24348056); aldehyde-alcohol dehydrogenase (Enterococcus faecalis V583) gi|29342944|gb|AA080708.1|(29342944); aldehyde-alcohol dehydrogenase (Listeria monocytogenes str. 4b F2365) gi|46881133|gb|AAT04430.1|(46881133); aldehyde-alcohol dehydrogenase (Listeria monocytogenes str. 1/2a F6854) gi|47097587|ref|ZP_00235115.1|(47097587); aldehyde-alcohol dehydrogenase (Listeria monocytogenes str. 4b H7858) gi|47094265|ref|ZP_00231973.1|(47094265); aldehyde-alcohol dehydrogenase (Listeria monocytogenes str. 4b H7858) gi|47017355|gb|EAL08180.1|(47017355); aldehyde-alcohol dehydrogenase (Listeria monocytogenes str. 1/2a F6854) gi|47014034|gb|EAL05039.1|(47014034); aldehyde-alcohol dehydrogenase (Streptococcus agalactiae 2603V/R) gi|22533058|gb|AAM98961.1|AE014194_6 (22533058)p; aldehyde-alcohol dehydrogenase (Yersinia pestis biovar Antigua str. E1979001) gi|166009278|ref|ZP_02230176.1|(166009278); aldehyde-alcohol dehydrogenase (Yersinia pestis biovar Orientalis str. IP275) gi|165938272|ref|ZP_02226831.1|(165938272); aldehyde-alcohol dehydrogenase (Yersinia pestis biovar Orientalis str. F1991016) gi|165927374|ref|ZP_02223206.1|(165927374); aldehyde-alcohol dehydrogenase (Yersinia pestis Angola) gi|162351931|gb|ABX85879.1|(162351931); aldehyde-alcohol dehydrogenase (Yersinia pseudotuberculosis IP 31758) gi|153949366|ref|YP_001400938.1|(153949366); aldehyde-alcohol dehydrogenase (Yersinia pseudotuberculosis IP 31758) gi|152960861|gb|ABS48322.1|(152960861); aldehyde-alcohol dehydrogenase (Yersinia pestis CA88-4125) gi|149365899|ref|ZP_01887934.1|(149365899); Acetaldehyde dehydrogenase (acetylating) (Escherichia coli CFT073) gi|26247570|ref|NP_753610.1|(26247570); aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase; acetaldehyde dehydrogenase (acetylating) (EC 1.2.1.10) (acdh); pyruvate-formate-lyase deactivase (pfl deactivase)) (Clostridium botulinum A str. ATCC 3502) gi|148287832|emb|CAL81898.1|(148287832); aldehyde-alcohol dehydrogenase (Includes: Alcohol dehydrogenase (ADH); Acetaldehyde dehydrogenase (acetylating) (ACDH); Pyruvate-formate-lyase deactivase (PFL deactivase)) gi|71152980|sp|P0A9Q7.21ADHE_ECOLI (71152980); aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase and acetaldehyde dehydrogenase, and pyruvate-formate-lyase deactivase (Erwinia carotovora subsp. atroseptica SCR11043) gi|50121254|ref|YP_050421.1|(50121254); aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase and acetaldehyde dehydrogenase, and pyruvate-formate-lyase deactivase (Erwinia carotovora subsp. atroseptica SCR11043) gi|49611780|emb|CAG75229.1|(49611780); Aldehyde-alcohol dehydrogenase (Includes: Alcohol dehydrogenase (ADH); Acetaldehyde dehydrogenase (acetylating) (ACDH)) gi|19858620|sp|P33744.3|ADHE_CLOAB (19858620); Aldehyde-alcohol dehydrogenase (Includes: Alcohol dehydrogenase (ADH); Acetaldehyde dehydrogenase (acetylating) (ACDH); Pyruvate-formate-lyase deactivase (PFL deactivase)) gi|71152683|sp|P0A9Q8.21ADHE_ECO5∂(71152683); aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase; acetaldehyde dehydrogenase (acetylating); pyruvate-formate-lyase deactivase (Clostridium difficile 630) gi|126697906|ref|YP_001086803.1|(126697906); aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase; acetaldehyde dehydrogenase (acetylating); pyruvate-formate-lyase deactivase (Clostridium difficile 630) gi|115249343|emb|CAJ67156.1|(115249343); Aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (acetylating) (ACDH); pyruvate-formate-lyase deactivase (PFL deactivase)) (Photorhabdus luminescens subsp. laumondii TTO1) gi|37526388|ref|NP_929732.1|(37526388); aldehyde-alcohol dehydrogenase 2 (includes: alcohol dehydrogenase; acetaldehyde dehydrogenase) (Streptococcus pyogenes str. Manfredo) gi|134271169|emb|CAM29381.1|(134271169); Aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (acetylating) (ACDH); pyruvate-formate-lyase deactivase (PFL deactivase)) (Photorhabdus luminescens subsp. laumondii TTO1) gi|36785819|emb|CAE14870.1|(36785819); aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase and pyruvate-formate-lyase deactivase (Clostridium difficile 630) gi|126700586|ref|YP_001089483.1|(126700586); aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase and pyruvate-formate-lyase deactivase (Clostridium difficile 630) gi|115252023|emb|CAJ69859.1|(115252023); aldehyde-alcohol dehydrogenase 2 (Streptococcus pyogenes str. Manfredo) gi|139472923|ref|YP_001127638.1|(139472923); aldehyde-alcohol dehydrogenase E (Clostridium perfringens str. 13) gi|18311513|ref|NP_563447.1|(18311513); aldehyde-alcohol dehydrogenase E (Clostridium perfringens str. 13) gi|18146197|dbj|BAB82237.1|(18146197); Aldehyde-alcohol dehydrogenase, ADHE1 (Clostridium acetobutylicum ATCC 824) gi|15004739|ref|NP_149199.1|(15004739); Aldehyde-alcohol dehydrogenase, ADHE1 (Clostridium acetobutylicum ATCC 824) gi|14994351|gb|AAK76781.1|AE001438_34 (14994351); Aldehyde-alcohol dehydrogenase 2 (Includes: Alcohol dehydrogenase (ADH); acetaldehyde/acetyl-CoA dehydrogenase (ACDH)) gi|2492737|sp|Q24803.1|ADH2_ENTHI (2492737); alcohol dehydrogenase (Salmonella enterica subsp. enterica serovar Typhi str. CT18) gi|16760134|ref|NP_455751.1|(16760134); and alcohol dehydrogenase (Salmonella enterica subsp. enterica serovar Typhi) gi|16502428|emb|CAD08384.1|(16502428)), each sequence associated with the accession number is incorporated herein by reference in its entirety.
Lactate Dehydrogenase (also referred to as D-lactate dehydrogenase and fermentive dehydrognase) is encoded in E. coli by ldhA and catalyzes the NADH-dependent conversion of pyruvate to D-lactate. ldhA homologs and variants are known. In fact there are currently 1664 bacterial lactate dehydrogenases available through NCBI. For example, such homologs and variants include, for example, D-lactate dehydrogenase (D-LDH) (Fermentative lactate dehydrogenase) gi|1730102|sp|P52643.1|LDHD_ECOLI (1730102); D-lactate dehydrogenase gi|1049265|gb|AAB51772.1|(1049265); D-lactate dehydrogenase (Escherichia coli APEC O1) gi|1176236551ref|YP_852568.1|(117623655); D-lactate dehydrogenase (Escherichia coli CFT073) gi|26247689|ref|NP_753729.1|(26247689); D-lactate dehydrogenase (Escherichia coli O157:H7 EDL933) gi|15801748|ref|NP_287766.1|(15801748); D-lactate dehydrogenase (Escherichia coli APEC 01) gi|115512779|gb|ABJ00854.1|(115512779); D-lactate dehydrogenase (Escherichia coli CFT073) gi|26108091|gb|AAN80291.1|AE016760_150 (26108091); fermentative D-lactate dehydrogenase, NAD-dependent (Escherichia coli K12) gi|16129341|ref|NP 415898.1|(16129341); fermentative D-lactate dehydrogenase, NAD-dependent (Escherichia coli UTI89) gi|91210646|ref|YP_540632.1|(91210646); fermentative D-lactate dehydrogenase, NAD-dependent (Escherichia coli K12) gi|1787645|gb|AAC74462.1|(1787645); fermentative D-lactate dehydrogenase, NAD-dependent (Escherichia coli W3110) gi|89108227|ref|AP_002007.1|(89108227); fermentative D-lactate dehydrogenase, NAD-dependent (Escherichia coli W3110) gi|1742259|dbj|BAA14990.1|(1742259); fermentative D-lactate dehydrogenase, NAD-dependent (Escherichia coli UTI89) gi|91072220|gb|ABE07101.1|(91072220); fermentative D-lactate dehydrogenase, NAD-dependent (Escherichia coli O157:H7 EDL933) gi|12515320|gb|AAG56380.1|AE005366_6 (12515320); fermentative D-lactate dehydrogenase (Escherichia coli O157:H7 str. Sakai) gi|13361468|dbj|BAB35425.1|(13361468); COG1052: Lactate dehydrogenase and related dehydrogenases (Escherichia coli 101-1) gi|83588593|ref|ZP_00927217.1|(83588593); COG1052: Lactate dehydrogenase and related dehydrogenases (Escherichia coli 53638) gi|75515985|ref|ZP_00738103.1|(75515985); COG1052: Lactate dehydrogenase and related dehydrogenases (Escherichia coli E22) gi|75260157|ref|ZP_00731425.1|(75260157); COG1052: Lactate dehydrogenase and related dehydrogenases (Escherichia coli F11) gi|75242656|ref|ZP_00726400.1|(75242656); COG1052: Lactate dehydrogenase and related dehydrogenases (Escherichia coli E110019) gi|75237491|ref|ZP_00721524.1|(75237491); COG1052: Lactate dehydrogenase and related dehydrogenases (Escherichia coli B7A) gi|75231601|ref|ZP_00717959.1|(75231601); and COG1052: Lactate dehydrogenase and related dehydrogenases (Escherichia coli B171) gi|75211308|ref|ZP_00711407.1|(75211308), each sequence associated with the accession number is incorporated herein by reference in its entirety.
Two membrane-bound, FAD-containing enzymes are responsible for the catalysis of fumarate and succinate interconversion; the fumarate reductase is used in anaerobic growth, and the succinate dehydrogenase is used in aerobic growth. Fumarate reductase comprises multiple subunits (e.g., frdA, B, and C in E. coli). Modification of any one of the subunits can result in the desired activity herein. For example, a knockout of frdB, frdC or frdBC is useful in the methods of the disclosure. Frd homologs and variants are known. For example, homologs and variants includes, for example, Fumarate reductase subunit D (Fumarate reductase 13 kDa hydrophobic protein) gi|67463543|sp|P0A8Q3.1|FRDD_ECOLI (67463543); Fumarate reductase subunit C (Fumarate reductase 15 kDa hydrophobic protein) gi|1346037|sp|P20923.2|FRDC_PROVU (1346037); Fumarate reductase subunit D (Fumarate reductase 13 kDa hydrophobic protein) gi|120499|sp|P20924.1|FRDD_PROVU (120499); Fumarate reductase subunit C (Fumarate reductase 15 kDa hydrophobic protein) gi|67463538|sp|P0A8Q0.1|FRDC_ECOLI (67463538); fumarate reductase iron-sulfur subunit (Escherichia coli) gi|145264|gb|AAA23438.1|(145264); fumarate reductase flavoprotein subunit (Escherichia coli) gi|145263|gb|AAA23437.1|(145263); Fumarate reductase flavoprotein subunit gi|37538290|sp|P17412.31FRDA_WOLSU (37538290); Fumarate reductase flavoprotein subunit gi|120489|sp|P00363.3|FRDA_ECOLI (120489); Fumarate reductase flavoprotein subunit gi|120490|sp|P20922.1|FRDA_PROVU (120490); Fumarate reductase flavoprotein subunit precursor (Flavocytochrome c) (Flavocytochrome c3) (Fcc3) gi|119370087|sp|Q07W07.2|FRDA_SHEFN (119370087); Fumarate reductase iron-sulfur subunit gi|81175308|sp|P0AC47.2|FRDB_ECOLI (81175308); Fumarate reductase flavoprotein subunit (Flavocytochrome c) (Flavocytochrome c3) (Fcc3) gi|119370088|sp|P0C278.1|FRDA_SHEFR (119370088); Frd operon uncharacterized protein C gi|140663|sp|P20927.1|YFRC_PROVU (140663); Frd operon probable iron-sulfur subunit A gi|140661|sp|P20925.1|YFRA_PROVU (140661); Fumarate reductase iron-sulfur subunit gi|120493|sp|P20921.2|FRDB_PROVU (120493); Fumarate reductase flavoprotein subunit gi|2494617|sp|O06913.2|FRDA_HELPY (2494617); Fumarate reductase flavoprotein subunit precursor (Iron(III)-induced flavocytochrome C3) (Ifc3) gi|13878499|sp|Q9Z4P0.1|FRD2_SHEFN (13878499); Fumarate reductase flavoprotein subunit gi|54041009|sp|P64174.1|FRDA_MYCTU (54041009); Fumarate reductase flavoprotein subunit gi|54037132|sp|P64175.1|FRDA_MYCBO (54037132); Fumarate reductase flavoprotein subunit gi|12230114|sp|Q9ZMP0.1|FRDA_HELPJ (12230114); Fumarate reductase flavoprotein subunit gi|1169737|sp|P44894.1|FRDA_HAEIN (1169737); fumarate reductase flavoprotein subunit (Wolinella succinogenes) gi|13160058|emb|CAA04214.2|(13160058); Fumarate reductase flavoprotein subunit precursor (Flavocytochrome c) (FL cyt) gi|25452947|sp|P83223.21FRDA_SHEON (25452947); fumarate reductase iron-sulfur subunit (Wolinella succinogenes) gi|2282000|emb|CAA04215.1|(2282000); and fumarate reductase cytochrome b subunit (Wolinella succinogenes) gi|2281998|emb|CAA04213.1|(2281998), each sequence associated with the accession number is incorporated herein by reference in its entirety.
Acetate kinase is encoded in E. coli by ackA. AckA is involved in conversion of acetyl-coA to acetate. Specifically, ackA catalyzes the conversion of acetyl-phophate to acetate. AckA homologs and variants are known. The NCBI database list approximately 1450 polypeptides as bacterial acetate kinases. For example, such homologs and variants include acetate kinase (Streptomyces coelicolor A3(2)) gi|21223784|ref|NP_629563.1|(21223784); acetate kinase (Streptomyces coelicolor A3(2)) gi|6808417|emb|CAB70654.1|(6808417); acetate kinase (Streptococcus pyogenes M1 GAS) gi|15674332|ref|NP_268506.1|(15674332); acetate kinase (Campylobacter jejuni subsp. jejuni NCTC 11168) gi|15792038|ref|NP_281861.1|(15792038); acetate kinase (Streptococcus pyogenes M1 GAS) gi|13621416|gb|AAK33227.1|(13621416); acetate kinase (Rhodopirellula baltica SH 1) gi|32476009|ref|NP_869003.1|(32476009); acetate kinase (Rhodopirellula baltica SH 1) gi|32472045|ref|NP_865039.1|(32472045); acetate kinase (Campylobacter jejuni subsp. jejuni NCTC 11168) gi|112360034|emb|CAL34826.1|(112360034); acetate kinase (Rhodopirellula baltica SH 1) gi|32446553|emb|CAD76388.1|(32446553); acetate kinase (Rhodopirellula baltica SH 1) gi|32397417|emb|CAD72723.1|(32397417); AckA (Clostridium kluyveri DSM 555) gi|153954016|ref|YP_001394781.1|(153954016); acetate kinase (Bifidobacterium longum NCC2705) gi|23465540|ref|NP_696143.1|(23465540); AckA (Clostridium kluyveri DSM 555) gi|1463468971gb|EDK33433.1|(146346897); Acetate kinase (Corynebacterium diphtheriae) gi|38200875|emb|CAE50580.1|(38200875); acetate kinase (Bifidobacterium longum NCC2705) gi|23326203|gb|AAN24779.1|(23326203); Acetate kinase (Acetokinase) gi|67462089|sp|P0A6A3.1|ACKA_ECOLI (67462089); and AckA (Bacillus licheniformis DSM 13) gi|52349315|gb|AAU41949.1|(52349315), the sequences associated with such accession numbers are incorporated herein by reference.
Phosphate acetyltransferase is encoded in E. coli by pta. PTA is involved in conversion of acetate to acetyl-CoA. Specifically, PTA catalyzes the conversion of acetyl-coA to acetyl-phosphate. PTA homologs and variants are known. There are approximately 1075 bacterial phosphate acetyltransferases available on NCBI. For example, such homologs and variants include phosphate acetyltransferase Pta (Rickettsia felis URRWXCa12) gi|67004021|gb|AAY60947.1|(67004021); phosphate acetyltransferase (Buchnera aphidicola str. Cc (Cinara cedri)) gi|116256910|gb|ABJ90592.1|(116256910); pta (Buchnera aphidicola str. Cc (Cinara cedri)) gi|116515056|ref|YP_802685.1|(116515056); pta (Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis) gi|25166135|dbj|BAC24326.1|(25166135); Pta (Pasteurella multocida subsp. multocida str. Pm70) gi|12720993|gb|AAK02789.1|(12720993); Pta (Rhodospirillum rubrum) gi|25989720|gb|AAN75024.1|(25989720); pta (Listeria welshimeri serovar 6b str. SLCC5334) gi|116742418|emb|CAK21542.1|(116742418); Pta (Mycobacterium avium subsp. paratuberculosis K-10) gi|41398816|gb|AAS06435.1|(41398816); phosphate acetyltransferase (pta) (Borrelia burgdorferi B31) gi|15594934|ref|NP_212723.1|(15594934); phosphate acetyltransferase (pta) (Borrelia burgdorferi B31) gi|2688508|gb|AAB91518.1|(2688508); phosphate acetyltransferase (pta) (Haemophilus influenzae Rd KW20) gi|1574131|gb|AAC22857.1|(1574131); Phosphate acetyltransferase Pta (Rickettsia bellii RML369-C) gi|91206026|ref|YP_538381.1|(91206026); Phosphate acetyltransferase Pta (Rickettsia bellii RML369-C) gi|91206025|ref|YP_538380.1|(91206025); phosphate acetyltransferase pta (Mycobacterium tuberculosis F11) gi|148720131|gb|ABR04756.1|(148720131); phosphate acetyltransferase pta (Mycobacterium tuberculosis str. Haarlem) gi|134148886|gb|EBA40931.1|(134148886); phosphate acetyltransferase pta (Mycobacterium tuberculosis C) gi|124599819|gb|EAY58829.1|(124599819); Phosphate acetyltransferase Pta (Rickettsia bellii RML369-C) gi|91069570|gb|ABE05292.1|(91069570); Phosphate acetyltransferase Pta (Rickettsia bellii RML369-C) gi|91069569|gb|ABE05291.1|(91069569); phosphate acetyltransferase (pta) (Treponema pallidum subsp. pallidum str. Nichols) gi|15639088|ref|NP_218534.1|(15639088); and phosphate acetyltransferase (pta) (Treponema pallidum subsp. pallidum str. Nichols) gi13322356|gb|AAC65090.1|(3322356), each sequence associated with the accession number is incorporated herein by reference in its entirety.
Pyruvate-formate lyase (Formate acetylytransferase) is an enzyme that catalyzes the conversion of pyruvate to acetly-coA and formate. It is induced by pfl-activating enzyme under anaerobic conditions by generation of an organic free radical and decreases significantly during phosphate limitation. Formate acetylytransferase is encoded in E. coli by pflB. PFLB homologs and variants are known. For examples, such homologs and variants include, for example, Formate acetyltransferase 1 (Pyruvate formate-lyase 1) gi|129879|sp|P09373.2|PFLB_ECOLI (129879); formate acetyltransferase 1 (Yersinia pestis C092) gi|16121663|ref|NP_404976.1|(16121663); formate acetyltransferase 1 (Yersinia pseudotuberculosis IP 32953) gi|51595748|ref|YP_069939.1|(51595748); formate acetyltransferase 1 (Yersinia pestis biovar Microtus str. 91001) gi|45441037|ref|NP_992576.1|(454-41037); formate acetyltransferase 1 (Yersinia pestis C092) gi|115347142|emb|CAL20035.1|(115347142); formate acetyltransferase 1 (Yersinia pestis biovar Microtus str. 91001) gi|45435896|gb|AAS61453.1|(45435896); formate acetyltransferase 1 (Yersinia pseudotuberculosis IP 32953) gi|51589030|emb|CAH20648.1|(51589030); formate acetyltransferase 1 (Salmonella enterica subsp. enterica serovar Typhi str. CT18) gi|16759843|ref|NP_455-460.1|(16759843); formate acetyltransferase 1 (Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150) gi|56413977|ref|YP_151052.1|(56413977); formate acetyltransferase 1 (Salmonella enterica subsp. enterica serovar Typhi) gi|16502136|emb|CAD05373.1|(16502136); formate acetyltransferase 1 (Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150) gi|56128234|gb|AAV77740.1|(56128234); formate acetyltransferase 1 (Shigella dysenteriae Sd197) gi|82777577|ref|YP_403926.1|(82777577); formate acetyltransferase 1 (Shigella flexneri 2a str. 2457T) gi|30062438|ref|NP_836609.1|(30062438); formate acetyltransferase 1 (Shigella flexneri 2a str. 2457T) gi|30040684|gb|AAP16415.1|(30040684); formate acetyltransferase 1 (Shigella flexneri 5 str. 8401) gi|110614459|gb|ABF03126.1|(110614459); formate acetyltransferase 1 (Shigella dysenteriae Sd197) gi|81241725|gb|ABB62435.1|(81241725); formate acetyltransferase 1 (Escherichia coli O157:H7 EDL933) gi|12514066|gb|AAG55388.1|AE005279_8 (12514066); formate acetyltransferase 1 (Yersinia pestis KIM) gi|22126668|ref|NP_670091.1|(22126668); formate acetyltransferase 1 (Streptococcus agalactiae A909) gi|76787667|ref|YP_330335.1|(76787667); formate acetyltransferase 1 (Yersinia pestis KIM) gi|21959683|gb|AAM86342.1|AE013882_3 (21959683); formate acetyltransferase 1 (Streptococcus agalactiae A909) gi|76562724|gb|ABA45308.1|(76562724); formate acetyltransferase 1 (Yersinia enterocolitica subsp. enterocolitica 8081) gi|123441844|ref|YP_001005827.1|(123441844); formate acetyltransferase 1 (Shigella flexneri 5 str. 8401) gi|110804911|ref|YP_688431.1|(110804911); formate acetyltransferase 1 (Escherichia coli UTI89) gi|91210004|ref|YP_539990.1|(91210004); formate acetyltransferase 1 (Shigella boydii Sb227) gi|82544641|ref|YP_408588.1|(82544641); formate acetyltransferase 1 (Shigella sonnei Ss046) gi|74311459|ref|YP_309878.1|(74311459); formate acetyltransferase 1 (Klebsiella pneumoniae subsp. pneumoniae MGH 78578) gi|152969488|ref|YP_001334597.1|(152969488); formate acetyltransferase 1 (Salmonella enterica subsp. enterica serovar Typhi Ty2) gi|29142384|ref|NP_805726.1|(29142384) formate acetyltransferase 1 (Shigella flexneri 2a str. 301) gi|24112311|ref|NP_706821.1|(24112311); formate acetyltransferase 1 (Escherichia coli O157:H7 EDL933) gi|15800764|ref|NP_286778.1|(15800764); formate acetyltransferase 1 (Klebsiella pneumoniae subsp. pneumoniae MGH 78578) gi|150954337|gb|ABR76367.1|(150954337); formate acetyltransferase 1 (Yersinia pestis CA88-4125) gi|149366640|ref|ZP_01888674.1|(149366640); formate acetyltransferase 1 (Yersinia pestis CA88-4125) gi|149291014|gb|EDM41089.1|(149291014); formate acetyltransferase 1 (Yersinia enterocolitica subsp. enterocolitica 8081) gi|122088805|emb|CAL11611.1|(122088805); formate acetyltransferase 1 (Shigella sonnei Ss046) gi|73854936|gb|AAZ87643.1|(73854936); formate acetyltransferase 1 (Escherichia coli UTI89) gi|91071578|gb|ABE06459.1|(91071578); formate acetyltransferase 1 (Salmonella enterica subsp. enterica serovar Typhi Ty2) gi|29138014|gb|AA069575.1|(29138014); formate acetyltransferase 1 (Shigella boydii Sb227) gi|812460521gb|ABB66760.1|(81246052); formate acetyltransferase 1 (Shigella flexneri 2a str. 301) gi|24051169|gb|AAN42528.1|(24051169); formate acetyltransferase 1 (Escherichia coli O157:H7 str. Sakai) gi|13360445|dbj|BAB34409.1|(13360445); formate acetyltransferase 1 (Escherichia coli O157:H7 str. Sakai) gi|15830240|ref|NP_309013.1|(15830240); formate acetyltransferase I (pyruvate formate-lyase 1) (Photorhabdus luminescens subsp. laumondii TTO1) gi|36784986|emb|CAE13906.1|(36784986); formate acetyltransferase I (pyruvate formate-lyase 1) (Photorhabdus luminescens subsp. laumondii TTO1) gi|37525558|ref|NP_928902.1|(37525558); formate acetyltransferase (Staphylococcus aureus subsp. aureus Mu50) gi|14245993|dbj|BAB56388.1|(14245993); formate acetyltransferase (Staphylococcus aureus subsp. aureus Mu50) gi|15923216|ref|NP_370750.1|(15923216); Formate acetyltransferase (Pyruvate Formate-Lyase) gi|81706366|sp|Q7A7X6.1|PFLB_STAAN (81706366); Formate acetyltransferase (Pyruvate formate-lyase) gi|81782287|sp|Q99WZ7.1|PFLB_STAAM (81782287); Formate acetyltransferase (Pyruvate formate-lyase) gi|81704726|sp|Q7A1W9.1|PFLB_STAAW (81704726); formate acetyltransferase (Staphylococcus aureus subsp. aureus Mui) gi|156720691|dbj|BAF77108.1|(156720691); formate acetyltransferase (Erwinia carotovora subsp. atroseptica SCR11043) gi|50121521|ref|YP_050688.1|(50121521); formate acetyltransferase (Erwinia carotovora subsp. atroseptica SCR11043) gi|49612047|emb|CAG75496.1|(49612047); formate acetyltransferase (Staphylococcus aureus subsp. aureus str. Newman) gi|150373174|dbj|BAF66434.1|(150373174); formate acetyltransferase (Shewanella oneidensis MR-1) gi|24374439|ref|NP_718482.1|(24374439); formate acetyltransferase (Shewanella oneidensis MR-1) gi|24349015|gb|AAN55926.1|AE0157303 (24349015); formate acetyltransferase (Actinobacillus pleuropneumoniae serovar 3 str. JL03) gi|165976461|ref|YP_001652054.1|(165976461); formate acetyltransferase (Actinobacillus pleuropneumoniae serovar 3 str. JL03) gi|165876562|gb|ABY69610.1|(165876562); formate acetyltransferase (Staphylococcus aureus subsp. aureus MW2) gi|21203365|dbj|BAB94066.1|(21203365); formate acetyltransferase (Staphylococcus aureus subsp. aureus N315) gi|13700141|dbj|BAB41440.1|(13700141); formate acetyltransferase (Staphylococcus aureus subsp. aureus str. Newman) gi|151220374|ref|YP_001331197.1|(151220374); formate acetyltransferase (Staphylococcus aureus subsp. aureus Mu3) gi|156978556|ref|YP_001440815.1|(156978556); formate acetyltransferase (Synechococcus sp. JA-2-3B′a(2-13)) gi|86607744|ref|YP_476506.1|(86607744); formate acetyltransferase (Synechococcus sp. JA-3-3Ab) gi|86605195|ref|YP_473958.1|(86605195); formate acetyltransferase (Streptococcus pneumoniae D39) gi|116517188|ref|YP_815928.1|(116517188); formate acetyltransferase (Synechococcus sp. JA-2-3B′a(2-13)) gi|86556286|gb|ABD01243.1|(86556286); formate acetyltransferase (Synechococcus sp. JA-3-3Ab) gi|86553737|gb|ABC98695.1|(86553737); formate acetyltransferase (Clostridium novyi NT) gi|118134908|gb|ABK61952.1|(118134908); formate acetyltransferase (Staphylococcus aureus subsp. aureus MRSA252) gi|49482458|ref|YP_039682.1|(49482458); and formate acetyltransferase (Staphylococcus aureus subsp. aureus MRSA252) gi|49240587|emb|CAG39244.1|(492-40587), each sequence associated with the accession number is incorporated herein by reference in its entirety.
Alpha isopropylmalate synthase (EC 2.3.3.13, sometimes referred to a 2-isopropylmalate synthase, alpha-IPM synthetase) catalyzes the condensation of the acetyl group of acetyl-CoA with 3-methyl-2-oxobutanoate (2-oxoisovalerate) to form 3-carboxy-3-hydroxy-4-methylpentanoate (2-isopropylmalate). Alpha isopropylmalate synthase is encoded in E. coli by leuA. LeuA homologs and variants are known. For example, such homologs and variants include, for example, 2-isopropylmalate synthase (Corynebacterium glutamicum) gi1452382|emb|CAA50295.1|(452382); 2-isopropylmalate synthase (Escherichia coli K12) gi|16128068|ref|NP_414616.1|(16128068); 2-isopropylmalate synthase (Escherichia coli K12) gi11786261|gb|AAC73185.1|(1786261); 2-isopropylmalate synthase (Arabidopsis thaliana) gi|15237194|ref|NP_197692.1|(15237194); 2-isopropylmalate synthase (Arabidopsis thaliana) gi|42562149|ref|NP_173285.21(42562149); 2-isopropylmalate synthase (Arabidopsis thaliana) gi|15221125|ref|NP_177544.1|(15221125); 2-isopropylmalate synthase (Streptomyces coelicolor A3 (2)) gi|32141173|ref|NP_733575.1|(32141173); 2-isopropylmalate synthase (Rhodopirellula baltica SH 1) gi|32477692|ref|NP_870686.1|(32477692); 2-isopropylmalate synthase (Rhodopirellula baltica SH 1) gi|32448246|emb|CAD77763.1|(32448246); 2-isopropylmalate synthase (Akkermansia muciniphila ATCC BAA-835) gi|166241432|gb|EDR53404.1|(166241432); 2-isopropylmalate synthase (Herpetosiphon aurantiacus ATCC 23779) gi|159900959|ref|YP_001547206.1|(159900959); 2-isopropylmalate synthase (Dinoroseobacter shibae DFL 12) gi|159043149|ref|YP_001531943.1|(159043149); 2-isopropylmalate synthase (Salinispora arenicola CNS-205) gi|159035933|ref|YP_001535186.1|(159035933); 2-isopropylmalate synthase (Clavibacter michiganensis subsp. michiganensis NCPPB 382) gi|148272757|ref|YP_001222318.1|(148272757); 2-isopropylmalate synthase (Escherichia coli B) gi|124530643|ref|ZP_01701227.1|(124530643); 2-isopropylmalate synthase (Escherichia coli C str. ATCC 8739) gi|124499067|gb|EAY46563.1|(124499067); 2-isopropylmalate synthase (Bordetella pertussis Tohama I) gi|33591386|ref|NP_879030.1|(33591386); 2-isopropylmalate synthase (Polynucleobacter necessarius STIR1) gi|164564063|ref|ZP_02209880.1|(164564063); 2-isopropylmalate synthase (Polynucleobacter necessarius STIR1) gi|164506789|gb|EDQ94990.1|(164506789); and 2-isopropylmalate synthase (Bacillus weihenstephanensis KBAB4) gi|163939313|ref|YP_001644197.1|(163939313), any sequence associated with the accession number is incorporated herein by reference in its entirety.
BCAA aminotransferases catalyze the formation of branched chain amino acids (BCAA). A number of such aminotranferases are known and are exemplified by ilvE in E. coli. Exemplary homologs and variants include sequences designated by the following accession numbers: ilvE (Microcystis aeruginosa PCC 7806) gi|159026756|emb|CA086637.1|(159026756); IlvE (Escherichia coli) gi|87117962|gb|ABD20288.1|(87117962); IlvE (Escherichia coli) gi167117960|gb|ABD20287.1|(87117960); IlvE (Escherichia coli) gi|87117958|gb|ABD20286.1|(87117958); IlvE (Shigella flexneri) gi|87117956|gb|ABD20285.1|(87117956); IlvE (Shigella flexneri) gi|87117954|gb|ABD20284.1|(87117954); IlvE (Shigella flexneri) gi|87117952|gb|ABD20283.1|(87117952); IlvE (Shigella flexneri) gi|87117950|gb|ABD20282.1|(87117950); IlvE (Shigella flexneri) gi|87117948|gb|ABD20281.1|(87117948); IlvE (Shigella flexneri) gi|87117946|gb|ABD20280.1|(87117946); IlvE (Shigella flexneri) gi|87117944|gb|ABD20279.1|(87117944); IlvE (Shigella flexneri) gi|87117942|gb|ABD20278.1|(87117942); IlvE (Shigella flexneri) gi|87117940|gb|ABD20277.1|(87117940); IlvE (Shigella flexneri) gi|87117938|gb|ABD20276.1|(87117938); IlvE (Shigella dysenteriae) gi|87117936|gb|ABD20275.1|(87117936); IlvE (Shigella dysenteriae) gi|87117934|gb|AB020274.1|(87117934); IlvE (Shigella dysenteriae) gi|87117932|gb|ABD20273.1|(87117932); IlvE (Shigella dysenteriae) gi|87117930|gb|ABD20272.1|(87117930); and IlvE (Shigella dysenteriae) gi|87117928|gb|ABD20271.1|(87117928), each sequence associated with the accession number is incorporated herein by reference.
Tyrosine aminotransferases catalyzes transamination for both dicarboxylic and aromatic amino-acid substrates. A tyrosine aminotransferase of E. coli is encoded by the gene tyrB. TyrB homologs and variants are known. For example, such homologs and variants include tyrB (Bordetella petrii) gi|163857093|ref|YP_001631391.1| (163857093); tyrB (Bordetella petrii) gi|163260821|emb|CAP43123.1| (163260821); aminotransferase gi|551844|gb|AAA24704.1| (551844); aminotransferase (Bradyrhizobium sp. BTAil) gi|146404387|gb|ABQ32893.1| (146404387); tyrosine aminotransferase TyrB (Salmonella enterica) gi|4775574|emb|CAB40973.21 (4775574); tyrosine aminotransferase (Salmonella typhimurium LT2) gi|16422806|gb|AAL23072.1| (16422806); and tyrosine aminotransferase gi|148085|gb|AAA24703.1| (148085), each sequence of which is incorporated herein by reference.
Pyruvate oxidase catalyzes the conversion of pyruvate to acetate and CO2. In E. coli, pyruvate oxidase is encoded by poxB. PoxB and homologs and variants thereof include, for example, pyruvate oxidase; PoxB (Escherichia coli) gi|685128|gb|AAB31180.1||bbm|348451|bbs|154716 (685128); PoxB (Pseudomonas fluorescens) gi|32815820|gb|AAP88293.1|(32815820); poxB (Escherichia coli) gi|25269169|emb|CAD57486.1|(25269169); pyruvate dehydrogenase (Salmonella enterica subsp. enterica serovar Typhi) gi|16502101|emb|CAD05337.1|(16502101); pyruvate oxidase (Lactobacillus plantarum) gi|41691702|gb|AAS10156.1| (41691702); pyruvate dehydrogenase (Bradyrhizobium japonicum) gi|20257167|gb|AAM12352.1|(20257167); pyruvate dehydrogenase (Yersinia pestis KIM) gi|22126698|ref|NP_670121.1|(22126698); pyruvate dehydrogenase (cytochrome) (Yersinia pestis biovar Antigua str. B42003004) gi|166211240|ref|ZP_02237275.1|(166211240); pyruvate dehydrogenase (cytochrome) (Yersinia pestis biovar Antigua str. B42003004) gi|166207011|gb|EDR51491.1|(166207011); pyruvate dehydrogenase (Pseudomonas syringae pv. tomato str. DC3000) gi|28869703|ref|NP_792322.1|(28869703); pyruvate dehydrogenase (Salmonella typhimurium LT2) gi|167642971ref|NP_459912.1|(16764297); pyruvate dehydrogenase (Salmonella enterica subsp. enterica serovar Typhi str. CT18) gi|16759808|ref|NP_455425.1|(16759808); pyruvate dehydrogenase (cytochrome) (Coxiella burnetii Dugway 5J108-111) gi|154706110|ref|YP_001424132.1|(154706110); pyruvate dehydrogenase (Clavibacter michiganensis subsp. michiganensis NCPPB 382) gi|148273312|ref|YP_001222873.1|(148273312); pyruvate oxidase (Lactobacillus acidophilus NCFM) gi|58338213|ref|YP_194798.1|(58338213); and pyruvate dehydrogenase (Yersinia pestis CO92) gi|16121638|ref|NP_404951.1|(16121638), the sequences of each accession number are incorporated herein by reference.
L-threonine 3-dehydrogenase (EC 1.1.1.103) catalyzes the conversion of L-threonine to L-2-amino-3-oxobutanoate. The gene tdh encodes an L-threonine 3-dehydrogenase. There are approximately 700 L-threonine 3-dehydrogenases from bacterial organism recognized in NCBI. Various homologs and variants of tdh include, for example, L-threonine 3-dehydrogenase gi1135560|sp|P07913.1|TDH_ECOLI (135560); L-threonine 3-dehydrogenase gi|166227854|sp|A4TSC6.1|TDH_YERPP (166227854); L-threonine 3-dehydrogenase gi|166227853|sp|A1JHX8.1|TDH_YERE8 (166227853); L-threonine 3-dehydrogenase gi|166227852|sp|A6UBM6.1|TDH_SINMW (166227852); L-threonine 3-dehydrogenase gi|166227851|sp|A1RE07.1|TDH_SHESW (166227851); L-threonine 3-dehydrogenase gi|166227850|sp|A0L2Q3.1|TDH_SHESA (166227850); L-threonine 3-dehydrogenase gi|166227849|sp|A4YCC5.1|TDH_SHEPC (166227849); L-threonine 3-dehydrogenase gi|166227848|sp|A3QJC8.1|TDH_SHELP (166227848); L-threonine 3-dehydrogenase gi|166227847|sp|A6WUG6.1|TDH_SHEB8 (166227847); L-threonine 3-dehydrogenase gi|166227846|sp|A3CYN0.1|TDH_SHEB5 (166227846); L-threonine 3-dehydrogenase gi|166227845|sp|A1S1Q3.1|TDH_SHEAM (166227845); L-threonine 3-dehydrogenase gi|166227844|sp|A4FND4.1|TDH_SACEN (166227844); L-threonine 3-dehydrogenase gi|166227843|sp|A1SVW5.1|TDH_PSYIN (166227843); L-threonine 3-dehydrogenase gi|166227842|sp|A5IGK7.1|TDH_LEGPC (166227842); L-threonine 3-dehydrogenase gi|166227841|sp|A6TFL2.1|TDH_KLEP7 (166227841); L-threonine 3-dehydrogenase gi|166227840|sp|A41Z92.1|TDH_FRATW (166227840); L-threonine 3-dehydrogenase gi|166227839|sp|A0Q5K3.1|TDH_FRATN (166227839); L-threonine 3-dehydrogenase gi|166227838|sp|A7NDM9.1|TDH_FRATF (166227838); L-threonine 3-dehydrogenase gi|166227837|sp|A7MID0.1|TDH_ENTS8 (166227837); and L-threonine 3-dehydrogenase gi|166227836|sp|A1AHF3.1|TDH_ECOK1 (166227836), the sequences associated with each accession number are incorporated herein by reference.
Acetohydroxy acid synthases (e.g. ilvH) and acetolactate synthases (e.g., alsS, ilvB, ilvI, ilv2, and modified forms such as ilv2Δ126) catalyze the synthesis of the branched-chain amino acids (valine, leucine, and isoleucine). IlvH encodes an acetohydroxy acid synthase in E. coli (see, e.g., acetohydroxy acid synthase AHAS III (IlvH) (Escherichia coli) gi|40846|emb|CAA38855.1|(40846), incorporated herein by reference). Acetolactate synthase can be derived from any number of organisms including Pichia stipitis (ilv2 or ilv6). The acetolactate synthase can be a modified acetolactate synthase that is optimized for cytoplasmic expression. Homologs and variants as well as operons comprising ilvH are known and include, for example, ilvH (Microcystis aeruginosa PCC 7806)gi|159026908|emb|CA089159.1|(159026908); IlvH (Bacillus amyloliquefaciens FZB42) gi|154686966|ref|YP_001422127.1|(154686966); IlvH (Bacillus amyloliquefaciens FZB42) gi|154352817|gb|ABS74896.1|(154352817); IlvH (Xenorhabdus nematophila) gi|131054140|gb|AB032787.1|(131054140); IlvH (Salmonella typhimurium) gi17631124|gb|AAF65177.1|AF117227_2 (7631124), ilvN (Listeria innocua) gi|16414606|emb|CAC97322.1|(16414606); ilvN (Listeria monocytogenes) gi|16411438|emb|CAD00063.1|(16411438); acetohydroxy acid synthase (Caulobacter crescentus) gi1408939|gb|AAA23048.1|(408939); acetohydroxy acid synthase I, small subunit (Salmonella enterica subsp. enterica serovar Typhi) gi|16504830|emb|CAD03199.1|(16504830); acetohydroxy acid synthase, small subunit (Tropheryma whipplei TWO8/27) gi|28572714|ref|NP_789494.1|(28572714); acetohydroxy acid synthase, small subunit (Tropheryma whipplei TWO8/27) gi|28410846|emb|CAD67232.1|(28410846); acetohydroxy acid synthase I, small subunit (Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150) gi|56129933|gb|AAV79439.1|(56129933); acetohydroxy acid synthase small subunit; acetohydroxy acid synthase, small subunit gi1551779|gb|AAA62430.1|(551779); acetohydroxy acid synthase I, small subunit (Salmonella enterica subsp. enterica serovat Typhi Ty2) gi|29139650|gb|AA071216.1|(29139650); acetohydroxy acid synthase small subunit (Streptomyces cinnamonensis) gi15733116|gb|AAD49432.1|AF175526_1 (5733116); acetohydroxy acid synthase large subunit; and acetohydroxy acid synthase, large subunit gi/4003341gb|AAA62429.1|(400334), the sequences associated with the accession numbers are incorporated herein by reference. Acetolactate synthase genes include alsS and ilvI. Homologs of ilvI and alsS are known and include, for example, acetolactate synthase small subunit (Bifidobacterium longum NCC2705) gi|23325489|gb|AAN24137.1|(23325489); acetolactate synthase small subunit (Geobacillus stearothermophilus) gi|19918933|gb|AAL99357.1|(19918933); acetolactate synthase (Azoarcus sp. BH72) gi|119671178|emb|CAL95091.1|(119671178); Acetolactate synthase small subunit (Corynebacterium diphtheriae) gi|38199954|emb|CAE49622.1|(38199954); acetolactate synthase (Azoarcus sp. BH72) gi|119669739|emb|CAL93652.1|(119669739); acetolactate synthase small subunit (Corynebacterium jeikeium K411) gi|68263981|emb|CAI37469:11 (68263981); acetolactate synthase small subunit (Bacillus subtilis) gi|1770067|emb|CAA99562.1|(1770067); Acetolactate synthase isozyme 1 small subunit (AHAS-I) (Acetohydroxy-acid synthase I small subunit) (ALS-I) gi|83309006|sp|P0ADF8.1|ILVN_ECOLI (83309006); acetolactate synthase large subunit (Geobacillus stearothermophilus) gi|19918932|gb|AAL99356.1|(19918932); and Acetolactate synthase, small subunit (Thermoanaerobacter tengcongensis MB4) gi|20806556|ref|NP_621727.1|(20806556), the sequences associated with the accession numbers are incorporated herein by reference. There are approximately 1120 ilvB homologs and variants listed in NCBI.
Acetohydroxy acid isomeroreductase is the second enzyme in parallel pathways for the biosynthesis of isoleucine and valine. IlvC encodes an acetohydroxy acid isomeroreductase in E. coli. Homologs and variants of ilvC are known and include, for example, acetohydroxyacid reductoisomerase (Schizosaccharomyces pombe 972h−) gi|1623123171ref|NP_001018845.21 (162312317); acetohydroxyacid reductoisomerase (Schizosaccharomyces pombe) gi|3116142|emb|CAA18891.1|(3116142); acetohydroxyacid reductoisomerase (Saccharomyces cerevisiae YJM789) gi|151940879|gb|EDN59261.1|(151940879); Ilv5p: acetohydroxyacid reductoisomerase (Saccharomyces cerevisiae) gi|609403|gb|AAB67753.1|(609403); ACL198Wp (Ashbya gossypii ATCC 10895) gi|45185490|ref|NP_983206.1|(45185490); ACL198Wp (Ashbya gossypii ATCC 10895) gi|44981208|gb|AAS51030.1|(44981208); acetohydroxy-acid isomeroreductase; Ilv5x (Saccharomyces cerevisiae) gi1957238|gb|AAB33579.1||bbm|369068|bbs|165406 (957238); acetohydroxy-acid isomeroreductase; Ilv5g (Saccharomyces cerevisiae) gi|957236|gb|AAB33578.1||bbm1369064|bbs|165405 (957236); and ketol-acid reductoisomerase (Schizosaccharomyces pombe) gi|2696654|dbj|BAA24000.1|(2696654), each sequence associated with the accession number is incorporated herein by reference. Accordingly, ketol-acid reductoisomerases are present in, for example, additional microorganisms such as the genus Pichia, Escherichia, and Corynebacterium. Such microorganism sources can be used for cloning of the ketol-acid reductoisomerase coding sequence and as a parental source of derivation of improved or modified ketol-acid reductoisomerases. For example, a modified acetohydroxyacid reductoisomerase includes the ILV5 Δ40 (KARI) derived from Pichia stipitis. 
Dihydroxy-acid dehydratases catalyzes the fourth step in the biosynthesis of isoleucine and valine, the dehydratation of 2,3-dihydroxy-isovaleic acid into alpha-ketoisovaleric acid. IlvD and ilv3 encode a dihydroxy-acid dehydratase. Homologs and variants of dihydroxy-acid dehydratases are known and include, for example, IlvD (Mycobacterium leprae) gi|2104594|emb|CAB08798.1|(2104594); dihydroxy-acid dehydratase (Tropheryma whipplei TWO8/27) gi|28410848|emb|CAD67234.1|(28410848); dihydroxy-acid dehydratase (Mycobacterium leprae) gi|13093837|emb|CAC32140.1|(13093837); dihydroxy-acid dehydratase (Rhodopirellula Baltica SH 1) gi|324478711emb|CAD77389.1|(32447871); and putative dihydroxy-acid dehydratase (Staphylococcus aureus subsp. aureus MRSA252) gi|49242408|emb|CAG41121.1|(492-42408), each sequence associated with the accession numbers are incorporated herein by reference. Modified dihydroxy-acid dehydratases can be utilized in the methods, microorganism and compositions of the disclosure. For example, ILVΔ34 derived from Pichia stipitis is useful in the methods, microorganisms and compositions of the disclosure.
2-ketoacid decarboxylases catalyze the conversion of a 2-ketoacid to the respective aldehyde. For example, 2-ketoisovalerate decarboxylase catalyzes the conversion of 2-ketoisovalerate to isobutyraldehyde. A number of 2-ketoacid decarboxylases are known and are exemplified by the pdc, pdc1, pdc5, pdc6, aro10, thI3, kdcA and kivd genes. Exemplary homologs and variants useful for the conversion of a 2-ketoacid to the respective aldehyde comprise sequences designated by the following accession numbers and identified enzymatic activity: gi|44921617|gb|AAS49166.1| branched-chain alpha-keto acid decarboxylase (Lactococcus lactis); gi|15004729|ref|NP_149189.1| Pyruvate decarboxylase (Clostridium acetobutylicum ATCC 824); gi|82749898|ref|YP_415639.11 probable pyruvate decarboxylase (Staphylococcus aureus RF122); gi|77961217|ref|ZP_00825060.1| COG3961: Pyruvate decarboxylase and related thiamine pyrophosphate-requiring enzymes (Yersinia mollaretii ATCC 43969); gi|71065418|ref|YP_264145.1| putative pyruvate decarboxylase (Psychrobacter arcticus 273-4); gi|16761331|ref|NP_456948.1| putative decarboxylase (Salmonella enterica subsp. enterica serovar Typhi str. CT18); gi|93005792|ref|YP_580229.1| Pyruvate decarboxylase (Psychrobacter cryohalolentis K5); gi|23129016|ref|ZP_00110850.1| COG3961: Pyruvate decarboxylase and related thiamine pyrophosphate-requiring enzymes (Nostoc punctiforme PCC 73102); gi|164170601gb|AAL18557.1|AF354297_1 pyruvate decarboxylase (Sarcina ventriculi); gi|15607993|ref|NP_215368.1|PROBABLE PYRUVATE OR INDOLE-3-PYRUVATE DECARBOXYLASE PDC (Mycobacterium tuberculosis H37Rv); gi|414068811ref|NP 959717.1| Pdc (Mycobacterium avium subsp. paratuberculosis K-10); gi|91779968|ref|YP_555176.1| putative pyruvate decarboxylase (Burkholderia xenovorans LB400); gi|15828161|ref|NP_302424.1| pyruvate (or indolepyruvate) decarboxylase (Mycobacterium leprae TN); gi|118616174|ref|YP_904506.1| pyruvate or indole-3-pyruvate decarboxylase Pdc (Mycobacterium ulcerans Agy99); gi|67989660|ref|NP_001018185.1| hypothetical protein SPAC3H8.01 (Schizosaccharomyces pombe 972h−); gi|21666011|gb|AAM73540.1|AF282847_1 pyruvate decarboxylase PdcB (Rhizopus oryzae); gi|69291130|ref|ZP_00619161.1| Pyruvate decarboxylase: Pyruvate decarboxylase (Kineococcus radiotolerans SRS30216); gi|66363022|ref|XP_628477.1| pyruvate decarboxylase (Cryptosporidium parvum Iowa II); gi|70981398|ref|XP_731481.1| pyruvate decarboxylase (Aspergillus fumigatus Af293); gi|121704274|ref|XP_001270401.1| pyruvate decarboxylase, putative (Aspergillus clavatus NRRL 1); gi|119467089|ref|XP_001257351.1| pyruvate decarboxylase, putative (Neosartorya fischeri NRRL 181); gi|26554143|ref|NP_758077.1| pyruvate decarboxylase (Mycoplasma penetrans HF-2); gi|21666009|gb|AAM73539.1|AF282846_1 pyruvate decarboxylase PdcA (Rhizopus oryzae).
Alcohol dehydrogenases (adh) catalyze the final step of amino acid catabolism, conversion of an aldehyde to a long chain or complex alcohol. Various adh genes are known in the art. As indicated herein adh1 homologs and variants include, for example, adh2, adh3, adh4, adh5, adh 6 and sfa1 (see, e.g., SFA (Saccharomyces cerevisiae) gi/288591|emb|CAA48161.1| (288591); the sequence associated with the accession number is incorporated herein by reference).
In one aspect, the disclosure provides a mutant CimA comprising at least about 70% identity to WT cimA from Mathanocaldococcus jannaschii (SEQ ID NO:89), wherein the mutant cimA comprises from about 9- to 22-fold higher production levels of 1-propanol and 1-butanol in an engineered pathway for their production when compared to the same pathway comprising a WT cimA. In another aspect, the mutant cimA comprises from about 1-10 mutated amino acids as compared to the WT cimA from Mathanocaldococcus jannaschii. In yet another embodiment, the mutant cimA comprises the sequence of SEQ ID NO:89 having a mutation at one or more of the following positions: Ile47, Lys435, Val441, His126 or Thr204. In yet another embodiment, the mutant cim A comprises a sequence as set forth SEQ ID NO:89 and having one or more of the following mutations: a Ile47Val, a Lys435Asn, a Val441Ala, a His126Gln or a Thr204Ala, an Arg53Ser, a Glu183Lys, a Leu253Ser, Lys265Arg, Phe349Cys, an Asp86Gly, and Asp328Val, a Glu114Val, a Leu238Ser, a Glu111Lys, a Glu121Val, a Met250Val or a Lys32Asn. In a specific embodiment, the cimA mutant comprises SEQ ID NO:89 and having a mutation at Ile47Val and one or more additional mutations as set forth above. For example, the cimA mutant can comprise SEQ ID NO:89 and an Ile47Val, His126Gln, and a Thr204Ala. The cimA mutatant comprising 147V, H126Q and T204A may further comprise one or more mutations selected from r53S, E183K, L253S, K265R, F349C, D141E, 1286V, L327H, D86G, D328V, E114V, L238S, E111K, E121V, M250V and K32N. The cimA mutant may further be truncated following residue 373 of the WT cimA. In yet another embodiment, the mutant cimA comprises a sequence as set forth in SEQ ID NO:89 and having 1-50, 1-20 or 1-10 conservative amino acid substitutions and having one or more of a Ile47Val, a His126Gln or a Thr204Ala mutation.
In one embodiment a microorganism of the disclosure can be characterized as an E. coli comprising rrnBT14DlacZWJ16 hsdR514 DaraBADAH33 DrhaBADLD78 (with F′ transduced from XL-1 blue to supply lacIq), ΔadhE, ΔldhA, ΔfrdBC, Δfnr, Δpta and ΔpflB and containing pSA55 and pSA69, wherein pSA55 is a ColE1 origin derived plasmid with kivd (Lactococcus lactis) and adh2 (Saccharomyces cerevisiae) genes under the control of the PLlacO1 and an ampicillin resistance gene and pSA69 is a p15A origin derived plasmid with alsS (Bacillus subtilis), ilvC (E. coli) and ilvD (E. coli) genes under the control of the PLlacO1 and a kanamycin resistance gene.
In another embodiment a microorganism of the disclosure can be characterized as an E. coli comprising rrnBT14DlacZWJ16 hsdR514DaraBADAH33 DrhaBADLD78 (with F′ transduced from XL-1 blue to supply lacIq), ΔmetA, Δtdh, ΔilvB, ΔilvI and ΔadhE with pCS49, pSA62 and pSA55I, wherein pSA55I comprises a ColE1 origin derived plasmid with kivd (Lactococcus lactis) and adh2 (Saccharomyces cerevisiae) genes under the control of the PLlacO1 and an ampicillin resistance gene with lad after the ampicillin resistance gene, pSA62 is a p15A origin derived plasmid with ilvA (E. coli) and leuABCD (E. coli) genes under the control of the PLlacO1 and a kanamycin resistance gene, and pCS49 is a pSC101* origin derived plasmid with thrA(fbr)BC (E. coli) genes under the control of the PLlacO1 and a spectinomycin resistance gene.
The disclosure also provides deposited microorganisms. The deposited microorganisms are exemplary only and, based upon the disclosure, one of ordinary skill in the art can modify additional parental organisms of different species or genotypes to arrive at a microorganism of the disclosure that produces n-butanol.
The disclosure provides a recombinant microorganism designated SA237 and having ATCC accession no. PTA-8945 as deposited with the ATCC on Feb. 7, 2008. The disclosure includes cultures of microorganisms comprising a population of a microorganism of ATCC accession no. PTA-8945, including mixed cultures. Also provided are polynucleotide fragments derived from ATCC accession no. PTA8945, which are useful in the preparation of a microorganism that produces alcohols for use as biofuels such as, for example, isobutanol at a yield of 0.12 to about 0.41 grams of isobutanol per gram of glucose. For examples such fragments can comprise a polynucleotide of about 1000 base pairs to several million base pairs. Also included are bioreactors comprising a population of the microorganism having ATCC accession no. PTA-8945 in the production of isobutanol or phenylethanol. One of ordinary skill in the art, using the deposited microorganism, can readily determine the sequence of the deposited organism or fragments thereof encoding any of the genes and polynucleotides described herein, including locations of knockouts or gene disruptions.
The disclosure also provides a recombinant microorganism designated CRS-BuOH23 and having ATCC accession no. PTA-8944 as deposited with the ATCC on Feb. 7, 2008. The disclosure includes cultures of microorganisms comprising a population of a microorganism of ATCC accession no. PTA-8944, including mixed cultures. Also provided are polynucleotide fragments derived from ATCC accession no. PTA-8944, which are useful in the preparation of a microorganism that produces n-butanol. For examples such fragments can comprise a polynucleotide of about 1000 base pairs to several million base pairs. Also included are bioreactors comprising a population of the microorganism having ATCC accession no. PTA-8944 in the production of n-butanol. One of ordinary skill in the art, using the deposited microorganism, can readily determine the sequence of the deposited organism or fragments thereof encoding any of the genes and polynucleotides described herein, including locations of knockouts or gene disruptions. Also provided are recombinant microorganisms comprising, for example, a mutant cimA polypeptide that produces an alcohol profile substantially equivalent or improved compared to that of an organism having accession no. PTA-8944 or PTA-8945.
It is understood that a range of microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of e.g., 1-propanol, 1-butanol, 2-methyl 1-butanol, or 3-methyl 1-butanol. It is also understood that various microorganisms can act as “sources” for genetic material encoding target enzymes suitable for use in a recombinant microorganism provided herein. The term “microorganism” includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms “microbial cells” and “microbes” are used interchangeably with the term microorganism.
The term “prokaryotes” is art recognized and refers to cells which contain no nucleus or other cell organelles. The prokaryotes are generally classified in one of two domains, the Bacteria and the Archaea. The definitive difference between organisms of the Archaea and Bacteria domains is based on fundamental differences in the nucleotide base sequence in the 16S ribosomal RNA.
The term “Archaea” refers to a categorization of organisms of the division Mendosicutes, typically found in unusual environments and distinguished from the rest of the procaryotes by several criteria, including the number of ribosomal proteins and the lack of muramic acid in cell walls. On the basis of ssrRNA analysis, the Archaea consist of two phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota. On the basis of their physiology, the Archaea can be organized into three types: methanogens (prokaryotes that produce methane); extreme halophiles (prokaryotes that live at very high concentrations of salt ((NaCl)); and extreme (hyper) thermophilus (prokaryotes that live at very high temperatures). Besides the unifying archaeal features that distinguish them from Bacteria (i.e., no murein in cell wall, ester-linked membrane lipids, etc.), these prokaryotes exhibit unique structural or biochemical attributes which adapt them to their particular habitats. The Crenarchaeota consists mainly of hyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeota contains the methanogens and extreme halophiles.
“Bacteria”, or “eubacteria”, refers to a domain of prokaryotic organisms. Bacteria include at least 11 distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic+non-photosynthetic Gram-negative bacteria (includes most “common” Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (11) Thermotoga and Thermosipho thermophiles.
“Gram-negative bacteria” include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium. 
“Gram positive bacteria” include cocci, nonsporulating rods, and sporulating rods. The genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces. 
The term “recombinant microorganism” and “recombinant host cell” are used interchangeably herein and refer to microorganisms that have been genetically modified to express or over-express endogenous polynucleotides, or to express non-endogenous sequences, such as those included in a vector, or which have a reduction in expression of an endogenous gene. The polynucleotide generally encodes a target enzyme involved in a metabolic pathway for producing a desired metabolite as described above. Accordingly, recombinant microorganisms described herein have been genetically engineered to express or over-express target enzymes not previously expressed or over-expressed by a parental microorganism. It is understood that the terms “recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism.
A “parental microorganism” refers to a cell used to generate a recombinant microorganism. The term “parental microorganism” describes a cell that occurs in nature, i.e. a “wild-type” cell that has not been genetically modified. The term “parental microorganism” also describes a cell that has been genetically modified but which does not express or over-express a target enzyme e.g., an enzyme involved in the biosynthetic pathway for the production of a desired metabolite such as 1-propanol, 1-butanol, 2-methyl 1-butanol, or 3-methyl 1-butanol. For example, a wild-type microorganism can be genetically modified to express or over express a first target enzyme such as thiolase. This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or over-express a second target enzyme e.g., hydroxybutyryl CoA dehydrogenase. In turn, the microorganism modified to express or over express e.g., thiolase and hydroxybutyryl CoA dehydrogenase can be modified to express or over express a third target enzyme e.g., crotonase. Accordingly, a parental microorganism functions as a reference cell for successive genetic modification events. Each modification event can be accomplished by introducing a nucleic acid molecule in to the reference cell. The introduction facilitates the expression or over-expression of a target enzyme. It is understood that the term “facilitates” encompasses the activation of endogenous polynucleotides encoding a target enzyme through genetic modification of e.g., a promoter sequence in a parental microorganism. It is further understood that the term “facilitates” encompasses the introduction of exogenous polynucleotides encoding a target enzyme in to a parental microorganism.
In another embodiment a method of producing a recombinant microorganism that converts a suitable carbon substrate to e.g., 1-propanol, 1-butanol, 2-methyl 1-butanol, or 3-methyl 1-butanol is provided. The method includes transforming a microorganism with one or more recombinant polynucleotides, in addition to a mutant citramalate synthase (e.g., cimA), encoding polypeptides that include, for example, acetohydroxy acid synthase (e.g., ilvIH operon), acetohydroxy acid isomeroreductase (e.g., ilvC), dihydroxy-acid dehydratase (e.g., ilvD), 2-keto-acid decarboxylase (e.g., PDC6, ARO10, THIS, kivd, or pdc), 2-isopropylmalate synthase (e.g., leuA), beta-isopropylmalate dehydrogenase (e.g., leuB), isopropylmalate isomerase (e.g., leuCD operon), threonine dehydrtase (e.g., ilvA), alpha-isopropylmalate synthase (e.g., cimA or mutant cimA), beta-isopropylmalate dehydrogenase (e.g., leuB), isopropylmalate isomerase (e.g., leuCD operon), threonine dehydratase (e.g., ilvA), acetolactate synthase (e.g., ilvMG or ilvNB), acetohydroxy acid isomeroreductase (e.g., ilvC), dihydroxy-acid dehydratase (e.g., ilvD), beta-isopropylmalate dehydrogenase (e.g., leuB), chorismate mutase P/prephenate dehydratase (e.g., pheA), chorismate mutase T/prephenate dehydrogenase (e.g., tyrA), 2-keto-acid decarboxylase (e.g., kivd, PDC6, or THIS), and alcohol dehydrogenase activity. Polynucleotides that encode enzymes useful for generating metabolites including homologs, variants, fragments, related fusion proteins, or functional equivalents thereof, are used in recombinant nucleic acid molecules that direct the expression of such polypeptides in appropriate host cells, such as bacterial or yeast cells. It is understood that the addition of sequences which do not alter the encoded activity of a polynucleotide, such as the addition of a non-functional or non-coding sequence, is a conservative variation of the basic nucleic acid. The “activity” of an enzyme is a measure of its ability to catalyze a reaction resulting in a metabolite, i.e., to “function”, and, may be expressed as the rate at which the metabolite of the reaction is produced. For example, enzyme activity can be represented as the amount of metabolite produced per unit of time or per unit of enzyme (e.g., concentration or weight), or in terms of affinity or dissociation constants.
A “protein” or “polypeptide”, which terms are used interchangeably herein, comprises one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds. An “enzyme” means any substance, composed wholly or largely of protein, that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions. The term “enzyme” can also refer to a catalytic polynucleotide (e.g., RNA or DNA). A “native” or “wild-type” protein, enzyme, polynucleotide, gene, or cell, means a protein, enzyme, polynucleotide, gene, or cell that occurs in nature.
It is understood that the polynucleotides described above include “genes” and that the nucleic acid molecules described above include “vectors” or “plasmids.” For example, a polynucleotide encoding a keto thiolase can be encoded by an atoB gene or homolog thereof, or a fadA gene or homolog thereof. Accordingly, the term “gene”, also called a “structural gene” refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions, including introns, 5′-untranslated region (UTR), and 3′-UTR, as well as the coding sequence. The term “nucleic acid” or “recombinant nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term “expression” with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein results from transcription and translation of the open reading frame sequence.
The term “operon” refers two or more genes which are transcribed as a single transcriptional unit from a common promoter. In some embodiments, the genes comprising the operon are contiguous genes. It is understood that transcription of an entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying the common promoter. Alternatively, any gene or combination of genes in an operon can be modified to alter the function or activity of the encoded polypeptide. The modification can result in an increase in the activity of the encoded polypeptide. Further, the modification can impart new activities on the encoded polypeptide. Exemplary new activities include the use of alternative substrates and/or the ability to function in alternative environmental conditions.
A “vector” is any means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are “episomes,” that is, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium.
“Transformation” refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including electroporation, microinjection, biolistics (or particle bombardment-mediated delivery), or agrobacterium mediated transformation.
Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given amino acid sequence of the disclosure. The native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with alternate amino acid sequences, and the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
The disclosure provides nucleic acid molecules in the form of recombinant DNA expression vectors or plasmids, as described in more detail below, that encode one or more target enzymes. Generally, such vectors can either replicate in the cytoplasm of the host microorganism or integrate into the chromosomal DNA of the host microorganism. In either case, the vector can be a stable vector (i.e., the vector remains present over many cell divisions, even if only with selective pressure) or a transient vector (i.e., the vector is gradually lost by host microorganisms with increasing numbers of cell divisions). The disclosure provides DNA molecules in isolated (i.e., not pure, but existing in a preparation in an abundance and/or concentration not found in nature) and purified (i.e., substantially free of contaminating materials or substantially free of materials with which the corresponding DNA would be found in nature) forms.
Provided herein are methods for the heterologous expression of one or more of the biosynthetic genes involved in 1-propanol, 1-butanol, 2-methyl 1-butanol, and/or 3-methyl 1-butanol, biosynthesis and recombinant DNA expression vectors useful in the method. Thus, included within the scope of the disclosure are recombinant expression vectors that include such nucleic acids. The term expression vector refers to a nucleic acid that can be introduced into a host microorganism or cell-free transcription and translation system. An expression vector can be maintained permanently or transiently in a microorganism, whether as part of the chromosomal or other DNA in the microorganism or in any cellular compartment, such as a replicating vector in the cytoplasm. An expression vector also comprises a promoter that drives expression of an RNA, which typically is translated into a polypeptide in the microorganism or cell extract. For efficient translation of RNA into protein, the expression vector also typically contains a ribosome-binding site sequence positioned upstream of the start codon of the coding sequence of the gene to be expressed. Other elements, such as enhancers, secretion signal sequences, transcription termination sequences, and one or more marker genes by which host microorganisms containing the vector can be identified and/or selected, may also be present in an expression vector. Selectable markers, i.e., genes that confer antibiotic resistance or sensitivity, are used and confer a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.
The various components of an expression vector can vary widely, depending on the intended use of the vector and the host cell(s) in which the vector is intended to replicate or drive expression. Expression vector components suitable for the expression of genes and maintenance of vectors in E. coli, yeast, Streptomyces, and other commonly used cells are widely known and commercially available. For example, suitable promoters for inclusion in the expression vectors of the disclosure include those that function in eukaryotic or prokaryotic host microorganisms. Promoters can comprise regulatory sequences that allow for regulation of expression relative to the growth of the host microorganism or that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus. For E. coli and certain other bacterial host cells, promoters derived from genes for biosynthetic enzymes, antibiotic-resistance conferring enzymes, and phage proteins can be used and include, for example, the galactose, lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla), bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can also be used. For E. coli expression vectors, it is useful to include an E. coli origin of replication, such as from pUC, p1P, p1, and pBR.
Thus, recombinant expression vectors contain at least one expression system, which, in turn, is composed of at least a portion of PKS and/or other biosynthetic gene coding sequences operably linked to a promoter and optionally termination sequences that operate to effect expression of the coding sequence in compatible host cells. The host cells are modified by transformation with the recombinant DNA expression vectors of the disclosure to contain the expression system sequences either as extrachromosomal elements or integrated into the chromosome.
A polynucleotide of the disclosure can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques and those procedures described in the Examples section below. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
It is also understood that an isolated nucleic acid molecule encoding a polypeptide homologous to the enzymes described herein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding the particular polypeptide, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into the polynucleotide by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In contrast to those positions where it may be desirable to make a non-conservative amino acid substitutions (see above), in some positions it is preferable to make conservative amino acid substitutions. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
In another embodiment a method for producing e.g., 1-propanol, 1-butanol, 2-methyl 1-butanol, or 3-methyl 1-butanol is provided. The method includes culturing a recombinant microorganism as provided herein in the presence of a suitable substrate and under conditions suitable for the conversion of the substrate to 1-propanol, 1-butanol, 2-methyl 1-butanol, or 3-methyl 1-butanol. The alcohol produced by a microorganism provided herein can be detected by any method known to the skilled artisan. Such methods include mass spectrometry as described in more detail below and as shown in FIG. 6. Culture conditions suitable for the growth and maintenance of a recombinant microorganism provided herein are described in the Examples below. The skilled artisan will recognize that such conditions can be modified to accommodate the requirements of each microorganism.
As previously discussed, general texts which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other relevant topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152, (Academic Press, Inc., San Diego, Calif.) (“Berger”); Sambrook et al., Molecular Cloning—A Laboratory Manual, 2d ed., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) (“Ausubel”). Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Qβ-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), e.g., for the production of the homologous nucleic acids of the disclosure are found in Berger, Sambrook, and Ausubel, as well as in Mullis et al. (1987) U.S. Pat. No. 4,683,202; Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press Inc. San Diego, Calif.) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Nat'l. Acad. Sci. USA 87: 1874; Lomeli et al. (1989) J. Clin. Chem 35: 1826; Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4:560; Barringer et al. (1990) Gene 89:117; and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the references cited therein, in which PCR amplicons of up to 40 kb are generated. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, e.g., Ausubel, Sambrook and Berger, all supra.
Appropriate culture conditions are conditions of culture medium pH, ionic strength, nutritive content, etc.; temperature; oxygen/CO2/nitrogen content; humidity; and other culture conditions that permit production of the compound by the host microorganism, i.e., by the metabolic action of the microorganism. Appropriate culture conditions are well known for microorganisms that can serve as host cells.
The disclosure is illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.
EXAMPLES
DNA polymerase KOD for PCR reactions was purchased from EMD Chemicals (San Diego, Calif.). All restriction enzymes and Antarctic phosphatase were from New England Biolabs (Ipswich, Mass.). Rapid DNA ligation kit was obtained from Roche (Manheim, Germany). Oligonucleotides were ordered from Operon (Huntsville, Ala.). All antibiotics and reagents in media were purchased from either Sigma Aldrich (St. Louis, Mo.) or Fisher Scientifics (Houston, Tex.). A list of oligonucleotides used is given in Table 10.
Table 9:








TABLE 9







Strains, plasmids, and oligonucleotide used











Strain
Genotype















BW25113
rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78




XL-1 Blue
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac (F′ proAB laclqZΔM15 Tn10 (TetR))



BW25113 F′
BW25113 (traD36, proAB+, laclq ZΔM15 (TetR))



CRS 21
BW25113F′ ΔmetA



CRS 22
BW25113F′ ΔmetA, tdh



CRS 23
BW25113F′ ΔmetA, tdh, ilvB



CRS 24
BW25113F′ ΔmetA, tdh, ilvB, ilvl



CRS 31
BW25113F′ ΔmetA, tdh, ilvB, ilvl, adhE



CRS-BuOH 2
BW25113F′ ΔmetA, tdh + pCS49/pSA62/pSA55I



CRS-BuOH 11
BW25113F′ ΔmetA, tdh, ilvB, ilvl + pCS49/pSA62/pSA55I



CRS-BuOH 12
BW25113F′ + pCS49/pSA62/pSA55I



CRS-BuOH 18
BW25113F′ ΔmetA, tdh, ilvB, ilvl + pCS49/pCS5I/pSA55I



CRS-BuOH 19
BW25113F′ ΔmetA, tdh, ilvB, ilvl + pCS49/pCS20/pSA55I



CRS-BuOH 20
BW25113F′ ΔmetA, tdh, ilvB, ilvl + pCS49/pCS50/pSA55I



CRS-BuOH 23
BW25113F′ ΔmetA, tdh, ilvB, ilvl, adhE + pCS49/pSA62/pSA55I



CRS-BuOH 31
BW25113F′ + pSA62/pSA55I



CRS-BuOH 32
BW25113F′ ΔmetA + pCS49/pSA62/pSA55I







 










Plasmid
Genotype















pZA31-luc
PLtetO1:: luc(VF); p15A ori; CmR




pZS24-MCS1
PLlac/ara-1:: MCS1; pSC101 ori; KanR



pCS20
PLlacO1:: tdcB(EC)-leUABCD(EC); p15A ori; KanR



pCS27
PLlacO1:: MCS1; p15A ori; KanR



pCS49
PLlacO1:: thrA*BC(EC ATCC 21277); pSC101 ori; SpecR



pCS50
PLlacO1:: tdcB (EC)-leuA*BCD(EC G462D mut); p15A ori; KanR



pCS51
PLlacO1:: ilvA (EC)-leuA*BCD(EC G462D mut); p15A ori; KanR



pSA55I
PLlaCO1:: kivd (LL)-adh2 (SC), lacl; ColE1 ori; AmpR



pCS59
PLlacO1:: thrABC (EC); pSC101 ori; SpecR



pSA62
PLlacO1:: ilvA (EC)-leuABCD(EC); p15A ori; KanR







 










Primer Name
Sequence 5′→3′















lacI SacI f
CTAGAGCTCGAAGGAGATATACCATGAAACCAGTAACGTTATACGATG





(SEQ ID NO: 83)



lacI SacI r
CTAGAGCTCTCACTGCCCGCTTTCCAGTC




(SEQ ID NO: 84)



tdcB f Acc65
CGAGCGGTACCATGCATATTACATACGATCTGCCGG




(SEQ ID NO: 85)



tdcB f SalI
ACGCAGTCGACTTAAGCGTCAACGAAACCGGTGATT




(SEQ ID NO: 86)



thrA f Acc65
TCAGGTACCATGCGAGTGTTGAAGTTCGGCGGTACAT




(SEQ ID NO: 87)



thrC r HindIII
TCAAAGCTTTTACTGATGATTCATCATCAATTTACGCAA




(SEQ ID NO: 88)










Bacterial strains. Escherichia coli BW25113 (rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33ΔrhaBADLD78) was designated as the wild-type (WT) (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97, 6640-6645, 2000) for comparison. In some experiments for isobutanol, JCL16 (rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD7B/F′ (traD36, proAB+, lacIq ZΔM15)) were used as wild-type (WT). Host gene deletions of metA, tdh, ilvB, ilvI, adhE, pta, ldhA, and pflB were achieved with P1 transduction using the Keio collection strains (Baba et al., Mol. Systems. Biol. 2, 2006) as donor. The kanR inserted into the target gene region was removed with pCP20 (Datsenko and Wanner, supra) in between each consecutive knock out. Then, removal of the gene segment was verified by colony PCR using the appropriate primers. XL-1 Blue (Stratagene, La Jolla, Calif.) was used to propagate all plasmids.
Plasmid construction. pSA40, pSA55, and pSA62 were designed and constructed as described elsewhere herein. The lad gene was amplified with primers lad SacI f and lad SacI r from E. coli MG 1655 genomic DNA. The PCR product was then digested with SacI and ligated into the pSA55 open vector cut with the same enzyme behind the promoter of the ampicillin resistance gene, creating pSA55I.
The gene tdcB was amplified with PCR using primers tdcB f Acc65 and tdcB r SalI from the genomic DNA of E. coli BW25113 WT. The resulting PCR product was gel purified and digested with Acc65 and SalI. The digested fragment was then ligated into the pSA40 open vector cut with the same pair of enzymes, creating pCS14.
To replace the replication origin of pCS14 from colE1 to p15A, pZA31-luc was digested with SacI and AvrII. The shorter fragment was gel purified and cloned into plasmid pCS14 cut with the same enzymes, creating pCS16.
The operon leuABCD was amplified using primers A106 and A109 and E. coli BW25113 genomic DNA as the template. The PCR product was cut with SalI and BglII and ligated into pCS16 digested with SalI and BamHI, creating pCS20.
To create an expression plasmid identical to pSA40 but with p15A origin, the p15A fragment obtained from digesting pZA31-luc with SacI and AvrII was cloned into pSA40 open vector cut with the same restriction enzymes, creating pCS27.
The leuA* G462D mutant was constructed using SOE (Splice Overlap extension) with primers G462Df and G462Dr and the E. coli BW25113 WT genomic DNA as a template to obtain leuA*BCD. Then the SOE product was digested and cloned into the restriction sites Acc65 and XbaI to create PZE_leuABCD. The resulting plasmid was next used as a template to PCR out the leuA*BCD using primers A106 and A109. The product was cut with SalI and BglII and ligated into pCS27 digested with SalI and BamHI, creating pCS48.
The gene ilvA was amplified from E. coli BW25113 WT genomic DNA with primers A110 and A112. Next, it was cut with Acc65 and XhoI and ligated into the pCS48 open vector digested with Acc65 and SalI, creating pCS51.
The gene tdcB from the genomic DNA of E. coli BW25113 WT was amplified with PCR using primers tdcB f Acc65 and tdcB r SalI. The resulting PCR product was gel purified, digested with Acc65 and SalI and then ligated into the pCS48 open vector cut with the same pair of enzymes, creating pCS50.
WT thrABC was amplified by PCR using primers thrA f Acc65 and thrC r HindIII. The resulting product was digested with Acc65 and HindIII and cloned into pSA40 cut with the same pair of enzymes, creating pCS41.
To replace the replication origin of pCS41 from colE1 to pSC101, pZS24-MCS1 was digested with SacI and AvrII. The shorter fragment was gel purified and cloned into plasmid pCS41 cut with the same enzymes, creating pCS59.
The feedback resistant mutant thrA* was amplified by PCR along with thrB and thrC from the genomic DNA isolated from the threonine over-producer ATCC 21277 using primers thrA f Acc65 and thrC r HindIII. The resulting product was digested with Acc65 and HindIII and cloned into pSA40 cut with the same pair of enzymes, creating pCS43.
To replace the replication origin of pCS43 from colE1 to pSC101, pZS24-MCS1 was digested with SacI and AvrII. The shorter fragment was gel purified and cloned into plasmid pCS43 cut with the same enzymes, creating pCS49.
Branched-chain amino-acid aminotransferase (encoded by ilvE) and tyrosine aminotransferase (encoded by tyrB) were deleted by P1 transduction from strains JWXXX and JWXXX (Baba et al.) respectively.
To clone the L-valine biosynthesis Ones i) ilvIHCD (EC) and ii) als (BS) along with ilvCD (EC), the low copy origin of replication (ori) from pZS24-MCS1 was removed by digestion with SacI and AvrII, and ligated into the corresponding sites of i) pSA54 and ii) pSA69 to create plasmid pIAA1 and pIAA11, respectively.
To clone kivd from L. lactis and ADH2 from S. cerevisiae, the ColE1 on of pSA55 was removed by digestion with SacI and AvrII and replaced with the p15A on of pSA54 digested with the same restriction enzymes to create pIAA13. To better control the expression of these genes, lad was amplified from E. coli MG1655 genomic DNA with KOD polymerase using primers lacISacIf and lacISacIr and ligated into the SacI site of pCS22 to be expressed along with the ampicillin resistance gene, bla, and create plasmid pIAA12.
In order to overexpress the leuABCD operon in BW25113/F′ from the chromosome, the native promoter and leader sequence was replaced with the PLlacO-1 promoter. The PLlacO-1 promoter was amplified from pZE12-luc with KOD polymerase using primers lacO1KanSOEf and lacO1LeuAlr. The gene encoding resistance to kanamycin, aph, was amplified from pKD13 using primers KanLeuO1f and KanlacO1SOEr. 1 μL of product from each reaction was added as template along with primers KanLeuO2f and lacO1LeuA2r, and was amplified with KOD polymerase using SOE. The new construct was amplified from the genomic DNA of kanamycin resistant clones using primers leuKOv1 and leuKOv2 and sent out for sequence verification to confirm the accuracy of cloning. To overexpress the leuABCD operon from plasmid, the p15A on from pSA54 was removed with SacI and AvrII and ligated into the corresponding sites of pCS22 (ColE1, CmR, PLlacO-1: leuABCD) to create plasmid pIAA2. In order for tighter expression, lad was amplified and ligated as described previously for pIAA12 into pCS22 to be expressed along with the chloroamphenicol resistance gene, cat, and create plasmid pIAA15. Plasmid pIAA16 containing leuA(G1385A) encoding for IPMS (G462D) was created by ligating the 5.5 kb fragment of pIAA15 digested with XhoI and NdeI and ligating it with the 2.3 kb fragment of pZE12-leuABCD (ColE1, AmpR, PLlacO-1: leuA(G1385A)BCD) cut with the same restriction enzymes. To control for expression level, the RBS was replaced in pIAA15 to match that of pIAA16. To do this, the 5.6 kb fragment of pIAA16 from digestion with HindIII and NdeI was ligated with the 2.2 kb fragment of pIAA15 digested with the same enzymes to create pIAA17.
Media and Cultivation. For initial production experiments, strains were grown for in a modified M9 medium (6 g Na2HPO4, 3 g KH2PO4, 1 g NH4Cl, 0.5 g NaCl, 1 mM MgSO4, 1 mM CaCl2, 10 mg Vitamin B1 per liter of water) containing 10 g/L of glucose, 5 g/L of yeast extract, and 1000× Trace Metals Mix A5 (2.86 g H3BO3, 1.81 g MnCl2.4H2O, 0.222 g ZnSO4.7H2O, 0.39 g Na2MoO4.2H2O, 0.079 g CuSO4.5H2O, 49.4 mg Co(NO3)2.6H2O per liter water) inoculated 1% from 3 mL overnight cultures in LB into 10 mL of fresh media in 125 mL screw cap flasks and grown at 37° C. in a rotary shaker for 4 hours. The culture was then induced with 1 mM IPTG and grown at 30° C. for 18 hours. Antibiotics were added as needed (ampicillin 100 μg/mL, chloroamphenicol 35 μg/mL, kanamycin 50 μg/mL).
For some alcohol fermentation experiments, single colonies were picked from LB plates and inoculated into 3 ml of LB media with the appropriate antibiotics (ampicillin 100 μg/ml, kanamycin 50 μg/ml, and spectinomycin 50 μg/ml). The overnight culture grown in LB at 37° C. in a rotary shaker (250 rpm) was then inoculated (1% vol/vol) into 20 ml of M9 medium (6 g Na2HPO4, 3 g KH2PO4, 0.5 g NaCl, 1 g NH4Cl, 1 mM MgSO4, 10 mg vitamin B1 and 0.1 mM CaCl2 per liter of water) containing 30 g/L glucose, 5 g/L yeast extract, appropriate antibiotics, and 1000× Trace Metal Mix A5 (2.86 g H3BO3, 1.81 g MnCl2.4H2O, 0.222 g ZnSO4.7H2O, 0.39 g Na2MoO4.2H2O, 0.079 g CuSO4.5H2O, 49.4 mg Co(NO3)2.6H2O per liter water) in 250 ml conical flask. The culture was allowed to grow at 37° C. in a rotary shaker (250 rpm) to an OD600 of 0.4 ̃0.6, then 12 ml of the culture was transferred to a 250 ml screw capped conical flask and induced with 1 mM IPTG. The induced cultures were grown at 30° C. in a rotary shaker (240 rpm). Samples were taken throughout the next three to four days by opening the screwed caps of the flasks, and culture broths were either centrifuged or filtered to retrieve the supernatant. In some experiments as indicated, 8 g/L of threonine was added directly into the cell culture at the same time of induction.
All α-keto acid experiments were done under oxygen ‘rich’ conditions unless otherwise noted. For oxygen rich experiments, 10 mL cultures in 250 mL baffled shake flasks were inoculated 1% from 3 mL overnight cultures in LB. For oxygen poor experiments, 10 mL cultures were inoculated in 125 mL screw caps as previously described. All cultures were grown at 37° C. for 4 hours and induced with 1 mM IPTG and harvested after 18 hrs of growth at 30° C.
Final production experiments were conducted as previously described, except that 20 mL modified M9 media containing 5 g/L of glucose was used in a 250 mL screw cap flask.
Metabolite Detections. The produced alcohol compounds were quantified by a gas chromatograph (GC) equipped with flame ionization detector. The system consisted of model 5890A GC (Hewlett-Packard, Avondale, Pa.) and a model 7673A automatic injector, sampler and controller (Hewlett-Packard). Supernatant of culture broth (0.1 ml) was injected in split injection mode (1:15 split ratio) using methanol as the internal standard.
The separation of alcohol compounds was carried out by A DB-WAX capillary column (30 m, 0.32 mm-i.d., 0.50 μm-film thickness) purchased from Agilent Technologies (Santa. Clara, Calif.). GC oven temperature was initially held at 40° C. for 5 min and raised with a gradient of 15° C./min until 120° C. It was then raised with a gradient 50° C./min until 230° C. and held for 4 min. Helium was used as the carrier gas with 9.3 psi inlet pressure. The injector and detector were maintained at 225° C. 0.5 ul supernatant of culture broth was injected in split injection mode with a 1:15 split ratio. Methanol was used as the internal standard.
For other secreted metabolites, filtered supernatant was applied (20 ul) to an Agilent 1100 HPLC equipped with an auto-sampler (Agilent Technologies) and a BioRad (Biorad Laboratories, Hercules, Calif.) Aminex HPX87 column (5 mM H2SO4, 0.6 ml/min, column temperature at 65° C.). Glucose was detected with a refractive index detector, while organic acids were detected using a photodiode array detector at 210 nm. Concentrations were determined by extrapolation from standard curves.
For other secreted metabolites, filtered supernatant was applied (0.02 ml) to an Agilent 1100 HPLC equipped with an auto-sampler (Agilent Technologies) and a BioRad (Biorad Laboratories, Hercules, Calif.) Aminex HPX87 column (0.5 mM H2SO4, 0.6 mL/min, column temperature at 65° C.). Glucose was detected with a refractive index detector while organic acids were detected using a photodiode array detector at 210 nm. Concentrations were determined by extrapolation from standard curves.
Expression of L-valine and L-leucine biosynthesis pathway genes leads to 3-methyl-1-butanol production. To produce 3-methyl-1-butanol in E. coli, the entire pathway from pyruvate to 3-methyl-1-butanol was overexpressed. ilvIHCD (E. coli), kivd (L. lactis), and ADH2 (S. cerevisiae) were all expressed from plasmid (pSA54 and pSA55) under control of the PLlacO-1 promoter. The leuABCD operon was overexpressed by replacing the upstream non-coding region of leuA with the PLlacO-1 promoter in JCL16. The strain was able to produce 56 mg/L of 3-methyl-1-butanol after 18 hr of induction with IPTG (FIG. 47A). In order to increase production of 3-methyl-1-butanol, ilvIH was replaced with alsS from B. subtilils. The replacement of ilvIH with alsS showed an increase in 3-methyl-1-butanol production (67 mg/L) (FIG. 47A). To increase the expression level of the leucine biosynthesis pathway, leuABCD was also cloned into a p15A derived plasmid and expressed under control of the PLlacO-1 promoter. Plasmid based expression of leuABCD increased 3-methyl-1-butanol production for strains containing either ilvIH (177 mg/L) or alsS (124 mg/L) (FIG. 47B), although overexpression of alsS lead to a dramatic increase in isobutanol production.
Host pathways competing for carbon and reducing power were deleted. The deletion of adhE, frdBC, ldhA, pta, fnr, and pflB was shown to increase production of isobutanol in E. coli relative to a wild-type (WT) background. When the 3-methyl-1-butanol pathway was transformed into this strain, the final titer of 3-methyl-1-butanol was 76 mg/L for the strain expressing alsS (FIG. 47B). Although 3-methyl-1-butanol accumulation diminished, alcohol production was dominated by isobutanol, with a final concentration of greater than 1.3 g/L. With an isobutanol titer greater than 10 times that of the target product, the process and metabolic pathway was examined to explain this result.
Deregulation of threonine biosynthesis. As shown in FIG. 50, 2-ketobutyrate and 2-ketovalerate are the precursor for 1-propanol and 1-butanol production, respectively. While 2-ketobutyrate is a common intermediate derived from threonine and a precursor for isoleucine biosynthesis, 2-ketovalerate is a rare metabolite used by the cell to synthesize the normative amino acid, norvaline. In order to increase the pool of 2-ketovalerate for 1-butanol, the gene ilvA and leuABCD from E. coli were over-expressed using plasmid pSA62 to i) direct higher metabolic flux towards 2-ketobutyrate and ii) utilize the norvaline synthetic chemistry as the major 2-ketovalerate production route (FIG. 50). Kivd and Adh2 were also over-expressed from pSA55I to convert the two keto acids into their corresponding alcohols.
Over-expression of ilvA and leuABCD in addition to kivd and ADH2 raised both the 1-propanol and 1-butanol levels by nearly 5 fold in BW WT, from a virtually non-detectable amount to about 60 mg/L and 30 mg/L respectively (FIG. 51). Nevertheless, native feedback regulation of amino acid biosynthesis, both at the transcriptional and enzymatic level, continued to act on the threonine production, resulting in the plateau of 1-propanol and 1-butanol after 24 hours and the steady increase in isobutanol and ethanol formation to discard the excess NADH.
To identify if threonine limitation was the major bottleneck, 8 g/L threonine was added to the E. coli culture at induction. The result verified the hypothesis: the accumulated propanol and butanol in 72 hours was raised to 2 g/L, which was about a ten fold increase for both alcohols. Since transcription attenuation and allosteric feedback inhibition of ThrA by threonine are the major regulatory mechanisms, expressing a feedback-resistant mutant of ThrA behind a non-native promoter would help deregulate threonine synthesis and therefore improve production of the downstream alcohols. A feedback resistant ThrA (designated as ThrA* a threonine hyper-producer ATCC 21277). The thrA*BC operon of this strain was then cloned and expressed from plasmid pCS49 under the control of PLlacO1 promoter. As a comparison, the WT thrABC operon was also cloned and expressed from pCS 59 under PLlacO1. With the addition of ThrA*BC over-expression, the production levels of both 1-propanol and 1-butanol were boosted three to four fold higher relative to the case without ThrA*BC (FIG. 51). Strains with WT ThrABC over-expressed showed a 10-20% drop of the two target alcohols compare to the level produced by the same strains with ThrA*BC over-expressed (FIGS. 51 and 54), both in the BW WT background and in BW ΔmetA, Δtdh, ΔilvI, ΔadhE. This demonstrated that the intracellular threonine accumulation was minor but still managed to affect the WT ThrA activity at a low level. As illustrated by the decrease of isobutanol production (FIG. 51), the presence of thrA*BC helped direct the metabolic flux more towards the threonine pathway, thus improved the overall 1-propanol and 1-butanol productivity.
Elimination of competing pathways. To further increase the propanol and butanol production titer, genes involved in the competing side reactions were removed to avoid the consumption or degradation of the desired intermediates. Since threonine production is the major check point in the synthesis of 2-ketobutyrate, homoserine O-succinyltransferase metA and threonine dehydrogenase tdh were first inactivated to minimize the loss of desired precursors to methionine biosynthesis and to block the catabolism of threonine into 2-amino-3-ketobutyrate. With metA and tdh disrupted, the combined production of 1-propanol and 1-butanol increased to about 1.2 g/L as seen from FIG. 52, with the major contribution primarily coming from 1-propanol. Less significant effect of these two gene deletions was seen in 1-butanol production, which may be attributed to the divergence of 2-ketobutyrate into the isoleucine pathway and/or the availability of acetyl-CoA. To further conserve acetyl-CoA and 2-ketobutyrate, the two key precursors for 1-butanol formation, the first enzymatic reaction in the biosynthesis of valine, leucine and isoleucine was disrupted. Elimination of the larger catalytic subunit (coded by ilvB) of the acetohydroxy acid synthase isozyme (AHAS I) and the catalytic subunit (coded by ilvI) of the AHAS III led to auxotroph of the above amino acids. These two additional deletions resulted in a two fold increase in the 1-butanol production (FIG. 52) while 1-propanol level stayed unchanged. It also nearly abolished the production of isobutanol and (2-, 3-) methyl-butanol by removal of their precursors. The minute accumulation of isobutanol might have resulted from the reverse reaction in the last step of valine synthesis catalyzed by IlvE, which took valine present in the media (supplemented with yeast extract) and converted it back into 2-keto-isovalerate.
To reduce ethanol production, the E. coli adhE gene was deleted. Although the adhE disruption did not improve the overall C3 and C4 alcohol production much, it did increase the specificity by lowering ethanol formation from 0.25 g/L down to approximately 0.1 g/L. With these genes eliminated from the genome, the final strain (ΔmetA, Δtdh, ΔilvB, ΔilvI, ΔadhE) showed a near 1:1 co-production of 1-propanol and 1-butanol with minor accumulation of ethanol and basal levels of isobutanol and (2-, 3-) methyl-butanol.
Assessment of alternative feedback resistant enzymes. Because the 1-propanol and 1-butanol production presented here relied heavily on the host's amino acid biosynthetic machinery, it is important to verify that the essential alcohol precursors downstream of threonine were not limited by the various amino acid regulatory mechanisms present in the cell, particularly the inhibition of enzymatic activity of IlvA and LeuA by isoleucine and leucine respectively.
TdcB, E. coli's catabolic threonine dehydratase, provided an alternative to IlvA for catalyzing the deamination of threonine into 2-ketobutyrate while being naturally insensitive to isoleucine feedback inhibition. To assess the benefit of this alternative enzyme towards the production, tdcB was over-expressed behind PLlacO1 with leuABCD on pCS20. Results showed that TdcB led to a 70% lower production of both target alcohols compared to IlvA (FIG. 53). It is possible that the minute amount of isoleucine brought about by the addition of yeast extract was insignificant to inhibit IlvA enzymatic activity. As a result, the insensitivity towards feedback inhibition of TdcB became less important than the activity of the enzyme itself under the given experimental condition.
Similarly, feedback inhibition on LeuA by leucine present in the yeast extract led to the construction and testing of the leuA* feedback insensitive mutant G462D. The point mutation on leuA* was introduced by site-directed mutagenesis using SOE and the resulting operon leuA*BCD was over-expressed on plasmid pCS51. As shown in FIG. 53, the feedback insensitive LeuA* failed to increase the production of 1-propanol and 1-butanol. Again, this demonstrated that the amount of leucine present in the cells was probably below the inhibitory level to cause an adverse effect on the LeuA enzymatic activity.
Co-production of 1-propanol and 1-butanol from CRS-BuOH 23 and its major by-products. Time course of the alcohol and metabolite productions in the final strain BW ΔmetA, Δtdh, ΔilvE, ΔilvI, ΔadhE with plasmids pCS49, pSA62 and pSA55I (CRS-BuOH 23) is shown in FIG. 54. Both propanol and butanol production steadily climbed up in almost a linear fashion throughout the 72 hour period and appeared to plateau by the end of the third day. The same behavior was also observed in the ethanol production. Ethanol formation in the ΔadhE-background might be due to the slight affinity of Kivd towards pyruvate. On the other hand, extracellular levels of the major metabolites acetate and lactate continued to increase significantly after the alcohol production period, which might be a result of excess acetyl-CoA and NADH respectively. As seen in FIG. 54, major consumption of glucose occurred in the alcohol production period and appeared to be independent of growth alone. After the first 24 hours, cells stopped growing and remained somewhat stationary during the next few days of alcohol production.
Elimination of fermentative products and its effect on the biofuel production. The major mixed-acid fermentation genes adhE, ldhA, pta, pflB were deleted in various combinations to further characterize the present C3/C4 alcohol production system. As shown in Table 12, ethanol generation was the primary sink for excess NADH and disruption of adhE led to a more prominent accumulation of lactate. When ldhA was deleted, approximately 1 g/L of ethanol was secreted while acetate production also increased. Decrease in glucose consumption was observed in both the pta and ldhA knock-out strains. While the deletion of adhE, ldhA, pta, and combinations of them all resulted in lower propanol and butanol production, pflB knock-out had less prominent effect in that regard. This showed that the bulk acetyl-CoA pool came from the activity of pyruvate dehydrogenase complex (PDHc) instead of the Pf1B complex.







TABLE 10







Effect of fermentation gene knock outs on target alcohols


and minor by-product levels








Production
Gene deleted in addition to ΔmetA,


level
tdh, ilvB, ilvI in BW25113F′ background













(mg/L)
—
ΔadhE
Δpta
ΔldhA
ΔpflB
ΔadhE, pta
















Propanol
1017.0
1218.5
576.8
649.0
788.5
710.0


Butanol
1006.5
1094.5
770.5
581.5
932.5
853.5


Ethanol
278.0
127.4
358.5
923.0
285.5
122.0


Pyruvate
17.9
16.7
23.8
40.2
9.9
8.4


Acetate
2882.8
2726.7
2895.5
4230.0
3390.0
2346.1


Lactate
0.0
494.6
0.0
0.0
0.0
0.0


Formate
0.0
0.0
0.0
0.0
0.0
0.0


Glucose
25.3
26.1
23.7
18.7
23.4
25.4


consumed


(g/L)









Cells were cultured for 72 hours as described in materials and methods. The data shown is the 72nd hour time point.

With over-expression of the heterologous kivd and adh2 and the E. coli ilvA, leuABCD, and thrA*BC, the disclosure demonstrates the production of 1-butanol and 1-propanol. The production of 1-butanol uses 2-ketovalerate, which inevitably involves the intermediate 2-ketobutyrate and the unnatural norvaline biosynthetic pathway. Since Kivd has similar affinity towards both 2-ketoacids and 2-ketobutyrate is a secondary substrate for LeuA, 1-propanol was co-produced with 1-butanol in similar amounts.
Deregulation of the threonine biosynthesis and removal of the diverging pathways catalyzed by metA and tdh successfully conserved the threonine pool and improved both 1-propanol and 1-butanol production titer. Lysine biosynthesis, on the other hand, although also branches off of the threonine pathway, was not eliminated for its essential intermediate diaminopimelate that participates in the synthesis of bacterial cell wall. Nevertheless, the significance of lysine auxotroph is contemplated. Threonine hyper-production using rational design has also hinted at the beneficial effect of lysA deletion. 1-butanol productivity was further optimized upon interruption of the valine, leucine, and isoleucine biosynthesis pathway by inactivating AHAS I and III, which led to a two-fold increase in 1-butanol but little effect on 1-propanol. This selective improvement was attributed to i) the increase in the availability of 2-ketobutyrate and acetyl-CoA and ii) the release of the essential enzymes LeuABCD from their natural substrates in the leucine pathway.
As seen from FIG. 54, an approximate 13-15 mg/L/hr production of each alcohol was observed under the experimental condition. Improvement of strain tolerance to 1-propanol and 1-butanol can lead towards higher productivity.
Transcriptional regulation and attenuation, which are two major mechanisms of amino acid regulation, had minimal effect on the key enzymes here since all essential genes were cloned and expressed behind a non-native promoter without the leader sequence. On the other hand, allosteric feedback inhibition of the enzymes by their own amino acid products could not be neglected, specifically IlvA and LeuA. TdcB is the biodegradative threonine dehydratase that provides metabolic energy for cells under anaerobic growth in the presence of excess amino acids and scarce glucose. Its expression is controlled transcriptionally through catabolite repression and is activated by its allosteric effector AMP by decreasing its Km for threonine. Since high concentrations of pyruvate and some 2-keto acids (including 2-ketobutyrate) were shown to inactivate TdcB enzymatically, build-up of these intermediates can have a detrimental impact on its enzymatic activity when there is not enough AMP to counter the negative effect. Also, the higher Km value for threonine associated with TdcB than IlvA in the absence of significant intracellular AMP level can result in a slower deamination rate, thus contribute to the poorer performance on the overall alcohol productivity as seen in FIG. 53 (Km=8 mM for purified E. coli IlvA in the absence of isoleucine and 20 mM for E. coli TdcB in the absence of AMP). As for the LeuA* mutant, it is possible that the mutation G462D selected towards 2-keto-isovalerate in the leucine biosynthetic pathway has led to a further decrease in its affinity towards 2-ketobutyrate.
Attempts to minimize fermentation by-products in the semi-aerobic culture environment has brought attention to the NADH(P)H balance in the present production system. Threonine biosynthesis requires the expenditure of three moles of NADPH and two moles of ATP while 1-butanol and 1-propanol production starting from 2-ketobutyrate consume a net zero mole of NADH and a net one mole of NADH respectively. Phosphoenolpyruvate carboxylase encoded by the gene ppc worked constitutively to replenish the oxaloacetate (OAA) being taken away by the threonine pathway and kept the TCA cycle going. Partial activities of the TCA cycle under the semi-aerobic condition led to excess reducing power; as a result, fermentative products such as ethanol and lactate were used to discard the extra NADH. As seen in Table 11, ethanol was a more favorable NADH sink for its consumption of an additional mole of NADH than the production of lactate. When adhE was disrupted, lactate secretion was much more prominent; on the other hand, when ldhA was deleted, about 1 g/L of ethanol was accumulated in the culture broth. With low activity of the TCA cycle due to gradual loss of aerobicity and inefficiency of the downstream pathways relative to glycolysis, acetate production appeared to become the major source of ATP generation and sink for excess acetyl-CoA and/or pyruvate. These could explain the negative correlation between butanol production and acetate secretion as illustrated by comparing the WT strain and CRS-BuOH 23 in FIGS. 51 and 54. As expected, elimination of pta reduced glucose consumption and growth in the particular culture system. However, it failed to eliminate the acetate production for reasons under investigation. From the absence of formate in the culture broth and its insignificant effect on butanol productivity of the pflB knock-out, it appears that PDHc was the main acetyl-CoA provider during the alcohol production period.
Initially the hypothesis was that isobutanol production was much higher than 3-methyl-1-butanol production due to competition of the substrate α-ketoisovalerate between the gene products of kivd and leuA. In order to investigate this hypothesis, the production of the α-keto acid precursors, the substrates for kivd, to isobutanol and 3-methyl-1-butanol were examined. To achieve this, leuABCD was expressed on a ColE1 derived plasmid along with alsS-ilvCD (pSC101 derived plasmid) under control of PLlacO-1, Under oxygen poor and oxygen rich conditions, the isobutanol precursor and leuA substrate, α-ketoisovalerate (KIV), was the main product (FIG. 48A). KIV was produced to a final concentration of 0.25 and 0.29 g/L, respectively, while the 3-methyl-1-butanol precursor, α-ketoisocaproate (KIC), was not detected (<5 mg/L). In hopes of increasing the KIC pool, the expression level of leuA was increased by changing the RBS from its native sequence to a more consensus sequence. Elevated expression of the leuA gene product increased the KIC concentration to 0.20 g/L, although KIV was still the main product (0.37 g/L) (FIG. 48A). This result suggests that the decreased 3-methyl-1-butanol production was not due completely to competition for KIV but rather from the low activity of the leuA gene product (isopropylmalate synthase).
Isopropylmalate synthase (IPMS) catalyzes the condensation of KIV with acetyl-CoA. The accumulation of α-ketoisovalerate could be due to feedback inhibition of IPMS by free L-leucine synthesized from α-ketoisocaproate. To relieve the feedback inhibition of the leuA gene product, two strategies were employed. A feedback insensitive mutant of IPMS (IPMS (G462D)) was employed. Second, the final step in the L-leucine synthesis pathway was inactivated by deleting ilvE (branched-chain amino acid transferase) and tyrB (tyrosine aminotransferase), two isozymes responsible for converting α-ketoisocaproate into L-leucine.
When IPMS (G462D) was expressed, the product distribution dramatically shifted toward KIC, with a final concentration of 1.61 g/L, while KIV accumulation decreased to 0.17 g/L (FIG. 48A). Inactivation of ilvE increased production of KIC to 0.53 g/L in the strain expressing WT IPMS, while deletion of ilvE and tyrB further increased accumulation of KIC to 1.23 g/L (FIG. 48B). The production of KIV in the ΔilvE and the ΔilvE ΔtyrB backgrounds remained similar to that of the ΔilvE+ tyrB+ strain, with final concentrations of 0.40 g/L and 0.37 g/L, respectively. By combining the ΔilvE and ΔilvE ΔtyrB host strains with the expression of IPMS (G462D), KIC increased to 2.31 g/L and 1.95 g/L, respectively (FIG. 48B).
With an increased production of KIC, the entire pathway for 3-methyl-1-butanol production from pyruvate was transformed using either WT IPMS or IPMS (G462D). Similar to the results seen for keto-acid production, the strain with WT IPMS still produced a significant amount of isobutanol (169 mg/L) in an ilvE+tyrB+ background, although 3-methyl-1-butanol was the main product (308 mg/L) (FIG. 49). As expected, when IPMS (G462D) was expressed in the ilvE+ tyrB+ background, 3-methyl-1-butanol was the main product, with a final titer of 459 mg/L, with isobutanol accumulating to only 15 mg/L (FIG. 49). The removal of feedback inhibition of IPMS by mutation changed the product distribution from 1.8:1 (3-methyl-1-butanol:isobutanol) using WT IPMS to greater than 30:1. Accumulation of other common metabolic byproducts including pyruvate, fumarate, and acetate was minimal (Table 11).







TABLE 11







Metabolic byproducts of 3-methyl-1-butanol producing strains









Metabolite Concentration (g/L)











Strain
Glucose
Pyruvate
Fumarate
Acetate















IAA88
Initial (0 hr)
4.7
ND
ND
ND


IPMS (WT)
Final (16 hr)
NDa
ND
ND
0.03


ilvE+ tyrB+
Maximum (hr)
4.7 (0 hr)
0.53 (4 hr)
0.02 (6 hr)
0.03 (16 hr)


IAA90
Initial (0 hr)
4.7
ND
ND
ND


IPMS (WT)
Final (16 hr)
ND
ND
ND
0.06


ilvE− tyrB−
Maximum (hr)
4.7 (0 hr)
0.66 (4 hr)
0.02 (6 hr)
0.07 (6 hr)


IAA89
Initial (0 hr)
4.7
ND
ND
ND


IPMS (G462D)
Final (16 hr)
ND
ND
ND
0.02


ilvE+ tyrB+
Maximum (hr)
4.7 (0 hr)
0.54 (4 hr)
0.02 (6 hr)
0.02 (16 hr)





aND = Not Detected


Other metabolites such as lactate and succinate were not detected






When the ΔilvE ΔtyrB strain expressing the WT leuA gene product was examined for 3-methyl-1-butanol production, the results mimicked that of the strain containing the mutant IPMS. 3-methyl-1-butanol accumulated to a final concentration of 553 mg/L, while isobutanol was present at only 42 mg/L (FIG. 49). This corresponds to a product distribution of 3-methyl-1-butanol to isobutanol of greater than 13:1.
The disclosure provide synthetic approaches for the production of high-order alcohols as next-generation biofuels. The examples provided herein utilize E. coli as a host cell metabolically-modified to include recombinant biosynthetic pathways. However, it is understood that other microorganisms, such as Saccharamyces cerevisiae, provide suitable host cells for including recombinant biosynthetic pathways therein. These host organisms have fast growth rates and are facultative anaerobes, allowing for a flexible and economical process design for large-scale production. However, importing non-native pathways from other organisms has disadvantages. The expression of the heterologous pathways may lead to a metabolic imbalance, while the accumulation of the heterologous metabolites may cause cytotoxicity. For example, the 1-butanol production pathway of C. acetobutylicum has three intermediate metabolites carrying coenzyme A (CoA). An excess expression of this pathway may cause the depletion of the free CoA pool and perturb the metabolism in E. coli. To achieve the high productivity of the target foreign products, it is desirable to seek pathways that are compatible to the host. Instead of direct transfer of the common pathway for 1-butanol production into the non-native host, E. coli, the modified microorganisms provided herein take advantage of the existing metabolic capability in E. coli and the broad substrate range of the last two steps in the Ehrlich pathway for 2-keto acid degradation.
2-Keto acids are intermediates in amino acid biosynthesis pathways. These metabolites can be converted to aldehydes by broad-substrate range 2-keto-acid decarboxylases (KDCs) and then to alcohols by alcohol dehydrogenases (ADHs). Using this strategy, two non-native steps are needed to produce biofuels by shunting intermediates from amino acid biosynthesis pathways to alcohol production (FIG. 1A). Amino acid biosynthesis pathways produce various 2-keto acids (FIG. 1B). In the present studies, six different 2-keto acids were used for alcohol production. The isoleucine biosynthesis pathway generates 2-ketobutyrate and 2-keto-3-methyl-valerate which can be converted to 1-propanol and 2-methyl-1-butanol, respectively. The valine biosynthesis pathway produces 2-keto-isovalerate which is the precursor for isobutanol. The leucine biosynthesis pathway generates 2-keto-4-methyl-pentanoate which is the substrate for 3-methyl-1-butanol. The phenylalanine biosynthesis pathway produces phenylpyruvate which can lead to 2-phenylethanol. The norvaline biosynthesis pathway, which is a side-reaction of the leucine biosynthesis, produces a substrate for 1-butanol, 2-ketovalerate.
The 2-keto acid decarboxylase activity can be provided by one of the following genes: PDC6 from Saccharomyces cerevisiae, kivd from Lactococcus lactis, and THI3 Saccharomyces cerevisiae (α-ketoisocaproate decarboxylase) and pdc Clostridium acetobutylicum. The alcohol dehydrogenase (Adh) activity can be provided by ADH2 from Saccharomyces cerevisiae. 
Valine is synthesized from two molecules of pyruvate in a pathway involving four reactions which are catalyzed by AHAS (the ilvBN gene product), isomeroreductase (the ilvC gene product), dihydroxyacid dehydratase (the ilvD gene product or ilv3 from Pichia stipitis), and transaminase B (the ilvE gene product). As in other organisms, the same enzymes also catalyze the synthesis of L-isoleucine from pyruvate and 2-ketobutyrate. The latter is formed from L-threonine by threonine dehydratase (the ilvA gene product). AHAS is the key enzyme of branched-chain amino acid synthesis. Valine causes feedback inhibition of AHAS I, encoded by ilvBN, and AHAS III, encoded by the acetohydroxy acid synthase operon ilvIH, and their small regulatory subunits, IlvN and IlvH, were shown to be necessary for valine sensitivity.
An enzyme in this alcohol production strategy is KDC, which is common in plants, yeasts and fungi but less so in bacteria. The aldehydes produced can then be converted to alcohols by an Adh, which is commonly found in many organisms. Some of the KDCs have broad-substrate ranges, while others are more specific. To test the capability of the endogenous 2-keto acids as a substrate for KDC in E. coli, five KDCs including Pdc6, Aro10, Thi3, from S. cerevisiae, Kivd from Lactococcus lactis, and Pdc from C. acetobutylycum were over-produced with alcohol dehydrogenase 2 (Adh2) of S. cerevisiae. E. coli cultures expressing these foreign genes were grown in a minimal media with 0.2 M glucose. GC-MS analysis (see Table 12) revealed that the strains expressing either kivd or ARO10 produced all of the expected alcohols. S. cerevisiae PDC6 and C. acetobutylum pdc were not as versatile, whereas S. cerevisiae THIS did not display the expected activity.
Table 12 shows alcohol productions with KDC and ADH in E. coli as follows:













KDC













Kivd
ARO10
PDC6
THI3
Pdc (C.A.)









plasmid












Product (μM)
pSA55
pSA56
pSA49
pSA57
pSA59















1-propanol
520
290
125
ND
ND


isobutanol
5242
2094
260
ND
 75


1-butanol
220
95
ND
ND
ND


2-methyl-1-butanol
766
652
56
ND
ND


3-methyl-1-butanol
1495
1099
92
ND
ND


2-phenylethanol
324
469
ND
ND
175









Aldehydes were detected in trace amounts, indicating sufficient activity of Adh2. These results demonstrate that Kivd is an active and versatile decarboxylase and, therefore, suited for these objectives.
The addition of various 2-keto acids (see Table 13) to the E. coli culture expressing kivd confirmed the specific production of the corresponding alcohols by 2- to 23-fold. The supply of 2-keto acids also decreased the production of the other alcohols dramatically. These results indicate that increasing the flux to the 2-keto acids could improve both the productivity and specificity of the alcohols production.
Table 13 shows alcohol productions with the supply of 2-keto acids as follows:




















2-keto-3-
2-keto-4-methyl-



Product (μM)
2-ketobutyrate
2-keto-isovalerate
2-ketovalerate
methyl-valerate
pentanoate
phenylpyruvate





















1-propanol
2138
ND
ND
ND
ND
8


isobutanol
98
10016
ND
ND
ND
64


1-butanol
492
ND
3926
ND
ND
23


2-methyl-1-butanol
1315
ND
ND
5284
ND
ND


3-methyl-1-butanol
ND
ND
52
ND
3756
105


2-phenylethanol
26
 109
66
ND
ND
7269









Existing E. coli metabolic pathways were genetically modified to increase the production of the specific 2-keto acid so that the desired alcohol is produced. To produce isobutanol, the ilvIHCD genes were amplified to enhance 2-ketoisovalerate biosynthesis (FIG. 1B). The ilvIH operon of Escherichia coli encodes acetohydroxy acid synthase, the first enzyme in the isoleucine, valine and leucine biosynthetic pathway. The acetohydroxy acid synthase III isozyme, which catalyzes the first common step in the biosynthesis of isoleucine, leucine, and valine in Escherichia coli K-12, is composed of two subunits, the ilvI (acetolactate synthase III large subunit) and ilvH (acetolactate synthase small subunit) gene products. The ilvIH operon of Escherichia coli encodes acetohydroxy acid synthase, the first enzyme in the isoleucine, valine and leucine biosynthetic pathway. The ilvC gene of Escherichia coli encodes acetohydroxy acid isomeroreductase, the second enzyme in the parallel isoleucine-valine biosynthetic pathway. The ilvD gene of Escherichia coli encodes dihydroxy-acid dehydratase, the third enzyme in the isoleucine-valine biosynthetic pathway.
An operon encoding the ilvI, ilvH, ilvC and ilvD genes under the control of PLlacO1 was constructed on a plasmid. The amplified Ilv pathway is then combined with the synthetic alcohol producing pathway (Kivd and Adh2) to achieve isobutanol production. As a result of the ilvIHCD pathway expression, this strain produced 23 mM isobutanol, which is a  ̃5-fold increase over the strain without the ilvIHCD pathway overexpression (see Table 14 below and FIG. 2A).
Table 14 shows alcohol productions with the ilvIHD pathway and overexpression as follows:













KDC














kivd
kivd
kivd
kivd
kivd
kivd









Strain














JCL16
JCL16
JCL16
JCL16
JCL88
JCL88









plasmid
















pSA54
pSA54
pSA54
pSA54



pSA55
pSA55
pSA55
pSA55
pSA55
pSA55









Time (hr)













Product (μM)
16
24
16
24
16
24
















ethanol
1277
1744
2047
2228
850
1430


1-propanol
321
420
612
808
210
493


isobutanol
2069
2172
12979
20813
13794
30910


1-butanol
67
112
146
198
89
127


2-methyl-1-
223
629
262
345
195
239


butanol


3-methyl-1-
1096
1696
1103
1577
727
1333


butanol


2-
545
1220
282
425
454
820


phenylethanol









These results demonstrate that the synthetic pathway was functional and capable of supplying the 2-ketoisovalerate required for the efficient production of isobutanol. To further increase the isobutanol production, one or more genes that contribute to byproduct formation, including adh, ldh, frd, fnr, pflB and/or pta, were deleted. These deletions could increase the level of pyruvate available for the ilvIHCD pathway. Indeed, this strain produced 30 mM isobutanol, indicating that these deletions were beneficial for isobutanol production. In addition, this strain converted glucose to isobutanol with a yield of 0.21 g of isobutanol per gram of glucose between 16 hr and 24 hr (FIG. 2A, right panel). Additional data illustrated in FIG. 42 further show that these deletions would also increase the level of pyruvate available for the acetolactate synthase pathway, affecting the isobutanol production with an increase from 4.5 g/L (61 mM) isobutanol for JCL16 to 13.2 g/L (356 mM) isobutanol for JCL260 after 64 h. This result demonstrates the potential of this strategy, since the yield has already reached 50% of the theoretical maximum without detailed optimization of the pathways and production conditions. This high yield is attributed to the full compatibility of the synthetic pathway with the host cell's physiology. Additional Yield Data is provided in the Table immediately below for strain SA237:


















Time






(hr-hr)
Yield (g/g)
Time (hr)
(g/L)







0-16
~0.32
0
0.00



16-40 
~0.4
16
~7.172



40-64 
~0.36
40
~12.01



64-88 
~0.32
64
~17.11



88-112
~0.33
88
~19.41



0-40
~0.34
112
~21.89



0-64
~0.35



0-88
~0.34



 0-112
~0.34











30 C.


M9 + 0.5%


YE


0.1 mM


IPTG







Ethanol production for 0-112 hrs


was~0.0037 g/g (240 mg/L)











Isobutanol (mM)

IsoBuOH













Time
#1
#2
#3
AVE
STDEV
(g/L)





 0


16
98.45
108.29
83.56
96.77
12.45
7.172345


40
158.21
173.42
154.3
161.98
10.10
12.00571


64
225.54
253.33
213.67
230.85
20.36
17.11035


88
256.23
281.21
248.11
261.85
17.25
19.40832


112 
296.43
312.56
277.12
295.37
17.74
21.89282














Time
Glucose (g/L)


















 0
36
36
36
36.00




16
13.2
12.5
14.1
13.27
0.80



40
1.2
0.9
1.5
1.20
0.30



40.1
31.2
30.9
31.5
31.20



64
17.4
15.6
18.9
17.30
1.65



88
10.3
7.8
12.5
10.20
2.35



112 
2.8
1.4
4.1
2.77
1.35










Initially 36 g/L glucose was present at T=0. At 40.1 hrs and additional 30 g of glucose was added to the culture. Glucose values at the different hours represent g/L at the specific time point (e.g., at 16 hrs. ̃22.8 g of glucose was metabolized).

A similar strategy was applied to generating a microorganism that produces 1-butanol. The 1-butanol producing pathway in its native producers is used during fermentative growth, and many of the enzymes in this pathway are oxygen-sensitive and CoA-dependent. The data indicates that by over-expressing kivd or ARO10 in E. coli, which does not have the 1-butanol fermentative pathway, the cell already produced a small amount of 1-butanol (Table 13) from glucose in a non-fermentative growth, suggesting the existence of corresponding 2-keto acid precursor, 2-ketovalerate. However, 2-ketovalerate is not a common metabolite in E. coli. To increase the amount of synthesized 2-ketovalerate, the broad-substrate specificity of the leuABCD pathway whose natural substrate is 2-ketoisovalerate (FIG. 1B) was utilized. By using a smaller substrate, 2-ketobutyrate, which has one less methyl group than 2-ketoisovalerate (FIG. 1A), 2-ketovalerate was synthesized in a manner similar to the transformations in leucine biosynthesis. 2-ketobutyrate can be generated from L-threonine by the threonine dehydratase, encoded by the ilvA gene, or from an alternative pathway starting from acetate and pyruvate identified in Leptospira interrogans serovar and Methanocaldococcus jannaschii. In the latter pathway, 2-ketobutyrate is generated from citramalate by the enzymes isopropylmalate isomerase (LeuCD) and beta-isopropylmalate dehydrogenase (LeuB).
To produce 1-butanol, an operon encoding the ilvA-leuABCD pathway under the control of PLlacO1 was constructed. Strain with ilvA-leuABCD pathway produced 0.6 mM 1-butanol, which was a  ̃3-fold increase compared with the strain without the ilvA-leuABCD pathway overexpression (see Table 15 below and FIG. 2B).
Table 15 shows alcohol productions with threonine pathway overexpression as follows:
















KDC
kivd
kivd
kivd
kivd








plasmid
pSA55
pSA55
pSA55
pSA54





pSA62
pSA62
pSA62



strain
JCL16
JCL16
JCL16
SA203



0.8% L-threonine
−
−
+
+







Product (μM)













ethanol
2450
2343
3243
1493



1-propanol
520
1356
7592
9849



isobutanol
2242
4322
1061
ND



1-butanol
220
583
3157
9232



2-methyl-1-
766
1444
2002
ND



butanol







3-methyl-1-
1495
4074
1349
ND



butanol







2-phenylethanol
324
358
269
 524









In addition, when the media was supplemented with 0.8% L-threonine, a dramatic increase of 1-butanol production to 3.2 mM was observed, indicating that 2-ketovalerate can be produced from L-threonine via an IlvA-mediated reaction (see FIG. 2C).
To further improve 1-butanol production, the ilvD gene was deleted. This gene encodes dihydroxy-acid dehydratase, an enzyme that produces both 2-ketoisovalerate (a precursor for leucine and valine) and 2-keto-3-methyl-valerate (a precursor for isoleucine). This deletion could be beneficial for two reasons. First, the deletion of ilvD eliminates the native substrate, 2-ketoisovalerate, for the leuABCD pathway, thus reducing the competitive substrate inhibition. Second, the deletion of ilvD eliminates of competing substrates for Kivd, 2-keto-3-methyl-valerate and 2-keto-4-methyl-pentanoate. As expected, deletion of ilvD improved 1-butanol production (FIG. 2C).
Since L-threonine hyperproduction in E. coli strains has been developed for commercial production, a threonine producing strain can be modified using the above strategy in order to manufacture an organism that produces 1-butanol. For further improvement, it would be necessary to increase the activity of the leuABCD pathway towards to the non-native substrate, 2-ketobutyrate, and to raise the specificity of Kivd for 2-ketovalerate. Since 2-ketobutyrate is also the substrate for 1-propanol (FIG. 1B), increasing 2-ketobutyrate availability also enhances the production of 1-propanol (FIG. 2B and FIG. 2C, right panel). Therefore, increasing the leuABCD activity and the specificity of KDC would be crucial for high-efficiency 1-butanol production.
Alcohol profiles and yield for 1-butanol and 1-propanol producing strain, CRS-BuOH23 is provided below:














time






period
PrOH
BuOH
total
EtOH



















 0-20
0.037
0.015
0.053
0.004


20-28
0.054
0.046
0.1
0.004


28-52
0.033
0.042
0.075
0.003


52-76
0.061
0.066
0.127
0.009


 76-100
−0.016
−0.014
−0.03
0.003


 0-76
0.043
0.038
0.081
0.005


 0-28
0.042
0.024
0.066
0.004


20-76
0.046
0.05
0.097
0.005


28-76
0.044
0.052
0.096
0.005





total = 1-propanol + 1-butanol;


Final EtOH: 116.2 mg/L






Alcohol profiles and yield for a 3-methyl-1-butanol producing strain is provided below:










3-methyl-1-butanol strain










3MB
Total Alcohol


Time (hr-hr)
Yield (g/g)
Yield (g/g)












0-4
0.000
0.000


4-6
0.040
0.040


6-8
0.180
0.180


 8-12
0.160
0.170


12-20
0.120
0.160


20-28
−0.050
−0.010


 0-28
0.100
0.120









Alcohol profiles and yield for 2 different 2-methyl-1-butanol strains are presented below:












2-methyl-1-butanol strain 1 (AFC-2MB-01BW25113: pAFC46:


kivd (L. lactis) AHD2 (S. cerevisiae) ilvA (C. glutamicum) pAFC3:


ilvGM (S. typhimurium) ilvCD (E. coli) PCS49: thrAFBRBC (E. coli))












2 MB






Yield
Total Alcohol Yield




Time (hr-hr)
(g/g)
(g/g)
EtOH Yield
EtOH mg/L





0-9
0.026
0.081
0.063
230.6


 9-15
0.050
0.171
0.019
156.1


15-18
0.043
0.164
0.016
196.0


18-21
0.038
0.163
0.016
226.2


21-24
0.037
0.157
0.017
271.6


24-27
0.034
0.144
0.017
288.7


27-33
0.046
0.146
0.022
376.4


33-39
0.031
0.123
0.018
329.5










2-methyl-1-butanol strain 2 (AFC-2MB-02: CRS22: BW25113 ΔmetA


Δtdh pAFC46: kivd (L. lactis) AHD2 (S. cerevisiae) ilvA


(C. glutamicum) pAFC3: ilvGM (S. typhimurium) ilvCD (E. coli)


PCS49: thrAFBRBC (E. coli))












2 MB





Time (hr-hr)
Yield
Total Alcohol Yield
EtOH Yield
EtOH mg/L





0-9
0.183
0.305
0.243
301.7


 9-15
0.089
0.161
0.014
133.4


15-18
0.083
0.164
0.014
166.5


18-21
0.080
0.169
0.016
214.8


21-24
0.073
0.161
0.016
256.7


24-27
0.065
0.149
0.017
315.1


27-33
0.058
0.138
0.017
326.5


33-39
0.064
0.151
0.018
344.8





total alcohol yield = 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol, 1-butanol and 1-propanol






Non-native hosts such as E. coli lack tolerance to high alcohols. Isobutanol is as toxic as 1-butanol to microorganisms, and the native 1-butanol producers can tolerate 1-butanol concentration up to about 2%. To show the potential for improving tolerance, in vitro evolution by the sequential transfer of cultures was used to enrich for strains with mutations which increase isobutanol tolerance. The data indicates that wild-type E. coli strain (JCL16) was inhibited by 1.5% isobutanol. However, after only 5 rounds of culture transfers with increasing isobutanol concentrations, mutants were found to grow in the presence of 2% isobutanol (FIG. 3). This level of solvent tolerance is comparable or better than the native producer of 1-butanol, suggesting that E. coli can adapt to high concentration of long chain alcohols. Other strategies such as gTME can be used for further improvement of tolerance.
The strategy described herein provides for biofuels production, both in E. coli and in other microorganisms. This strategy takes advantage of amino acid production technology and channels the amino acid intermediates to the 2-keto acid degradation pathway for alcohol production. The strategy avoids the CoA-mediated chemistry which is used in 1-butanol production in the native producer, and enables the synthesis of other higher and complex alcohols in an industrial scale. Specific strategies for producing other alcohols can be readily devised based on the synthetic pathways provided herein. For example, the production of 2-phenylethanol can be achieved by shunting the phenylalanine pathway, which has been efficiently amplified in E. coli. These strategies can also be readily implemented in yeast or other industrial microorganisms. In the case of isobutanol production, the complete pathway is CoA-independent and requires only pyruvate as a precursor. This feature avoids the mitochondria compartmentalization issue of acetyl-CoA when implementing the strategy in yeast. This strategy for production of isobutanol and 1-butanol offers the same theoretical yields (0.41 g/g glucose) as that from the native producer of 1-butanol.
Gene deletion was accomplished using methods known to the skilled artisan. Briefly, BW25113 (rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78) was used as the parent (e.g., wild-type) microorganism. The adhE, ldhA, frdBC, fnr, pflB and ilvD sequences were deleted as described (Datsenko and Wanner, Proc Natl Acad Sci U.S.A 97:6640 (2000)). The pta sequence deletion was made by P1 transduction with JW2294 (Baba et al., Mol Syst Biol 2:E1-E11 (2006)) as the donor. F′ was transferred from XL-1 blue to supply lacIq. A list of the strains used in the present studies is provided in Table 16. In particular for the deletion of pta nucleotides 2,412,772-2,414,893 were removed. For the frdBC deletion nucleotides 4,377,400-4,378,540 were removed. For the deletion of adhE nucleotides 1,294,669-1,297,344 were removed. For the ldhA deletion nucleotides 1,439,878-1,440,867 were removed. For the fnr deletion nucleotides 1,396,798-1,397,550 were removed. For the pflB deletion nucleotides 950,508-952,784 were removed.








TABLE 16





Strain
Relevant genotype







BW25113
rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78


JCL16
BW25113, F′ (traD36, proAB+, lacIq ZDM15)


JCL88
JCL16, ΔadhE, ΔldhA-fnr, ΔfrdBC, Δpta


JCL93
JCL16, ΔadhE, ΔldhA, ΔfrdBC


SA203
JCL16 ΔilvD


JCL260
JCL16, ΔadhE, ΔldhA-fnr, ΔfrdBC, ΔpflB, Δpta


JCL167
JCL16, ΔadhE, ΔldhA-fnr, ΔfrdBC


JCL274
JCL16, ΔadhE, ΔldhA, ΔfrdBC, Δpta


JCL168
JCL16, ΔadhE, ΔldhA-fnr, ΔfrdBC, ΔpflB


JCL171
JCL16, ΔadhE, ΔldhA, ΔfrdBC, ΔpflB, Δpta









pSA46 includes an PDC6 sequence. Genomic DNA of Saccharomyces cerevisiae (ATCC) was used as PCR template with a pair of primers A65 and A66 (see Table 17). PCR products were digested with Acc65I and SphI and cloned into pZE12-luc (3) cut with the same enzyme.
pSA49 includes an ADH2 sequence. To clone ADH2, genomic DNA of Saccharomyces cerevisiae (ATCC) was used as PCR template with a pair of primers A67 and A68 (see Table 17). PCR products were digested with SphI and XbaI and cloned into pSA46 cut with the same enzyme.
pSA53 was created by replacing the replication origin of pSA49 with p15A, pZA31-luc was digested with SacI and AvrII. Shorter fragment was purified and cloned into plasmid pSA49 cut with the same enzymes.
pSA55 includes a kivd sequence obtained by using the genomic DNA of Lactococcus lactis as a PCR template with a pair of primers A96 and A97 (see Table 17). PCR products were digested with Acc65I and SphI and cloned into pSA49 cut with the same enzyme.
pSA56 includes an ARO10 sequence. Genomic DNA of Saccharomyces cerevisiae (ATCC) was used as a PCR template with a pair of primers A98 and A99 (see Table 17). PCR products were digested with Acc65I and SphI and cloned into pSA49 cut with the same enzyme.
pSA57 includes a THI3 sequence. Genomic DNA of Saccharomyces cerevisiae (ATCC) was used as a PCR template with a pair of primers A100 and A101 (see Table 17). PCR products were digested with Acc65I. pSA49 was digested with SphI and blunted with Klenow Fragment, followed by digestion with Acc65I. This backbone was ligated with PCR products.
pSA58 includes a pdc sequence obtained from Clostridium acetobutylicum. Genomic DNA was used as a PCR template with a pair of primers A102 and A103 (see Table 17). PCR products were digested with Acc65I and SphI and cloned into pSA49 cut with the same enzyme.
To create pSA40, the PLtetO1 sequence of pZE21-MCS1 was replaced with PLlacO1. pZE12-luc was digested with AatII and Acc65I and the shorter fragment was purified and cloned into plasmid pZE21-MCS1 cut with the same enzymes.
pSA45 includes an ilvC sequence. The ilvC sequence was obtained using the genomic DNA of E. coli MG1655 as a PCR template with a pair of primers A71 and A72 (see Table 17). PCR products were digested with SalI and XmaI and cloned into pSA40 cut with the same enzyme.
pSA47 includes an ilvD sequence. The ilvD sequence was obtained using the genomic DNA of E. coli MG1655 as a PCR template with a pair of primers A74 and A84 (see Table 17). PCR products were digested with BspEI and MluI and cloned into pSA45 cut with SalI and MluI.
pSA51 includes ilvI and ilvH sequences. Genomic DNA of Escherichia coli MG1655 was used as PCR template with a pair of primers A70 and A83 (see Table 17). PCR products were digested with BsaI and SalI and cloned into pSA40 cut with Acc65I and SalI.
pSA52 includes ilvC and ilvD sequences downstream of ilvH. pSA47 was digested with SalI and MluI. The shorter fragment was purified and cloned into plasmid pSA51 cut with the same enzymes.
pSA54 was created by transferring the p15A replication origin from pZA31-luc, digested with SacI and AvrII, to plasmid pSA52.
pSA59 includes leuABCD sequence. The genomic DNA of E. coli MG1655 was used as PCR template with a pair of primers A106 and A109 (see Table 17). PCR products were digested with SalI and BglII and cloned into pSA40 cut with SalI and BamHI.
pSA60 includes ilvA sequence. The genomic DNA of E. coli MG1655 was used as PCR template with a pair of primers A104 and A105 (see Table 17). PCR products were digested with Acc65I and XhoI and cloned into pSA59 cut with Acc65I and SalI.
pSA62 was created by cloning the replication origin p15A from pZA31-luc into plasmid pSA60. A partial description of exemplary plasmids provided herein are listed in Table 18.
pSA66 includes 3′ fragment of an alsS sequence. The alsS sequence was obtained using the genomic DNA of Bacillus subtilis as a PCR template with a pair of primers A123 and A124 (see Table 17). PCR products were digested with Acc65I and SalI and cloned into pSA40 cut with the same enzyme.
pSA67 includes alsS sequence. The 5′ fragment of the alsS sequence was obtained using the genomic DNA of Bacillus subtilis as a PCR template with a pair of primers A125 and A126 (see Table 17). PCR products were digested with BsrGI and XbaI and cloned into pSA66 cut with Acc65I and XbaI.
pSA68 includes ilvC and ilvD sequence downstream of alsS. pSA47 was digested with SalI and MluI. The shorter fragment was purified and cloned into plasmid pSA67 cut with the same enzymes.
pSA69 was created by transferring the p15A replication origin from pZA31-luc, digested with SacI and AvrII, to plasmid pSA68.









TABLE 18






Plasmid
Relevant genotype








pSA54
Origin of p15A; PLlacO1::ilvIHCD; kanr



pSA55
Origin of ColE1; PLlacO1::kivd-ADH2;




ampr



pSA69
Origin of p15A; PLlacO1::alsS-ilvCD;




kanr









An exemplary list of oligonucleotides useful for PCR and cloning procedures is provided in Table 17. It is understood that the exemplary oligonucleotides can be modified according to the particular sequence targeted for PCR and/or cloning procedures.









TABLE 17





name
sequence
(SEQ ID NO:)







 










A65
CGAGCGGTACCATGTCTGAAATTACTCTTGGAAAAT
 (1).



 


A66
GCCTGCGCATGCTTATTGTTTGGCATTTGTAGCGGCA
 (2)


 


A67
GCCTGCGCATGCAGGAGATATACCATGTCTATTCCAGAAACTCAAAAAG
 (3)


 


A68
GCTCTAGATTATTTAGAAGTGTCAACAACGTAT
 (4)


 


A70
ACGCAGTCGACTCAACGCATTATTTTATCGCCGCGC
 (5)


 


A71
ACGCAGTCGACGAGGAATCACCATGGCTAACTACTT
 (6)


 


A72
AATAACCCGGGTTAACCCGCAACAGCAATACGTTTC
 (7)


 


A74
CGAGCACGCGTTTAACCCCCCAGTTTCGATTTATCG
 (8)


 


A83
GCCACCGGTCTCCGTACCATGGAGATGTTGTCTGGAGCCGAGA
 (9)


 


A84
GGCTCCGGAAGGAGATATACCATGCCTAAGTACCGTTCCGCCACCA
(10)


 


A96
CGAGCGGTACCATGTATACAGTAGGAGATTACCTAT
(11)


 


A97
GCCTGCGCATGCTTATGATTTATTTTGTTCAGCAAAT
(12)


 


A98
CGAGCGGTACCATGGCACCTGTTACAATTGAAAAGT
(13)


 


A99
GCCTGCGCATGCCTATTTTTTATTTCTTTTAAGTGCCGC
(14)


 


A100
CGAGCGGTACCATGAATTCTAGCTATACACAGAGAT
(15)


 


A101
GGTCAGTATCCAACTTGATTTTTTTTTAGAAG
(16)


 


A102
CGAGCGGTACCATGAAGAGTGAATACACAATTGGAAG
(17)


 


A103
GCCTGCGCATGCCTAATTATTTTGATTTGCAAAACGT
(18)


 


A104
CGAGCGGTACCATGGCTGACTCGCAACCCCTGTCCG
(19)


 


A105
CCGCTCGAGCTAACCCGCCAAAAAGAACCTCAAC
(20)


 


A106
ACGCAGTCGACAAGAGACAAGGACCCAAACCATGAGCCAG
(21)


 


A109
GGAAGATCTTTAATTCATAAACGCAGGTTGTTTTGC
(22)


 


A123
GCCACCCGTCTCCGTACCATGTTGACAAAAGCAACAAAAGAAC
(23)


 


A124
ACGCAGTCGACCTAGAGAGCTTTCGTTTTCATGAGT
(24)


 


A125
CGAGCTGTACAATGTTGACAAAAGCAACAAAAGAAC
(25)


 


A126
TCTCTAGAAAGGGTACCGGCAGCTTG
(26)









In an exemplary procedure for the production of isobutanol, the host strain JCL260 was transformed with the plasmids pSA69 and pSA55. The transformation was plated on LB+0.5% glucose+50 mg/L kanamycin+200 mg/L ampicillin. Fresh transformants were used to inoculate 2 mL cultures in LB+0.5% glucose+50 mg/L kanamycin+200 mg/L ampicillin. The cultures were incubated at 37° C. and 290 rpm overnight.
200 μL from the overnight culture were used to inoculate 20 mL of M9+1× Trace Metal Mix A5+7.2% glucose+3% tryptone+30 mg/L kanamycin+100 mg/L ampicillin in 250 mL screw cap flasks. The cultures were incubated at 37° C./260 rpm. At OD600 of between 1 and 2 ( ̃5 h) the cultures were induced with 0.1 mM IPTG and incubated at 30° C./260 rpm for 24 hours.
The above solutions should be sterilized separately by filtration (thiamine) or autoclaving. The medium is sterilized by filtration.
Production of isobutanol was measured by gas chromatography and the results are illustrated in FIGS. 42-44. In FIG. 42, cultivation was performed as follows: M9 medium containing 7.2% glucose, 0.5% yeast extract, 100 μg/ml ampicillin, 30 μg/ml kanamycin and 1000× Trace Metal Mix A5 was prepared. Preculture of various knockout strains containing pSA55 and pSA69 in test tubes containing 3 ml of LB medium were performed at 37° C. overnight on a rotary shaker (250 rpm). Overnight cultures were diluted 1:100 into 20 ml of fresh medium in a 250 ml screw cap conical flask. Cells were grown to an OD600 of  ̃2.0 at 37° C., followed by adding 0.1 mM IPTG. After IPTG addition, cells were cultured at 30° C. for 64 hr on a rotary shaker (250 rpm). Screw cap was opened at 16 hr, 40 hr and 64 hr for sampling. The cultivation samples were analyzed by GC/FID. In FIG. 43, cultivation was performed as follows: M9 medium containing 3.6% glucose, 100 μg/ml ampicillin, 30 μg/ml kanamycin and 1000× Trace Metal Mix A5 was prepared. Precultures of SA237 (JCL260 containing pSA55 and pSA69) in test tubes containing 3 ml of LB medium were performed at 37° C. overnight on a rotary shaker (250 rpm). Overnight cultures were diluted 1:100 into 20 ml of fresh medium in a 250 ml screw cap conical flask (red) or a 250 ml conical flask (blue). Cells were grown to an OD600 of  ̃0.3 at 37° C., followed by adding 0.1 mM IPTG. After IPTG addition, the cultures were incubated in 30° C. for 72 hr on a rotary shaker (250 rpm). Isobutanol concentration, OD600 and pH of the cultivation samples were measured at 24 hr, 48 hr and 72 hr. In FIG. 45, cultivation was performed as follows: For the cultivation, M9 medium containing 3.6% glucose, 100 μg/ml ampicillin, 30 μg/ml kanamycin and 1000× Trace Metal Mix A5 was prepared. To this cultivation medium, 0.8% casamino acids (red), 2% tryptone (green) or 0.5% yeast extract (blue) was added. Preculture of SA237 (JCL260 containing pSA55 and pSA69) in test tubes containing 3 ml of LB medium were performed at 37° C. overnight on a rotary shaker (250 rpm). Overnight culture was diluted 1:100 into 20 ml of fresh medium in a 250 ml screw cap conical flask. Cells were grown to an OD600 of  ̃0.8 at 37° C., followed by adding 0.1 mM IPTG. After IPTG addition, the cultures were incubated in 30° C. for 198 hr on a rotary shaker (250 rpm). Isobutanol concentration, glucose concentration, OD600 and pH of the cultures were measured at each time points. At 40 hr, 1 ml of 36% glucose was added to all cultures. At 112 hr, 5 ml of fresh medium was added to the casamino acids-containing culture (red) and the tryptone-containing culture (green). At 112 hr, 5 ml of fresh medium and 2 ml of 36% glucose were added to the yeast extract-containing culture (blue).
The following methods and reagents were used in the development of a mutant cimA:
Restriction enzymes and Antarctic phosphatase were from New England Biolabs (Ipswich, Mass.). Rapid DNA ligation kit was from Roche (Manheim, Germany). KOD DNA polymerase was from EMD Chemicals (San Diego, Calif.). Oligonucleotides were from Operon (Huntsville, Ala.).
Strains and plasmids. A list of strains and plasmids used is given in Table 19.







TABLE 19







Strains used








Strain
Relevant genotype





BW25113
rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78


JCL16
BW25113/F′ [traD36, proAB+, lacIq ZΔM15]


KS145
Same as JCL16 but with ΔilvI, ΔilvB


SA405
Same as JCL16 but with ΔilvA, ΔtdcB


SA408
Same as SA405 but with ΔilvI


SA459
Same as JCL299 but with ΔilvI, ΔilvB


pSA55
ColE1 ori; AmpR; PLlacOl: kivd-ADH2


pSA63
p15A ori; KanR; PLlacOl: cimA-leuABCD


pSA90
p15A ori; KanR; PLlacOl: cimA1-leuABCD


pSA121
p15A ori; KanR; PLlacOl: cimA2-leuABCD


pSA123
p15A ori; KanR; PLlacOl: cimAΔ1117-1473-leuABCD


pSA124
p15A ori; KanR; PLlacOl: cimA2Δ1117-1473-leuABCD


pSA142
p15A ori; KanR; PLlacOl: cimA3.7-leuABCD









To clone cimA, genomic DNA of M. jannaschii was used as PCR template with a pair of primers A113 (5′-CGAGCGGTACCATGATGGTAAGGATATTTGATACAA-3′ (SEQ ID NO:90)) and A114 (5′-ACGCAGTCGACTTAATTCAATAACATATTGATTCCT-3′ (SEQ ID NO:91)). PCR products were digested with Acc65I and SalI and cloned into pSA59 cut with the same enzymes, creating pSA61. To replace replication origin with p15A, pZA31-luc was digested with SacI and AvrII. Shorter fragment was purified and cloned into plasmid pSA61 cut with the same enzymes, creating pSA63.
To remove the non-coding region in pSA121, pSA121 was used as PCR template with a pair of primers A113 and A227 (5′-ACGCAGTCGACCTACAATTTTCCAGTAACTTCTCTA-3′ (SEQ ID NO:92)). PCR products were digested with Acc65I and SalI and cloned into pSA63 cut with the same enzymes, creating pSA125.
Medium and cultivation for 1-propanol and 1-butanol production. M9 medium containing 36 g/L glucose, 5 g/L yeast extract, 100 μg/ml ampicillin, 30 μg/ml kanamycin and 1000th dilution of Trace Metal Mix A5 (2.86 g H3BO3, 1.81 g MnCl2.4H2O, 0.222 g ZnSO4.7H2O, 0.39 g Na2MoO4.2H2O, 0.079 g CuSO4.5H2O, 49.4 mg Co(NO3)2.6H2O per liter water) was used for cell growth. Preculture in test tubes containing 3 ml of medium was performed at 37° C. overnight on a rotary shaker (250 rpm). Overnight culture was diluted 1:100 into 20 ml of fresh medium in a 250 ml screw cap conical flask. Cells were grown at 37° C. for 3 hr, followed by adding 0.1 mM IPTG. Cultivation was performed at 30° C. on a rotary shaker (250 rpm). Gas chromatography-flame ionization detector and high-performance liquid chromatography analyses were performed.
Directed evolution. Error-prone PCR was carried out using pSA63 (Table 19) as a template. A plasmid library of cimA variants was constructed on pSA63 by ligating the error-prone PCR product digested with Acc65I and SalI. 10 μl of the ligation reaction was used to transform 100 μl of XL10 Gold cells (Stratagene). The resultant library size was calculated ( ̃1×106 colonies) and the plasmid library was amplified on LB agar plates containing 30 μg/ml kanamycin. SA405 or SA408 was transformed with the plasmid library. The cells were incubated in 20 ml of M9 medium containing 10 g/L glucose, 100 μg/ml ampicillin and 30 μg/ml kanamycin, with shaking at 30° C. for 3 days. Plasmids were purified from the resulting cultures. DNA shuffling was carried out, KOD DNA polymerase was used for fragment amplification. A plasmid library construction and liquid culture selection were carried out. The resulting cultures were spread out on M9 agar plates containing 10 g/L glucose, 100 μg/ml ampicillin and 30 μg/ml kanamycin. The plates were incubated at 30° C. for 3 days.
Protein purification. The wild-type CimA and CimA3.7 were synthesized from pSA153 and pSA154 in E. coli strain BL21 Star (DE3) (Invitrogen, Carlsbad, Calif.), followed by purification with Ni-nitrilotriacetic acid spin columns (Qiagen, Valencia, Calif.). Protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, Calif.).
Citramalate synthase assay. The CimA enzyme activity was assayed by monitoring the production of CoA over time. Purified proteins (0.1 μM) were dissolved in 150 μl of TES buffer (0.1 M [pH 7.5]) containing various concentrations of acetyl-CoA and pyruvate. The production of CoA was confirmed to be linear over 1 h. After incubation at various temperatures for 1 h, 50 μl of 10 mM 5,5′-dithio-bis(2-nitrobenzoic acid) in 0.1 M Tris-HCl (pH 8.0) was added to measure the appearance of the free SH group of the released CoA SH. The absorbance at 412 nm was recorded. The concentrations of CoASH produced were calculated from a standard curve generated with various concentrations of 2-mercaptoethanol.
Transfer the citramalate pathway to E. coli. To produce 1-propanol and 1-butanol by the citramalate pathway, cimA (M. jannaschii) and leuABCD (E. coli) were cloned and expressed under the control of the IPTG inducible PLlacO1 promoter on a p15A derived plasmid (pSA63, FIG. 56A). An E. coli strain auxotrophic for L-isoleucine (ΔilvI and ΔilvB) was transformed with pSA63 and pSA55 (PLlacO1:: kivd-ADH2). The deletion of ΔilvI and ΔilvB is beneficial for two reasons (see, e.g., FIGS. 13A and B). First, the deletion eliminates the native substrate, 2-ketoisovalerate, for the leuABCD pathway, thus reducing the competitive substrate inhibition. Second, these deletions eliminate the production of 2-keto-3-methyl-valerate and 2-keto-4-methyl-pentanoate, which are competing substrates for Kivd. This strain produced about 350 mg/L 1-propanol and about 21 mg/L 1-butanol after 64 hr (FIG. 56B)
To test the activity of the pathway, an E. coli strain SA405 was used, which is deficient in ilvA and tdcB. This strain is auxotrophic for L-isoleucine as it cannot synthesize 2-ketobutyrate unless the citramalate pathway is active. Thus, the growth rate of the cell should reflect the activity of the citramalate pathway. Growth rates were compared for SA405 (ΔilvA and ΔtdcB) transformed with pSA63 (harboring wild-type cimA) (FIG. 56A), pCS27 (without cimA and leuABCD), or the wildtype strain (JCL16) (FIG. 56B). SA405 cells not expressing CimA were unable to grow without L-isoleucine (FIG. 56B). The citramalate pathway rescued the growth of SA405 under the same condition, although the growth rate of SA405 with the citramalate pathway was lower than that of JCL16 or SA405 with L-isoleucine (FIG. 56B). These result shows that the wild-type CimA is able to function in the synthetic pathway and produce 1-propanol and 1-butanol.
Improvement of 1-propanol and 1-butanol production by directed evolution strategy. A strategy of evolving CimA for alcohol production takes advantage of the fact that the product of the citramalate pathway, 2-ketobutyrate, is a precursor for isoleucine (FIGS. 13A and B). By deleting ilvA and tdcB from the host chromosome, the cell cannot synthesize 2-ketobutyrate unless the citramalate pathway is active. Thus, the growth rate of the cell should reflect the activity of the citramalate pathway, allowing a growth-based selection strategy.
The partial rescue of the isoleucine auxotroph allows the evolution of CimA based on growth improvement. In the first round of mutation, cimA variants were generated by error-prone PCR and mutants with increased growth were enriched in liquid media. Plasmids from the pool of fast-growing variants were then purified and used as templates for DNA shuffling in a second round of evolution. After these two rounds of selection, five variants of cimA were randomly picked and tested for 1-propanol and 1-butanol production. The cimA variant that leads to the highest 1-propanol production, and thus the largest 2-ketobutyrate pool and highest cimA activity, was designated cimA1 (FIG. 57A). This mutant was found to contain three amino acid substitutions (Ile47Val, Lys435Asn, and Val441A1a) and was subjected to the next two generations of mutagenesis, selection, and screening.
The next two rounds of selection were performed using the same scheme as the first two except that the selection pressure was increased by introducing an ilvI knockout in addition to ΔilvA and ΔtdcB in the host. IlvI is the large subunit of acetohydroxy acid synthase (AHAS) III. The other isozyme (AHAS I), encoded by the ilvBN genes, has a higher specificity to pyruvate than to 2-ketobutyrate. Endogenous concentrations of 2-ketobutyrate ( ̃10 μm) are much lower than Km value for 2-ketobutyrate of AHAS I ( ̃5 mM). This suggests that this deletion decreases the flux from 2-ketobutyrate to isoleucine, and thus requires more 2-ketobutyrate to synthesize isoleucine through the less efficient isozyme.
After the fourth round, eight colonies were randomly picked. The CimA variant that produced the highest amount of alcohols (denoted CimA2, FIG. 57) was sequenced. In addition to the amino acid substitutions in CimA1, CimA2 contains 2 amino acid substitutions (His126Gln and Thr204A1a) and a frame shift mutation at 1117 bp, creating a CimA variant missing the C-terminal domain from the 373rd residue.
The cimA2 mutant contains a stop codon at 1117 bp, indicating that this operon contains  ̃350 bp of non-coding region between the cimA and the leuA gene located on the synthetic operon. It has been known that large non-coding region decreases mRNA stability and translational efficiency. To eliminate the possibility of an expression deficiency of leuABCD downstream of cimA, the non-coding region from the plasmid (denoted CimA2A, FIG. 57) was removed. CimA2Δ was subjected to the next two generations of mutagenesis, selection and screening.
The next two rounds of selection were performed using the same scheme as the last two rounds. After the sixth round, 9 colonies were randomly picked for sequencing (FIG. 57). The selected cimA mutants were recloned into pSA63 to remove the possibility of extra mutations in the plasmid. All mutations were outside of the active site.
1-Propanol and 1-butanol production with the selected CimA variants. 1-Propanol and 1-butanol production was tested with the selected CimA variants in the strain with ΔilvI and ΔilvB. The ilvB and ilvI genes codes for subunits for AHASI and AHASIII which participate in the production of 2-ketoisovalerate and 2-keto-3-methyl-valerate (FIGS. 13A and B). The formation of these 2-keto acids reduces the yield of desired alcohols. As shown in FIG. 57, the production of 1-propanol from the strain with CimA1 increased 2.3-fold compared to the strain with the wild-type CimA. The production of 1-propanol and 1-butanol from the strain with CimA2 increased 3.9 and 4.3-fold, respectively, compared to the strain with the wild-type CimA. These results indicate that the selection method is effective in isolating CimA variants with increased activity.
To confirm the effect of the non-coding region, the production from the strain with CimA2 or CimA2Δ (FIG. 57) were compared. The production of 1-propanol and 1-butanol from the strain with CimA2Δ increased 2.4- and 1.6-fold, respectively, compared to the strain with CimA2 with the non-coding region. As a control, a truncated version of the wild-type cimA (denoted WTA) was also constructed. However, this construct showed diminished 1-propanol and 1-butanol production (FIG. 57). This result indicates that the truncated version of CimA requires other mutations (Ile47Val, His126Gln and Thr204A1a) for enhanced activity.
The strains expressing the CimA3 mutants showed similar production levels of 1-propanol with the strain expressing CimA2Δ (FIG. 57). However, 1-butanol production increased compared to the strain with CimA2Δ (FIG. 57). The production of 1-propanol and 1-butanol from the strain expressing the CimA3.7 variant increased 9.2- and 21.9-fold, respectively, compared to the strain with wild-type CimA. In addition to the amino acid substitutions in CimA2Δ, CimA3.7 contains 2 amino acid substitutions (Glu114Val and Leu238Ser).
Time profiles of alcohol production with CimA3.7. Both 1-propanol and 1-butanol production increased in a linear fashion up to 40 hours, after which the production rate appeared to decrease (FIGS. 58A and B). This strain produced more than 3.5 g/L 1-propanol and 524 mg/L 1-butanol after 92 hours. The formation of ethanol may be due to the native production by adhE, or by the decarboxylation of pyruvate by Kivd (FIG. 58C). To minimize ethanol formation, adhE was deleted from the host strain. However, initial results showed this deletion impaired cell growth and decreased both 1-propanol and 1-butanol production, suggesting that ethanol production may be required to maintain metabolic balance. Cell growth stopped after 10 hours and remained stationary during alcohol production (FIG. 58D). This result indicated overexpression of the citramalate pathway coupled with 1-propanol and 1-butanol production can be tolerated by E. coli. The rate of glucose consumption decreased after 40 hr, which is consistent with the alcohol production rates (FIG. 58E).
The data demonstrate success in 1-propanol and 1-butanol production with directed evolution coupled with L-isoleucine biosynthesis. The data demonstrate that the 2-keto acid based pathway for alcohol production enables a growth-based selection.
CimA is a homologue of LeuA. The activities of CimA and LeuA are regulated by the corresponding amino acid end products L-isoleucine or L-leucine, respectively. It has been shown that L-leucine binds to C-terminal domain in LeuA (FIGS. 58 and 59). CimA2 is missing this C-terminal domain shown by homology alignment (FIG. 59B) to be involved in feedback inhibition, suggesting that CimA2 may be insensitive to feedback inhibition by L-isoleucine (FIG. 59; see also FIG. 57C-D). However, this truncated form needed additional mutations to exhibit high activity. This led to speculation that the structural stability or the dimer formation rate was improved by some of these mutations.
The CimA employed in the directed evolution process described herein was isolated from M. jannaschii, an extremely thermophilic archaeon. The acquired mutations are not located near the catalytic center, suggesting these mutations may stabilize its active structure in 30° C. It has been shown that the thermophilic proteins have higher residue volume, higher residue hydrophobicity, more charged amino acids, and fewer uncharged polar residues than mesophilic proteins. Twelve out of eighteen substitutions identified in CimA3s resulted in the selection of amino acids with smaller sidechains. Eight substitutions resulted in the selection of lower residue hydrophobicity, while the replacement of native residues with uncharged polar residues was observed six times. Additionally, eight substitutions resulted in the replacement of a charged residue with a non-charged residue. Most of the substitutions observed were consistent with the differences between the thermophilic and mesophilic proteins.
The citramalate pathway was then used to enhance the production of 1-propanol and 1-butanol (FIG. 13). An E. coli strain (KS145) auxotrophic for L-isoleucine, leucine, and valine (ΔilvI and ΔilvB). was transformed with pSA63 (or other plasmids containing variants of cimA) and pSA55 (PLlacO1::kivd-ADH2). The deletions of ilvI and ilvB (FIG. 13) were introduced for two reasons. First, the deletions eliminated the native substrate, 2-ketoisovalerate, for the leuABCD pathway, thus reducing the competitive substrate inhibition. Second, these deletions eliminated the production of 2-keto-3-methyl-valerate and 2-keto-4-methyl-pentanoate, which are competing substrates for Kivd. The strain expressing the wild-type cimA gene (KS145/pSA63/pSA55) produced 302 mg/liter 1-propanol and 18 mg/liter 1-butanol after 40 h (FIG. 60). KS145 with pSA55 only, where Kivd utilizes endogenous 2-keto acids, produced 40 mg/liter 1-propanol and 10 mg/liter 1-butanol under the same condition. KS145 without pSA55 and pSA63 produced neither 1-propanol nor 1-butanol (FIG. 60). Note that the yeast extract was added to the medium to boost the cell density. However, without glucose added to the medium the cells produce no alcohols (FIG. 60), indicating that these products were derived from glucose, but not from yeast extract.
1-Propanol and 1-butanol production was tested with the selected CimA variants in KS145 by replacing pSA63 with corresponding plasmids (Table 19) containing various cimA mutants. As shown in Table 20, the production of 1-propanol from the strain with CimA1 increased 2.3-fold compared to the strain with the wild-type CimA. The production of 1-propanol and 1-butanol from the strain with CimA2 increased 3.9- and 4.3-fold, respectively, compared to the strain with the wild-type CimA. These results indicate that our selection method is effective in isolating CimA variants which lead to higher alcohol production titers.







TABLE 20







1-Propanol and 1-butanol production with the selected CimA


mutants.










Production (mg/liter) ofa:












Protein
1-Propanol
1-Butanol






WT
302 (1)
18 (1)  



CimA1
  695 (2.3)
22 (1.2)



CimA2
1,178 (3.9)
77 (4.3)



CimA2Δ
2,416 (8.0)
106 (5.9) 



WTΔ
  60 (0.2)
NDb



CimA3.1
2,471 (8.2)
373 (20.7)



CimA3.2
2,567 (8.5)
254 (14.1)



CimA3.3
2,392 (7.9)
124 (6.9) 



CimA3.4
2,452 (8.1)
114 (6.2) 



CimA3.5
2,328 (7.7)
140 (7.8) 



CimA3.6
2,353 (7.8)
146 (8.0) 



CimA3.7
2,778 (9.2)
393 (21.9)



CimA3.8
2,481 (8.2)
391 (21.7)



CimA3.9
2,446 (8.1)
199 (11.0)





aCultures were grown at 30° C. for 40 h. The titers of 1-propanol and 1-butanol production from KS145 with pSA55 and pSA63 (cimA-leuABCD) and its variations are shown. The change (fold) relative to the strain containing wild-type CimA (WT) is in shown parentheses.


bND, not detectable.






To confirm the effect of the noncoding region created by mutation in CimA2, we compared the production from the strain with CimA2 with that from the strain with CimA2Δ (Table 20). The production of 1-propanol and 1-butanol from the strain with CimA2Δ increased 2.4- and 1.6-fold, respectively, compared to the strain with CimA2. As a control, a truncated version of the wild-type cimA (denoted WTΔ) without the acquired amino acid substitutions in CimA2 was generated. However, this construct showed diminished 1-propanol and 1-butanol production (Table 20). This result indicates that the truncated version of CimA requires other mutations (Ile47Val, His126Gln, and Thr204A1a) for enhanced activity.
The strains expressing the CimA3.1 to CimA3.9 mutants showed production levels of 1-propanol similar to those of the strain expressing CimA2Δ (Table 20). However, 1-butanol production increased compared to that in the strain with CimA2Δ (Table 20). The production of 1-propanol and 1-butanol from the strain expressing the CimA3.7 variant increased 9.2- and 21.9-fold, respectively, compared to that in the strain with wild-type CimA. In addition to the amino acid substitutions in CimA2Δ, CimA3.7 contains two new amino acid substitutions (Glu114Val and Leu238Ser).
Time profiles of alcohol production with CimA3.7. Since CimA3.7 is the best alcohol producer, the production profiles of KS145/pSA55/pSA142 (containing cimA3.7) were characterized in shake flasks. Cell growth stopped after 10 h and remained stationary during alcohol production (FIG. 58A). The growth with IPTG was similar to that without IPTG, indicating that overexpression of this pathway had almost no effect on cell growth. Both 1-propanol and 1-butanol production increased in a linear fashion up to 40 h, after which the production rate appeared to decrease (FIGS. 58B and C). This strain produced more than 3.5 g/liter 1-propanol and 524 mg/liter 1-butanol after 92 h. The formation of ethanol may be due to the native production by adhE or by the decarboxylation of pyruvate by Kivd (FIG. 58D). This result indicates that overexpression of the citramalate pathway coupled with 1-propanol and 1-butanol production can be tolerated by E. coli. The rate of glucose consumption decreased after 40 h, which is consistent with the alcohol production rates (FIG. 58E). Acetate and lactate are the major organic acids produced at a significant level (FIG. 58F).
The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the devices, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
2-Methyl-1-butanol production via combination of threonine and citramalate pathways: Using wild-type E. coli BW25113 as a host and two plasmids for the expression of pathway genes (pAFC78 [pLlacO1::kivd (L. lactis) ADH2 (S. cerevisiae) ilvA (C. glutamicum), pLlacO1::cimA (M. jannaschii) leuBCD (E. coli), ColE1 origin, AmpR] and pAFC75 [pLlacO1::thrAFBRBC (E. coli), pLlacO1::ilvGM (S. typhimurium) ilvCD (E. coli), P15A origin, CmR]), this strain was cultured using a 1% inoculum from an LB overnight into a 250 mL screw-top culture flask containing 10 mL of M9 medium with 72 g/L glucose, 5 g/L yeast extract, and 1:1000 dilution of A9 metal mix as well as 200 mg/L ampicillin and 20 mg/L chloramphenicol. The culture was grown at 37° C. for 2.5 hr and then induced with 0.1 mM IPTG and then grown at 30° C. for upwards of 72 hr. The pH of the culture was maintained near 7 by adjusting with appropriate amounts of 10N NaOH every 12 hr. The final titer resulting from this culture is about 3.0 to 3.2 g/L 2-methyl-1-butanol.
A summary of the data is presented in FIG. 61. The other data shows the production achieved using only the threonine pathway for 2-methyl-1-butanol production under similar culture conditions. The host strain for this data is E. coli BW25113 ΔmetA Δtdh.
Direct comparison of threonine, citramalate, and combination pathways for 2-methyl-1-butanol production: Using E. coli BW25113 ΔthrB to knockout endogenous threonine production for the sake of comparison, several plasmids were used to express the threonine and citramalate pathways in various combinations. The plasmids used are as follows: pAFC67 [pLlacO1::kivd (L. lactis) ADH2 (S. cerevisiae) ilvA (C. glutamicum), pLlacO1: ilvGM (S. typhimurium) ilvCD (E. coli), ColE1 origin, AmpR] was used in all cultures for conversion of pyruvate and 2-ketobutyrate into 2-methyl-1-butanol; pAFC88 [pLtetO1:: thrAFBRBC (E. coli), tetR, pSC101 origin, SpecR] and pAFC89 [pLtetO1::cimA (M. jannaschii) leuBCD (E. coli), tetR, pSC101 origin, SpecR] were used separately to generate 2-ketobutyrate by the threonine and citramalate pathways, respectively; pAFC94 [pLtetO1:: thrAFBRBC (E. coli), tetR, P15A origin, CmR] and pAFC95 [pLtetO1::cimA (M. jannaschii) leuBCD (E. coli), tetR, P15A origin, CmR] were used in combination with either pAFC88 or pAFC89 to show the effect of further threonine pathway expression (pAFC88+pAFC94), further citramalate pathway expression (pAFC89+pAFC95), or the combination of the pathways (pAFC88+pAFC95, pAFC89+pAFC94). These strains were cultured using a 1% inoculum from an LB overnight into a 125 mL screw-top culture flask containing 5 mL of M9 medium with 15 g/L glucose, 5 g/L yeast extract, and 1:1000 dilution of A9 metal mix as well as 200 mg/L ampicillin, 50 mg/L of spectinomycin, and 20 mg/L chloramphenicol as needed. The cultures were grown at 37° C. for 2 hr and then induced with 0.1 mM IPTG. For the cultures expressing pAFC67 and either pAFC88 or pAFC89, the inducer anhydrous tetracycline (aTC) was also added at levels varying from 0.01 to 1.0 μg/mL to show a titration of the threonine or citramalate pathway, respectively. Strains expression three plasmids (e.g. pAFC67+pAFC88+pAFC95) were induced only at 1.0 μg/mL of aTC. Upon induction the cultures were grown at 30° C. for 24 hr. Under these conditions, the titer of 2-methyl-1-butanol obtained from the threonine pathway was between about 650 and 750 mg/L, the titer from the citramalate pathway was between about 525 and 625 mg/L, while the titer obtained from a combination of the two pathways was between about 1300 and 1400 mg/L. The combination of threonine and citramalate pathways therefore constitutes an improvement of approximately 100% over either pathway expressed individually. The data is summarized in FIG. 62.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

## Claims

1. A recombinant microorganism selected from a genus of Escherichia, Corynebacterium, Lactobacillus, Lactococcus, Salmonella, Enterobacter, Enterococcus, Erwinia, Pantoea, Morganella, Pectobacterium, Proteus, Serratia, Shigella, Klebsiella, Citrobacter, Saccharomyces, Dekkera, Kluyveromyces, and Pichia that produces an alcohol selected from the group consisting of:
(a) 1-propanol,
(b) 1-butanol, and
(c) 2-methyl 1-butanol,

wherein the alcohol is produced from a metabolite comprising 2-ketobutyrate;
wherein the recombinant microorganism comprises a recombinant citramalate synthase (cimA) having at least 95% identity to SEQ ID NO:89 and having citramalate synthase activity;
wherein the recombinant microorganism comprises a 2-keto-acid decarboxylase having at least 95% identity to SEQ ID NO:28, 32, 34 or 36 and having 2-keto-acid decarboxylase activity;
wherein the recombinant microorganism comprises a recombinant thrA*, a recombinant thrB from E. coli, and a recombinant thrC from E. coli, wherein the recombinant thrA* is a feedback-resistant mutant of thrA from E. Coli, and wherein the recombinant thrA* encodes a homoserine dehydrogenase, the recombinant thrB encodes a homoserine kinase and the recombinant thrC encodes a threonine synthase; and
wherein the recombinant microorganism is recombinantly engineered to express or overexpress the citramalate synthase, the 2-keto-acid decarboxylase, the homoserine dehydroqenase, the homoserine kinase, and the threonine synthase as compared to a parental microorganism lacking said engineering when grown under the same conditions and wherein:
(i) the microorganism expresses an isopropylmalate isomerase (LeuCD) having at least 95% identity to SEQ ID NO:54 and 56, an isopropylmalate dehydrogenase (LeuB) having at least 90% identity to SEQ ID NO:52; a 2-isopropylmalate synthase (LeuA) having at least 95% identity to SEQ ID NO:50; and an alcohol dehydrogenase (Adh2) having at least 90% identity to SEQ ID NO:38, for production of 1-butanol;
(ii) the microorganism expresses an isopropylmalate isomerase (LeuCD) having at least 95% identity to SEQ ID NO:54 and 56, an isopropylmalate dehydrogenase (LeuB) having at least 90% identity to SEQ ID NO:52, and an alcohol dehydrogenase (Adh2) having at least 90% identity to SEQ ID NO:38, for production of 1-propanol; or
(iii) the microorganism expresses an isopropylmalate isomerase (LeuCD) having at least 95% identity to SEQ ID NO:54 and 56, an isopropylmalate dehydrogenase (LeuB) having at least 90% identity to SEQ ID NO:52, an acetohydroxy acid synthase (IlvHI) having at least 95% identity to SEQ ID NO:40 and 42, an acetohydroxy acid isomeroreductase (IlvC) having at least 95% identity to SEQ ID NO:44 and an alcohol dehydrogenase (Adh2) having at least 90% identity to SEQ ID NO:38, for production of 2-methyl 1-butanol.

2. The recombinant microorganism of claim 1, wherein the microorganism comprises reduced ethanol production compared to the parental microorganism.

3. The recombinant microorganism of claim 1, wherein the microorganism comprises a reduction or inhibition in the conversion of acetyl-coA to ethanol.

4. The recombinant microorganism of claim 1, wherein the recombinant microorganism comprises a reduction of an ethanol dehydrogenase thereby providing a reduced ethanol production in the microorganism.

5. The recombinant microorganism of claim 4, wherein the microorganism is derived from E. coli.

6. The recombinant microorganism of claim 5, wherein the ethanol dehydrogenase is adhE.

7. The recombinant microorganism of claim 6, wherein the microorganism comprises a deletion or knockout of adhE.

8. The recombinant microorganism of claim 1, wherein the parental microorganism comprises a citramalate synthase.

9. The recombinant microorganism of claim 1, wherein the parental microorganism comprises a 2-keto-acid decarboxylase.

10. The recombinant microorganism of claim 1, wherein the 2-keto-acid decarboxylase is encoded by a polynucleotide selected from the group consisting of pdc from Clostridium acetobutylicum, aro10 from Saccharomyces cerevisiae, thi3 from Saccharomyces cerevisiae, and kivd from Lactococcus lactis, and a homolog or variant of any of the foregoing having at least 95% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:31, SEQ ID NO:33, and SEQ ID NO:27 and wherein the polynucleotide encodes a polypeptide having 2-keto acid decarboxylase activity.

11. The recombinant microorganism of claim 10, wherein the 2-keto-acid decarboxylase is encoded by a polynucleotide derived from a kivd gene of Lactococcus lactis and having a sequence as set forth in SEQ ID NO:28, or homolog thereof having at least 95% identity to SEQ ID NO:28.

12. The recombinant microorganism of claim 1, wherein the recombinant microorganism comprises a deletion or knockout in a gene encoding an enzyme that catalyzes the conversion of homoserine to o-succinylhomoserine and/or a deletion or knockout in a gene encoding an enzyme that catalyzes the catabolism of threonine to 2-amino-3-ketobutyrate.

13. The recombinant microorganism of claim 12, wherein the recombinant microorganism further comprises one or more deletions or knockouts in a gene encoding an enzyme that (1) catalyzes the conversion of acetyl-coA to ethanol, (2) catalyzes the conversion of pyruvate to lactate, (3) catalyzes the conversion of fumarate to succinate, (4) catalyzes the conversion of acetyl-coA and phosphate to coA and acetyl phosphate, (5) catalyzes the conversion of acetyl-coA and formate to coA and pyruvate, (6) catalyzes the condensation of the acetyl group of acetyl-CoA with 3-methyl-2-oxobutanoate (2-oxoisovalerate), (7) catalyzes the isomerization between 2-isopropylmalate and 3-isopropylmalate, (8) catalyzes the conversion of alpha-keto acid to branched chain amino acids, wherein the gene is IlvE from E. coli, IlvE from S. flexneri, or IlvE from S. dysenteriae, and/or tyrB from E. coli, tyrB from B. petrii, or tyrB from S. enterica, (9) catalyzes the synthesis of Phe, Tyr, Asp or Leu, wherein the gene is aspC from E. coli, and (10) catalyzes the conversion of pyruvate to acetyl-coA.

14. A recombinant microorganism selected from a genus of Escherichia, Corynebacterium, Lactobacillus, Lactococcus, Salmonella, Enterobacter, Enterococcus, Erwinia, Pantoea, Morganella, Pectobacterium, Proteus, Serratia, Shigella, Klebsiella, Citrobacter, Saccharomyces, Dekkera, Kluyveromyces, and Pichia, wherein the microorganism has been recombinantly engineered to express or overexpress (a)-(i) as compared to a parental microorganism lacking said engineering when grown under the same conditions:
a) citramalate synthase (cimA) having at least 95% identity to SEQ ID NO:89 and having citramalate synthase activity;
b) acetohydroxy acid synthase having at least 95% identity to SEQ ID NO:40 and 42;
c) acetohydroxy acid isomeroreductase having at least 95% identity to SEQ ID NO:44;
d) dihydroxy-acid dehydratase having at least 95% identity to SEQ ID NO:46;
e) 2-keto-acid decarboxylase having at least 95% identity to SEQ ID NO:28, 32, 34 or 36 and having 2-keto-acid decarboxylase activity;
f) alcohol dehydrogenase having at least 90% identity to SEQ ID NO:38;
g) feedback resistant mutant of thrA from E. coli having homoserine dehydrogenase activity;
h) E. coli thrB; and
i) E. coli thrC;
wherein the recombinant microorganism produces 2-methyl 1-butanol.

15. A method for producing 1-propanol, 1-butanol, or 2-methyl 1-butanol, the method comprising:
a) providing a recombinant microorganism of any one of claim 1 or 14;
b) culturing the microorganism of a) in the presence of a suitable substrate or metabolic intermediate and under conditions suitable for the conversion of the substrate to 1-propanol, 1-butanol, or 2-methyl 1-butanol; and
c) substantially purifying the 1-propanol, 1-butanol, or 2-methyl 1-butanol.