[0001] This application claims priority to U.S. Provisional Application Serial No. 61/454,943, filed March 21 , 201 1 , and is a continuation-in-part application of U.S. Appiiation Serial No. 13/228,342, filed September 8, 201 1 , which is a divisional of U.S. Application Serial No. 12/953,884, filed November 24, 2010, now U.S. Patent No. 8,017,376, which claims the benefit of U.S. Provisional Application Serial No. 61/263,952, filed November 24, 2009, and U.S. Provisional Application Serial No. 61/350,209, filed June 1 , 2010, all of which are herein incorporated by reference in their entireties for all purposes.

ACK OWLEDGME T OF GOVERNMENTAL SUPPORT

[0002] This invention was made with government support under Contract No. ΠΡ- 0823122, awarded by the National Science Foundation, and under Contract No. EP- D-09-023, awarded by the Environmental Protection Agency. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] Recombinant microorganisms and methods of producing such organisms are provided. Also provided are methods of producing beneficial metabolites including fuels, chemicals, and amino acids by contacting a suitable substrate with recombinant microorganisms and enzymatic preparations therefrom.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0004] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: GEVO_041_15WO_SeqList_ST25.txt, date recorded: October 20, 201 1 , file size: 748 kilobytes).

BACKGROUND

[0005] Dihydroxyacid dehydratase (DHAD) is an enzyme that catalyzes the

1

364561 vl/CO conversion of 2,3-dihydroxyisovaierate to a-ketoisovalerate and of 2,3-dihydroxy-3- methylvalerate to 2-keto-3-methyIvaIerate. This enzyme plays an important role in a variety of biosynthetic pathways, including pathways producing valine, isoleucine, leucine and pantothenic acid (vitamin B5). DHAD also catalyzes the conversion of 2,3-dihydroxyisovalerate to a-ketoisovaierate as part of isobutanol biosynthetic pathways disclosed in commonly owned and co-pending US Patent Publication Nos. 2009/0226991 and 2010/0143997. In addition, biosynthetic pathways for the production of 3-rnethyi-1 -butanol and 2-methyl-1 -butanoi use DHAD to convert 2,3- dihydroxyisovalerate to a-ketoisovaierate and 2,3-dihydroxy-3~methylvalerate to 2- keto-3-methylvalerate, respectively (Atsumi et al., 2008, Nature 451 (7174): 86-9), [0006] DHAD is an essential enzyme in all of these biosynthetic pathways, hence, it is desirable that recombinant microorganisms engineered to produce the above- mentioned compounds exhibit optimal DHAD activity. The optimal level of DHAD activity will typically have to be at levels that are significantly higher than those found in non-engineered microorganisms in order to sustain commercially viable productivities, yields, and titers. The present application addresses this need by engineering recombinant microorganisms to improve their DHAD activity.

SUMMARY OF THE INVENTION

[0007] The present inventors have discovered that overexpression of the iron sulfur ("FeS" or "Fe/S") cluster assembly genes NFS1 and ISD11 in a recombinant yeast microorganism improves DHAD activity. Thus, the invention relates to recombinant yeast ceils engineered to provide increased heterologous or native expression of NFS1 and/or ISD11 or homologs thereof. In general, cells that overexpress NFS1 and/or ISD11 or homologs thereof exhibit an enhanced ability to produce beneficial metabolites such as isobutanol, 3-methyl- -butanoi, 2~methyl-1 ~ butanoi, 1 -butanoi, valine, isoleucine, leucine, and pantothenic acid.

[0008] One aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more cysteine desulfurase (Nfs1 ) proteins or homologs thereof. In one embodiment, the Nfs1 protein is selected from SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231 , SEO ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241 , SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, and SEQ ID NO: 249, or

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364561 vl/CO homoiogs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Nfsi proteins or homoiogs thereof is a native polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Nfsi proteins or homoiogs thereof is a heterologous polynucleotide.

[0009] Another aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more iron-sulfur protein biogenesis desulfurase-interacting (Isd1 1 ) proteins or homoiogs thereof. In one embodiment, the !sd1 1 protein is selected from SEQ ID NO: 251 , SEO ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEO ID NO: 259, SEQ ID NO: 261 , SEQ ID NO: 283, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 289, SEQ ID NO: 271 , and SEQ ID NO: 273, or homoiogs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Isd1 1 proteins or homoiogs thereof is a native polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Isd1 1 proteins or homoiogs thereof is a heterologous polynucleotide.

[0010] Another aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more Nfsi proteins or homoiogs thereof and one or more polynucleotides encoding one or more encoding one or more Isd1 1 proteins or homoiogs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Nfsi proteins or one or more of the polynucleotides encoding said one or more Isd1 1 proteins or homoiogs thereof is a native polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Nfsi proteins or one or more of the polynucleotides encoding said one or more Isd1 1 proteins or homoiogs thereof is a heterologous polynucleotide.

[0011] Another aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein the activity of one or more Nfsi proteins or homoiogs thereof and/or one or more Isd1 1 proteins or homoiogs thereof is increased. In one embodiment, one or more of the Nfsi proteins and/or one or more of the Isd1 1 proteins is encoded by a native polynucleotide. In another embodiment, one or more of the Nfsi proteins and/or one or more of the Isd1 1 proteins is encoded by a heterologous polynucleotide.

3

364561 vl/CO [0012] In various embodiments described herein, the recombinant microorganisms of the invention that comprise a DHAD-requiring biosynthetic pathway may also be engineered to overexpress one or more polynucleotides encoding one or more Aft proteins or homo!ogs thereof as described below.

[0013] In various embodiments described herein, the DHAD-requiring biosynthetic pathway may be selected from isobutanol, 3-methy!-1 -butanol, 2-methyl- 1 -butanoi, valine, isoieucine, leucine, and/or pantothenic acid biosynthetic pathways. In various embodiments described herein, the DHAD enzyme which acts as part of an isobutanol, 3-methyl-1 -butano!, 2-methyi-1 -butano!, valine, isoieucine, leucine, and/or pantothenic acid biosynthetic pathway may be localized to the cytoso!. in alternative embodiments, the DHAD enzyme which acts as part of an isobutanol, 3- methyl-1 -butanoi, 2-methy!-1 -butanol, valine, isoieucine, leucine, and/or pantothenic acid biosynthetic pathway may be localized to the mitochondria. In additional embodiments, a DHAD enzyme which acts as part of an isobutanol, 3-methyl-1 - butanoi, 2-methy!-1 -butanol, valine, isoieucine, leucine, and/or pantothenic acid biosynthetic pathway is localized to the cytosol and the mitochondria.

[0014] As described herein, an exemplary DHAD-requiring biosynthetic pathway is an isobutanol producing biosynthetic pathway, i.e., an isobutanol producing metabolic pathway. Accordingly, one specific aspect of the invention is directed to a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway and wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more Nfs1 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Nfs1 proteins or homologs thereof is a native polynucleotide, !n another embodiment, one or more of the polynucleotides encoding said one or more Nfs1 proteins or homologs thereof is a heterologous polynucleotide.

[0015] Another aspect of the invention is directed to a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway and wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more Isd1 1 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Isd1 1 proteins or homologs thereof is a native polynucleotide. In another embodiment, one or more of the polynucleotides

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364561 vl/CO encoding said one or more Isd1 1 proteins or homologs thereof is a heterologous polynucleotide.

[0016] Another aspect of the invention is directed to a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway and wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more Nfs1 proteins or homologs thereof and one or more polynucleotides encoding one or more encoding one or more isdl 1 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Nfs1 proteins or one or more of the polynucleotides encoding said one or more !sd1 1 proteins or homologs thereof is a native polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Nfs1 proteins or one or more of the polynucleotides encoding said one or more !sd 1 1 proteins or homologs thereof is a heterologous polynucleotide.

[0017] Another aspect of the invention is directed to a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway and wherein the activity of one or more Nfs1 proteins or homologs thereof and/or one or more Isdl 1 proteins or homologs thereof is increased. In one embodiment, one or more of the Nfs1 proteins and/or one or more of the Isd1 1 proteins is encoded by a native polynucleotide. In another embodiment, one or more of the Nfs1 proteins and/or one or more of the Isdl 1 proteins is encoded by a heterologous polynucleotide.

[0018] The present inventors have also discovered that overexpression of the transcriptional activator genes AFT1 and/or AFT2 or homologs thereof in a recombinant yeast microorganism improves DHAD activity. Thus, the invention relates to recombinant yeast ceils engineered to provide increased heterologous or native expression of AFT1 and/or AFT2 or homologs thereof. In general, cells that overexpress AFT1 and/or AFT2 or homologs thereof exhibit an enhanced ability to produce beneficial metabolites such as isobutanol, 3-methyl-l -butanoi, 2-methyl-1 - butanoi, valine, isoleucine, leucine, and pantothenic acid.

[0019] One aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more Aft proteins or homologs thereof. In one embodiment, the Aft protein is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ

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364561 yl/CO ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEO ID NO: 18, SEQ ID NO: 20, SEO ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 209, SEQ ID NO: 21 1 , SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221 , SEQ ID NO: 223, and SEQ ID NO: 225. In another embodiment, one or more of the polynucleotides encoding said one or more Aft proteins or homologs thereof is a native polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said one or more Aft proteins or homologs thereof is a heterologous polynucleotide.

[0020] In a specific embodiment according to this aspect, the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said microorganism has been engineered to overexpress a polynucleotide encoding Aft1 (SEQ ID NO: 2) and/or Aft2 (SEQ ID NO: 4) or a homolog thereof. In one embodiment, the polynucleotide encoding the Aft protein or homolog thereof is native to the recombinant microorganism. In another embodiment, the polynucleotide encoding the Aft protein or homolog thereof is heterologous to the recombinant microorganism.

[0021] Another aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein the activity of one or more Aft proteins or homologs thereof is increased. In one embodiment, the Aft protein is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 209, SEQ ID NO: 21 1 , SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221 , SEQ ID NO: 223, and SEQ ID NO: 225. In one embodiment, the polynucleotide encoding the Aft protein or homolog thereof is native to the recombinant microorganism. In another embodiment, the polynucleotide encoding the Aft protein or homolog thereof is heterologous to the recombinant microorganism.

[0022] Another aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said microorganism has been engineered to overexpress one or more polynucleotides encoding one or more proteins or homologs thereof regulated by an Aft protein or homolog thereof. In one embodiment, the proteins regulated by an Aft protein or

6

364561 yl/CO homolog thereof are selected from FET3, FET4, FET5, FTR1, FTH1, SMF3, MRS4, CCC2, COT1, ATX1, FRE1, FRE2, FRE3, FRE4, FRE5, FRE6, FIJI, FIT2, FIT3, ARN1, ARN2, ARN3, ARN4, ISU1, ISU2, TIS11, HMX1, AKR1, PCL5, YOR387C, YHL035C, YMR034C, iCY2, PRY1, YDL124W, BNA2, ECM4, LAP 4, YOL083W, YGR146C, B!Q5, YDR271C, OYE3, CTH1, CTH2, MRS3, MR84, HSP26, YAP2, VMR1, ECL1, OSW1, NFT1, ARA2, TAF1/TAF130/TAF145, YOR225W, YKR1Q4W, YBR012C, and YMR041C or homo!ogs thereof. In a specific embodiment, the protein regulated by an Aft protein or homolog thereof is ENB1 . In another specific embodiment, the protein regulated by an Aft protein or homologs thereof is FET3. !n yet another specific embodiment, the protein regulated by an Aft protein or homolog thereof is SMF3. In one embodiment, all genes demonstrated to increase DHAD activity and/or the production of a metabolite from a DHAD-requiring biosynthetic pathway are overexpressed. Where none of the AFT regulon genes expressed alone are effective in increasing DHAD activity and/or the production of a metabolite from a DHAD-requiring biosynthetic pathway, then 1 , 2, 3, 4, 5, or more of the genes in the AFT regulon may be overexpressed together,

[0023] In various embodiments described herein, the DHAD-requiring biosynthetic pathway may be selected from isobutanol, 3-methy!-1 -butanol, 2~methyl- 1 -butano!, valine, isoieucine, leucine, and/or pantothenic acid biosynthetic pathways. In various embodiments described herein, the DHAD enzyme which acts as part of an isobutanol, 3-methyl-1 -butano!, 2-methy!-1 -butano!, valine, isoieucine, leucine, and/or pantothenic acid biosynthetic pathway may be localized to the cytosol. in alternative embodiments, the DHAD enzyme which acts as part of an isobutanol, 3- methyl-1 -butanoi, 2-methyi-1 -butanoi, valine, isoieucine, leucine, and/or pantothenic acid biosynthetic pathway may be localized to the mitochondria. In additional embodiments, a DHAD enzyme which acts as part of an isobutanol, 3-methyl-1 - butanoi, 2-methy!-1 -butano!, valine, isoieucine, leucine, and/or pantothenic acid biosynthetic pathway is localized to the cytosol and the mitochondria.

[0024] In one embodiment, the invention is directed to a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway and wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more Aft proteins or homologs thereof. In one embodiment, the Aft protein is selected from SEQ ID NO: 2, SEO ID NO: 4, SEQ ID NO: 6, SEO ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID

7

364561 yl/CO NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 209, SEQ ID NO: 21 1 , SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221 , SEQ ID NO: 223, and SEQ ID NO: 225.

[0025] In a specific embodiment, the invention is directed to a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway and wherein said microorganism is engineered to overexpress a polynucleotide encoding Aft1 (SEQ ID NO: 2) or a homolog thereof. In another specific embodiment, the invention is directed to a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway and wherein said microorganism is engineered to overexpress a polynucleotide encoding Aft2 (SEQ ID NO: 4) or a homolog thereof. In yet another embodiment, the invention is directed to a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway and wherein said microorganism is engineered to overexpress a polynucleotide encoding Aft1 (SEQ ID NO: 2) or a homolog thereof and Aft2 (SEQ ID NO: 4) or a homolog thereof.

[0026] In each of the aforementioned aspects and embodiments, the Aft protein may be a constitutively active Aft protein or a homolog thereof, in one embodiment, the constitutively active Aft protein or homolog thereof comprises a mutation at a position corresponding to the cysteine 291 residue of the native S. cerevisiae Aft1 (SEQ ID NO: 2). In a specific embodiment, the cysteine 291 residue is replaced with a phenylalanine residue. In another embodiment, the constitutively active Aft protein or homolog thereof comprises a mutation at a position corresponding to the cysteine 187 residue of the native 8. cerevisiae Aft2 (SEQ ID NO: 2). In a specific embodiment, the cysteine 187 residue is replaced with a phenylalanine residue.

[0027] In another embodiment, the invention is directed to a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, wherein said microorganism has been engineered to overexpress one or more polynucleotides encoding one or more proteins or homologs thereof regulated by an Aft protein or homolog thereof. In one embodiment, the proteins regulated by Aft or a homolog thereof are selected from FET3, FET4, FET5, FTR1, FTH1, SMF3, MRS4, CCC2, COT1, ATX1, FRE1, FRE2 _; FRE3, FRE4, FRE5, FRE6, Fill FIT2, FIT3, ARN1, ARN2, ARN3, ARN4,

8

364561 yl/CO ISU1, !SU2, TIS11, HMX1, AKR1, PCL5, YOR387C, YHL035C, YMR034C, !CY2, PRY1, YDL124W, BNA2, ECM4, LAP4, YOL083W, YGR146C, β/05, YDR271C, OYE3, CTH1, CTH2, MRS3, MRS4, HSP26, YAP2, VMR1, ECU, Q8W1, NFT1, ARA2, TAF1/TAF130/TAF145, YOR225W, YKR104W, YBR012C, and YMR041C or homologs thereof. In a specific embodiment, the protein regulated by an Aft protein or homolog thereof is ENB1 . In another specific embodiment, the protein regulated by an Aft protein or homologs thereof is FET3. In yet another specific embodiment, the protein regulated by an Aft protein or homolog thereof is SMF3. In one embodiment, ail genes demonstrated to increase DHAD activity and/or the production of a metabolite from a DHAD-requiring biosynthetic pathway are overexpressed. Where none of the AFT regulon genes expressed alone are effective in increasing DHAD activity and/or the production of a metabolite from a DHAD-requiring biosynthetic pathway, then 1 , 2, 3, 4, 5, or more of the genes in the AFT regulon may be overexpressed together.

[0028] In various embodiments described herein, the recombinant microorganism comprises an isobutanol producing metabolic pathway. In one embodiment, the isobutanol producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanol. In another embodiment, the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.

[0029] In one embodiment, one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol. In one embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol. In another

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364561 yl/CO embodiment, the recombinant microorganisms comprise an isobutano! producing metabolic pathway with at least two isobutano! pathway enzymes localized in the cytosoi. In yet another embodiment, the recombinant microorganisms comprise an isobutano! producing metabolic pathway with at least three isobutano! pathway enzymes localized in the cytosoi. In yet another embodiment, the recombinant microorganisms comprise an isobutano! producing metabolic pathway with at least four isobutano! pathway enzymes localized in the cytosoi. In an exemplary embodiment, the recombinant microorganisms comprise an isobutano! producing metabolic pathway with five isobutano! pathway enzymes localized in the cytosoi. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with all isobutanol pathway enzymes localized in the cytosoi. In a further exemplary embodiment, at least one of the pathway enzymes localized to the cytosoi Is a cytosoiicaily active DHAD enzyme as disclosed herein.

[0030] In various embodiments described herein, the isobutanol pathway genes may encode enzyme(s) selected from the group consisting of acetolactate synthase (ALS), ketoi-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2- keto-acid decarboxylase, e.g., keto-isovalerate decarboxylase (KIVD), and alcohol dehydrogenase (ADH).

[0031] Another aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said microorganism has been engineered to overexpress a polynucleotide encoding Grx3 and/or Grx4 or a homolog thereof. In one embodiment, the polynucleotide encoding the Grx protein or homolog thereof is native to the recombinant microorganism. In another embodiment, the polynucleotide encoding the Grx protein or homolog thereof is heterologous to the recombinant microorganism.

[0032] In various embodiments described herein, the recombinant microorganism may be engineered reduce the concentration of reactive oxygen species (ROS) in the recombinant microorganism. Thus, the recombinant microorganisms may be engineered to express one or more proteins that reduce the concentration of reactive oxygen species (ROS) in said cell. The proteins to be expressed for reducing the concentration of reactive oxygen species may be selected from cataiases, superoxide dismutases, metailothioneins, and methionine suiphoxide reductases. In a specific embodiment, said cataiase may be encoded by one of more of the genes selected from the group consisting of the E. coll genes katG and katE, the S.

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364561 yl/CO cerevisiae genes CTT1 and CTA1, or homologs thereof. In another specific embodiment, said superoxide dismutase is encoded by one of more of the genes selected from the group consisting of the £. coii genes sod A, sodB, sodC, the S, cerevisiae genes SOD1 and SOD2, or homologs thereof. In another specific embodiment, said mefallothionein is encoded by one of more of the genes selected from the group consisting of the S. cerevisiae CUP1-1 and CUP1-2 genes or homologs thereof. In another specific embodiment, said metailothionein is encoded by one or more genes selected from the group consisting of the Mycobacterium tuberculosis MymT gene and the Synechococcus PCC 7942 SmtA gene or homologs thereof. In another specific embodiment, said methionine suiphoxide reductase is encoded by one or more genes selected from the group consisting of the S. cerevisiae genes MXR1 and MXR2, or homologs thereof.

[0033] In some embodiments, the recombinant microorganism may be engineered to increase the level of available glutathione in the recombinant microorganism. Thus, the recombinant microorganisms may be engineered to express one or more proteins that increase the level of available glutathione in the ceil. In one embodiment, the proteins are selected from glutaredoxin, glutathione reductase, glutathione synthase, and combinations thereof. In a specific embodiment, said glutaredoxin is encoded by one of more of the genes selected from the group the S. cerevisiae genes GRX2, GRX4, GRX6, and GRX7, or homologs thereof. In another specific embodiment, said glutathione reductase is encoded by the S. cerevisiae genes GLR1 or homologs thereof. In another specific embodiment, said glutathione synthase is encoded by one of more of the genes selected from the S. cerevisiae genes GSH1 and GSH2, or homologs thereof. In some embodiments, two enzymes are expressed to increase the level of available glutathione in the ceil. In one embodiment, the enzymes are γ-glutamyi cysteine synthase and glutathione synthase, !n a specific embodiment, said glutathione synthase is encoded by one of more of the genes selected from the group the S. cerevisiae genes GSH1 and GSH2, or homologs thereof.

[0034] In some embodiments, it may be desirable to overexpress one or more functional components of the thioredoxin system, as overexpression of the functional components of the thioredoxin system can increase the amount of bioavaiiable thioredoxin. In one embodiment, the functional components of the thioredoxin system may be selected from a thioredoxin and a thioredoxin reductase. In a specific embodiment, said thioredoxin is encoded by the S. cerevisiae TRX1 and

11

364561 yl/CO TRX2 genes or horno!ogs thereof. In another specific embodiment, said thioredoxin reductase is encoded by S. cerevisiae TRR1 gene or homologs thereof. In additional embodiments, the recombinant microorganism may further be engineered to overexpress the mitochondrial thioredoxin system. In one embodiment, the mitochondrial thioredoxin system is comprised of the mitochondrial thioredoxin and mitochondrial thioredoxin reductase. In a specific embodiment, said mitochondrial thioredoxin is encoded by the S. cerevisiae TRX3 gene or homologs thereof. In another specific embodiment, said mitochondrial thioredoxin reductase is encoded by the S. cerevisiae TRR2 gene or homologs thereof.

[0035] In various embodiments described herein, it may be desirable to engineer the recombinant microorganism to overexpress one or more mitochondrial export proteins. In a specific embodiment, said mitochondrial export protein may be selected from the group consisting of the S. cerevisiae ATM1, the S. cerevisiae ERV1, and the S. cerevisiae BAT1, or homologs thereof.

[0036] In addition, the present invention provides recombinant microorganisms that have been engineered to increase the inner mitochondrial membrane electrical potential, ΔΨ _Μ . In one embodiment, this is accomplished via overexpression of an ATP/ADP carrier protein, wherein said overexpression increases ATP ^4" import into the mitochondrial matrix in exchange for ADP ^3" . In a specific embodiment, said ATP/ADP carrier protein is encoded by the S. cerevisiae AAC1, AAC2, and/or AAC3 genes or homologs thereof. In another embodiment, the inner mitochondrial membrane electrical potential, ΔΨ _Μ is increased via a mutation in the mitochondrial ATP synthase complex that increases ATP hydrolysis activity. In a specific embodiment, said mutation is an ATP1 -1 1 1 suppressor mutation or a corresponding mutation in a homologous protein.

[0037] In various embodiments described herein, it may further be desirable to engineer the recombinant microorganism to express one or more enzymes in the cytosol that reduce the concentration of reactive nitrogen species (RNS) and/or nitric oxide (NO) in said cytosol. In one embodiment, said one or more enzymes are selected from the group consisting of nitric oxide reductases and g!utathione-S- nitrosothiol reductase. In a specific embodiment, said nitric oxide reductase is encoded by one of more of the genes selected from the group consisting of the £. coli gene norV and the Fusarium oxysporum gene P~450dNIR _s or homologs thereof. In another specific embodiment, said g!utathione~S-nitrosothiol reductase is encoded by the S. cerevisiae gene SFA 1 or homologs thereof. In one embodiment, said

12

364561 yl/CO glutathione-S-nitrosothio! reductase gene SFA1 is overexpressed. In another specific embodiment, said one or more enzymes is encoded by a gene selected from the group consisting of the E. coii gene ytfE, the Staphylococcus aureus gene scdA, and Neisseria gonorrhoeae gene dnrN, or homoiogs thereof.

[0038] Also provided herein are recombinant microorganisms that demonstrate increased the levels of sulfur-containing compounds within yeast cells, including the amino acid cysteine, such that this sulfur is more available for the production of iron- sulfur cluster-containing proteins in the yeast cell. In one embodiment, the recombinant microorganism has been engineered to overexpress one or more of the genes selected from the S. cerevisiae genes MET1, MET2, MET3, MET5, MET8, MET10, MET 14, MET16, MET17, OM2, HOM3, HOM6, CYS3, CYS4, SUL1, and SUL2, or homoiogs thereof. The recombinant microorganism may additionally or optionally also overexpress one or more of the genes selected from the S. cerevisiae genes YCT1, MUP1, GAP1, AGP1, GNP1, BAP1, BAP2, TAT1, and TAT2, or homoiogs thereof.

[0039] In various embodiments described herein, the recombinant microorganism may exhibit at least about 5 percent greater dihydroxyacid dehydratase (DHAD) activity as compared to the parental microorganism. In another embodiment, the recombinant microorganism may exhibit at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 85 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 100 percent, at least about 200 percent, or at least about 500 percent greater dihydroxyacid dehydratase (DHAD) activity as compared to the parental microorganism.

[0040] In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Saccharomyces sensu stricto microorganisms, Crabtree-negative yeast microorganisms, Crabfree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

[0041] In some embodiments, the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces clade.

13

364561 vl/CO [0042] In some embodiments, the recombinant microorganisms may be Saccharomyces sensu stricio microorganisms. In one embodiment, the Saccharomyces sensu stricto is selected from the group consisting of S, cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum. S. carocanis and hybrids thereof.

[0043] In some embodiments, the recombinant microorganisms may be Crabtree- negative recombinant yeast microorganisms. In one embodiment, the Crabtree- negative yeast microorganism is classified into a genera selected from the group consisting of Kluyveromyces, Pichia, Issatchenkia, Hansenula, or Candida. In additional embodiments, the Crabtree-negative yeast microorganism is selected from Kluyveromyces lactis, Kluyveromyces marxianus. Pichia anomala, Pichia stipitis, Hansenula anomala, Candida utiiis and Kluyveromyces waiiii.

[0044] In some embodiments, the recombinant microorganisms may be Crabtree- posi ive recombinant yeast microorganisms. In one embodiment, the Crabtree- positive yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia and Schizosacchammyces, In additional embodiments, the Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, Saccharomyces kiuyveri, Kluyveromyces thermotolerans, Candida glabrata, Z. bailli, Z. rouxii, Debaryomyces hansenii, Pichia pastonus, Schizosacchammyces pombe, and Saccharomyces uvarum.

[0045] In some embodiments, the recombinant microorganisms may be post- WGD (whole genome duplication) yeast recombinant microorganisms. In one embodiment, the post-VVGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces or Candida. In additional embodiments, the post-WGD yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabrata.

[0046] In some embodiments, the recombinant microorganisms may be pre-WGD (whole genome duplication) yeast recombinant microorganisms. In one embodiment, the pre-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Pachysoien, Yarrowia and Schizosacchammyces. In additional embodiments, the pre-WGD yeast is

14

364561 yl/CO selected from the group consisting of Saccharomyces kiuyveri, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Kluyveromyces waltli, Kluyveromyces iactis, Candida tropicalis, Pichia pastoris, Pichia anomala, Pichia stipitis, Isstachenkia orientalis, Issatchenkia occidentalis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, and Schizosaccharomyces pombe.

[0047] In some embodiments, the recombinant microorganisms may be microorganisms that are non-fermenting yeast microorganisms, including, but not limited to those, classified into a genera selected from the group consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida. In a specific embodiment, the non-fermenting yeast is C. xestobii.

[0048] In another aspect, the present invention provides methods of producing beneficial metabolites including fuels, chemicals, and amino acids using a recombinant microorganism as described herein. In one embodiment, the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the metabolite is produced and optionally, recovering the metabolite. In one embodiment, the microorganism produces the metabolite from a carbon source at a yield of at least about 5 percent theoretical. In another embodiment, the microorganism produces the metabolite at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 80 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5 percent theoretical. The metabolite may be derived from any DHAD-requiring biosynthetic pathway, including, but not limited to, biosynthetic pathways for the production of isobutanol, 3-methyl-1 -butanol, 2-methyl- 1 -butanoi, valine, isoieucine, leucine, and pantothenic acid.

[0049] In one embodiment, the recombinant microorganism converts the carbon source to isobutanol under aerobic conditions. In another embodiment, the recombinant microorganism converts the carbon source to isobutanol under microaerobic conditions. In yet another embodiment, the recombinant microorganism converts the carbon source to isobutanol under anaerobic conditions.

15

364561 vl/CO BRIEF DESCRIPTION OF DRAWINGS

[0050] Illustrative embodiments of the invention are illustrated in the drawings, in which:

[0051] Figure 1 illustrates an exemplary embodiment of an isobutanol pathway.

[0052] Figure 2 illustrates an exemplary embodiment of an NADH-dependent isobutanol pathway.

[0053] Figure 3 illustrates a phylogenetic tree of DHAD proteins. Numbers at nodes indicate bootstrap values. EcJ!vD is a known 4Fe-4S DHAD enzyme from Escherichia coll.

[0054] Figure 4 illustrates a S. cerevisiae AFT1-1 ^UP allelic exchange construct.

[0055] Figure 5 illustrates a S. cerevisiae AFT2-1 ^UP allelic exchange construct.

[0056] Figure 6 illustrates a linear DNA fragment containing the K. marxianus AFT, the L lactis DHAD, and a G418 resistance marker.

[0057] Figure 7 illustrates a linear DNA fragment containing the L. lactis DHAD and a G418 resistance marker.

[0058] Figure 8 illustrates a p!asrnid map for pGV2196, comprising TEF1 and TDH3 promoters for use in the overexpression of NFS1 and/or ISD11

[0059] Figure 9 illustrates a plasmid map for pGV2964, incorporated into S. cerevisiae strain GEVO8014.

[0060] Figure 10 illustrates the total isobutanol titers [g/L] of yeast strains engineered to overexpress NFS1 and/or ISD11.

[0061] Figure 11 illustrates the isobutanol specific titers [g/g CDW] of yeast strains engineered to overexpress NFS1 and/or ISD11.

[0062] Figure 12 illustrates the total isobutanol titer [g/L] of a yeast strain engineered to overexpress NFS1 and ISD11 as compared to a control strain with an empty vector.

[0063] Figure 13 illustrates the specific isobutanol titer [g/g CDW] of a yeast strain engineered to overexpress NFS1 and ISD11 as compared to a control strain with an empty vector.

[0064] Figure 14 illustrates the total isobutanol titers [g/L] of yeast strains engineered to overexpress NFS1 and/or ISD11 at 24 hr and 48 hr.

[0065] Figure 15 illustrates the specific isobutanol titers [g/g CDW] of yeast strains engineered to overexpress NFS1 and/or ISD11 at 24 hr and 48 hr.

[0066] Figure 16 illustrates the results of DHAD activity assays in yeast strains

16

364561 vl/CO engineered to overexpress NFS1 and/or ISD11.

DETAILED DESCRIPTION

[0067] 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.

[0068] Unless defined otherwise, ail 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.

[0069] 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.

[0070] 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 ceils" and "microbes" are used interchangeably with the term microorganism.

[0071] The term "genus" is defined as a taxonomic group of related species according to the Taxonomic Outline of Bacteria and Archaea (Garrity, G.M., Lilburn, T.G., Cole, J.R., Harrison, S.H., Euzeby, J., and Tindail, B.J. (2007) The Taxonomic Outline of Bacteria and Archaea. TOBA Release 7.7, March 2007. Michigan State University Board of Trustees.

[0072] The term "species" is defined as a collection of closely related organisms with greater than 97% 16S ribosomai RNA sequence homology and greater than 70% genomic hybridization and sufficiently different from ail other organisms so as to be recognized as a distinct unit.

[0073] The terms "recombinant microorganism," "modified microorganism," and "recombinant host ceil" are used interchangeably herein and refer to microorganisms that have been genetically modified to express or to overexpress endogenous polynucleotides, to express heterologous polynucleotides, such as those included in

17

364561 vl/CO a vector, in an integration construct, or which have an alteration in expression of an endogenous gene. By "alteration" it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration. For example, the term "alter" can mean "inhibit," but the use of the word "alter" is not limited to this definition. It is understood that the terms "recombinant microorganism" and "recombinant host ceil" refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0074] 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 level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the ceil, or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (see Sambrook et a/., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. See Sambrook et a!., 1989, supra.

[0075] The term "overexpression" refers to an elevated level (e.g., aberrant level) of mRNAs encoding for a protein(s) (e.g. an Nfs1 or Isd1 1 protein or homoiog thereof), and/or to elevated levels of protein(s) (e.g. Nfs1 or !sd1 1 ) in cells as compared to similar corresponding unmodified ceils expressing basal levels of mRNAs (e.g., those encoding Nfs1 or Isd1 1 proteins) or having basal levels of proteins. In particular embodiments, Nfs1 and/or isd1 1 , or homologs thereof, may be overexpressed by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 2-

18

364561 vl/CO fold, 15-fold or more In microorganisms engineered to exhibit increased Nfs1 and/or Isd1 1 mRNA, protein, and/or activity.

[0076] As used herein and as would be understood by one of ordinary skill in the art, "reduced activity and/or expression" of a protein such as an enzyme can mean either a reduced specific catalytic activity of the protein (e.g. reduced activity) and/or decreased concentrations of the protein in the cell (e.g. reduced expression).

[0077] The term "wild-type microorganism" describes a ceil that occurs in nature, i.e., a cell that has not been genetically modified. A wild-type microorganism can be genetically modified to express or overexpress a first target enzyme. This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or overexpress a second target enzyme. In turn, the microorganism modified to express or overexpress a first and a second target enzyme can be modified to express or overexpress a third target enzyme.

[0078] 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 overexpression 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 heterologous polynucleotides encoding a target enzyme in to a parental microorganism.

[0079] The term "engineer" refers to any manipulation of a microorganism that results in a detectable change in the microorganism, wherein the manipulation includes but is not limited to inserting a polynucleotide and/or polypeptide heterologous to the microorganism and mutating a polynucleotide and/or polypeptide native to the microorganism.

[0080] The term "mutation" as used herein indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include, for example, point mutations, deletions, or insertions of single or multiple residues in a polynucleotide, which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, an insertion, or a

19

364561 yl/CO deletion of part or all of a gene, in addition, in some embodiments of the modified microorganism, a portion of the microorganism genome has been repiaced with a heteroiogous polynucleotide. In some embodiments, the mutations are naturally- occurring . In other embodiments, the mutations are the results of artificial selection pressure, In still other embodiments, the mutations in the microorganism genome are the result of genetic engineering.

[0081] The term "biosynthetic pathway", also referred to as "metabolic pathway", refers to a set of anabolic or cataboiic biochemical reactions for converting 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.

[0082] As used herein, the term "isobutanoi producing metabolic pathway" refers to an enzyme pathway which produces isobutanoi from pyruvate.

[0083] The term "NADH-dependent" as used herein with reference to an enzyme, e.g. , KAR! and/or ADH, refers to an enzyme that catalyzes the reduction of a substrate coupled to the oxidation of NADH with a catalytic efficiency that is greater than the reduction of the same substrate coupled to the oxidation of NADPH at equal substrate and cofactor concentrations.

[0084] The term "exogenous" as used herein with reference to various molecules, e.g. , polynucleotides, polypeptides, enzymes, etc., refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

[0085] On the other hand, the term "endogenous" or "native" as used herein with reference to various molecules, e.g. , polynucleotides, polypeptides, enzymes, etc., refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or ceil in nature.

[0086] The term "heterologous" as used herein in the context of a modified host ceil refers to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true: (a) the molecule(s) is/are foreign ("exogenous") to (i.e., not naturally found in) the host cell; (b) the molecuie(s) is/are naturally found in (e.g., is "endogenous to") a given host microorganism or host cell but is either produced in an unnatural location or in an unnatural amount in the cell; and/or (c) the molecu!e(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in

20

364561 yl/CO nucleotide or amino acid sequence from the endogenous nucleotide or amino acid as found endogenously is produced in an unnatural (e.g., greater than naturally found) amount in the cell.

[0087] The term "feedstock" is defined as a raw material or mixture of raw materials supplied to a microorganism or fermentation process from which other products can be made. For example, a carbon source, such as biomass or the carbon compounds derived from biomass are a feedstock for a microorganism that produces a biofuel in a fermentation process. However, a feedstock may contain nutrients other than a carbon source.

[0088] 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 recombinant microorganism as described herein.

[0089] The term "C2-compound" as used as a carbon source for engineered yeast microorganisms with mutations in ail pyruvate decarboxylase (PDC) genes resulting in a reduction of pyruvate decarboxylase activity of said genes refers to organic compounds comprised of two carbon atoms, including but not limited to ethanol and acetate.

[0090] The term "fermentation" or "fermentation process" is defined as a process in which a microorganism is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, wherein the microorganism converts raw materials, such as a feedstock, into products.

[0091] The term "volumetric productivity" or "production rate" is defined as the amount of product formed per volume of medium per unit of time. Volumetric productivity is reported in gram per liter per hour (g/L/h).

[0092] The term "specific productivity" or "specific production rate" is defined as the amount of product formed per volume of medium per unit of time per amount of ceils. Specific productivity is reported in gram (or milligram) per gram ceil dry weight per hour (g/g h).

[0093] The term "yield" is defined as the amount of product obtained per unit

21

364561 yl/CO weight of raw materia! and may be expressed as g product per g substrate (g/g). Yield may be expressed as a percentage of the theoretical yield. "Theoretical yield" is defined as the maximum amount of product that can be generated per a given amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isobutanol is 0.41 g/g. As such, a yield of isobutanol from glucose of 0.39 g/g would be expressed as 95% of theoretical or 95% theoretical yield.

[0094] The term "titer" is defined as the strength of a solution or the concentration of a substance in solution. For example, the titer of a biofuel in a fermentation broth is described as g of biofuel in solution per liter of fermentation broth (g/L).

[0095] "Aerobic conditions" are defined as conditions under which the oxygen concentration in the fermentation medium is sufficiently high for an aerobic or facultative anaerobic microorganism to use as a terminal electron acceptor.

[0096] In contrast, "anaerobic conditions" are defined as conditions under which the oxygen concentration in the fermentation medium is too low for the microorganism to use as a terminal electron acceptor. Anaerobic conditions may be achieved by sparging a fermentation medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism as a terminal electron acceptor. Alternatively, anaerobic conditions may be achieved by the microorganism consuming the available oxygen of the fermentation until oxygen is unavailable to the microorganism as a terminal electron acceptor. Methods for the production of isobutanol under anaerobic conditions are described in commonly owned and copending publication, US 2010/0143997, the disclosures of which are herein incorporated by reference in its entirety for ail purposes.

[0097] "Aerobic metabolism" refers to a biochemical process in which oxygen is used as a terminal electron acceptor to make energy, typically in the form of ATP, from carbohydrates. Aerobic metabolism occurs e.g. via glycolysis and the TCA cycle, wherein a single glucose molecule is metabolized completely into carbon dioxide in the presence of oxygen.

[0098] In contrast, "anaerobic metabolism" refers to a biochemical process in which oxygen is not the final acceptor of electrons contained in NADH. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than oxygen serve as the terminal electron acceptor, and substrate level phosphorylation, in which the electrons from NADH are utilized to generate a reduced product via a "fermentative pathway."

22

364561 yl/CO [0099] In "fermentative pathways", NAD(P)H donates Its electrons to a molecule produced by the same metabolic pathway that produced the electrons carried In NAD(P)H, For example, in one of the fermentative pathways of certain yeast strains, NAD(P)H generated through glycolysis transfers its electrons to pyruvate, yielding ethanol. Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain. For example, above certain glucose concentrations, Crabtree-positive yeasts produce large amounts of ethanol under aerobic conditions.

[00100] The term "byproduct" or "by-product" means an undesired product related to the production of an amino acid, amino acid precursor, chemical, chemical precursor, biofuel, or biofuel precursor.

[00101] The term "substantially free" when used in reference to the presence or absence of a protein activity (3-KAR enzymatic activity, ALDH enzymatic activity, PDC enzymatic activity, GPD enzymatic activity, etc.) means the level of the protein is substantially less than that of the same protein in the wild-type host, wherein less than about 50% of the wild-type level is preferred and less than about 30% is more preferred. The activity may be less than about 20%, less than about 10%, less than about 5%, or less than about 1 % of wild-type activity. Microorganisms which are "substantially free" of a particular protein activity (3-KAR enzymatic activity, ALDH enzymatic activity, PDC enzymatic activity, GPD enzymatic activity, etc.) may be created through recombinant means or identified in nature.

[00102] The term "non-fermenting yeast" is a yeast species that fails to demonstrate an anaerobic metabolism in which the electrons from NADH are utilized to generate a reduced product via a fermentative pathway such as the production of ethanol and CO2 from glucose. Non-fermentative yeast can be identified by the "Durham Tube Test" (J.A. Barnett, R.W. Payne, and D. Yarrow. 2000. Yeasts Characteristics and Identification. 3 ^rd edition, p. 28-29. Cambridge University Press, Cambridge, UK.) or by monitoring the production of fermentation productions such as ethanol and CO2.

[00103] The term "polynucleotide" is used herein interchangeably with the term "nucleic acid" and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or

23

364561 yl/CO antisense ribonucleic acid (RNA) of any length, including siRNA. The term "nucleotide" refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids. The term "nucleoside" refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term "nucleotide analog" or "nucleoside analog" refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called nucleotidic oligomer or oligonucleotide.

[00104] It is understood that the polynucleotides described herein include "genes" and that the nucleic acid molecules described herein include "vectors" or "plasmids." 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, S'-untranslated region (UTR), and 3 ^! -UTR, as well as the coding sequence.

[00105] The term "operon" refers to 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.

[00106] 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, piasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial

24

364561 yl/CO 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 po!y-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.

[00107] "Transformation" refers to the process by which a vector is introduced into a host ceil. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including chemical transformation (e.g. lithium acetate transformation), e!ectroporation, microinjection, bioiistics (or particle bombardment- mediated delivery), or agrobacterium mediated transformation.

[00108] The term ^!! enzyme ^!! as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide, but can include enzymes composed of a different molecule including polynucleotides.

[00109] The term "protein," "peptide," or "polypeptide" as used herein indicates an organic polymer composed of two or more amino acidic monomers and/or analogs thereof. As used herein, the term "amino acid" or "amino acidic monomer" refers to any natural and/or synthetic amino acids including glycine and both D or L optical isomers. The term "amino acid analog" refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group. Accordingly, the term polypeptide includes amino acidic polymer of any length including full length proteins, and peptides as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer or oligopeptide.

[00110] The term "homolog," 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, homoiogs will have functional, structural or genomic similarities. Techniques are known by which homoiogs of an enzyme or gene can readily be cloned using genetic probes and

25

364561 vl/CO PGR. Identity of cloned sequences as hornolog can be confirmed using functional assays and/or by genomic mapping of the genes.

[00111] A protein has "homology" or is "homologous" to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. 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).

[00112] The term "analog" or "analogous" refers to nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.

Enhancing DHAD Activity by Increasing Nfs1/lsd1 1 Expression and/or Activity

[00113] The present inventors have found that altering the expression of the NFS1 and ISD11 genes of S. cerevisiae increases DHAD activity and contributes to increased isobutanoi titers, productivity, and yield in strains comprising DHAD as part of an isobutanol-producing metabolic pathway. The observed increases in

DHAD activity resulting from the increased expression of NFS1 and/or ISD11 therefore has broad applicability to any DHAD-requiring biosynthetic pathway, as

DHAD activity is often a rate-limiting component of such pathways.

[00114] Accordingly, one aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more Nfs1 proteins or homoiogs thereof and/or one or more polynucleotides encoding one or more Isd1 1 proteins or homoiogs thereof.

[00115] As used herein, a "DHAD-requiring biosynthetic pathway" refers to any metabolic pathway which utilizes DHAD to convert 2,3-dihydroxyisovalerate to a- kefoisovalerate or 2,3-dihydroxy-3-methylvalerate to 2-keto-3-methyivaierate. Examples of DHAD-requiring biosynthetic pathways include, but are not limited to, isobutanoi, 3-methyi-1 -butanoi, 2-methy!-1 -butanol, valine, isoieucine, leucine, and pantothenic acid (vitamin B5) metabolic pathways. The metabolic pathway may

26

364561 vl/CO naturally occur in a microorganism {e.g., a natural pathway for the production of valine) or arise from the introduction of one or more heterologous polynucleotides through genetic engineering. In one embodiment, the recombinant microorganisms expressing the DHAD-requiring biosynthetic pathway are yeast ceils. Engineered biosynthetic pathways for synthesis of isobutanol are described in commonly owned and co-pending applications US 12/343,375 (published as US 2009/0228991 ), US 12/610,784 (published as US 2010/0143997), US 12/696,645, US 12/820,505 (published as US 201 1 /0020889), US 12/855,276, US 12/953,884, PCT/US09/62952 (published as WO/2010/051527), and PCT/US09/69390 (published as WO/2010/075504), ail of which are herein incorporated by reference in their entireties for ail purposes. Additional DHAD-requiring biosynthetic pathways have been described for the synthesis of valine, leucine, and isoleucine (See, e.g. , WO/2001/021772, and McCourf et aL, 2006, Amino Acids 31 : 173-210), pantothenic acid (See, e.g., WO/2001/021772), 3-methyl-1 -butanol (See, e.g., WO/2008/098227, Atsumi ef a/., 2008, Nature 451 : 86-89, and Connor et a/., 2008, Appl. Environ. Microbiol. 74: 5769-5775), and 2-methyl-1 -butanol (See, e.g., WO/2008/098227, WO/2009/076480, and Atsumi et a!., 2008, Nature 451 : 86-89).

[00116] As used herein, the terms "DHAD" or "DHAD enzyme" or "dihydroxyacid dehydratase" are used interchangeably to refer to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to ketoisovaierate and/or the conversion of 2,3-dihydroxy-3-methyivaierate to 2-keto-3-methy!va!erate. DHAD sequences are available from a vast array of microorganisms, including, but not limited to, L. lactis, E. coli, S. cerevisiae, B. subtilis, Streptococcus pneumoniae, and Streptococcus mutans. A representative list of DHAD enzymes that can benefit from the methods described herein, such as the increased expression of NFS1 and/or ISD11 or homoiogs thereof, include, but are not limited to those, disclosed in commonly owned and co-pending U.S. Patent Publication No. 201 1/0076733. Such DHAD enzymes may be cytosolically localized or mitochondrially localized. A representative listing of DHAD enzymes exhibiting cytosolic localization and activity are disclosed in commonly owned and co-pending U.S. Patent Publication No. 201 1 /0076733.

[00117] A person skilled in the art, equipped with this disclosure, will appreciate suitable methods for increasing the expression (i.e. overexpressing) NFS1 or a homoiog thereof and/or ISD11 or a homoiog thereof. For instance, in one embodiment, NFS1 or a homoiog thereof and/or ISD11 or a homoiog thereof may be

27

364561 yl/CO overexpressed from a plasmid. In another embodiment, one or more copies of the NFS1 gene or a homolog thereof and/or one or more copies of the ISD11 gene or a homolog thereof is inserted into the chromosome under the control of a constitutive promoter. In addition, a skilled person in the art, equipped with this disclosure, will recognize that the amount of NFS1 and/or ISD11 overexpressed may vary from one yeast to the next. For example, the optimal level of overexpression may be one, two, three, four or more copies in a given yeast. As would be understood in the art, naturally occurring homologs of NFS1 and ISD11 in yeast other than S. cerevisiae can similarly be overexpressed using the methods of the present invention. NFS1 and ISD11 homologs and methods of identifying such NFS1 and ISD11 homologs are described herein.

[00118] A person skilled in the art will be able to utilize publicly available sequences to construct relevant recombinant microorganisms with altered expression of NFS1 and/or ISD11 homologs. A listing of a representative number of NFS1 and ISD11 homologs known in the art and useful in the construction of recombinant microorganisms engineered for increased DHAD activity are listed in the attached Sequence Listing. One skilled in the art, equipped with this disclosure, will appreciate other suitable homologs for the generation of recombinant microorganisms with increased DHAD activity. Sequences of NFS1 and ISD11 genes found in sub-species or variants of a given species may not be identical. While if is preferred to overexpress a NFS1 and/or ISD11 gene{s) native to the subspecies or variant, NFS1 and/or ISD11 gene{s) may be interchangeably expressed across subspecies or variants of the same species or of different species.

[00119] In various exemplary embodiments, increasing the expression of NFS1 or a homolog thereof alone, ISD11 or a homolog thereof alone, or NFS1 or homologs thereof in combination with ISD11 or homologs thereof will increase DHAD activity and the production of beneficial metabolites from DHAD-requiring biosynthetic pathways. As described herein, the increased activity of DHAD in a recombinant microorganism is a favorable characteristic for the production of beneficial metabolites including isobutanol, 3-methy!-1 -butanol, 2-methy!-1 -butanol, valine, isoleucine, leucine, and pantothenic acid derived from DHAD-requiring biosynthetic pathways. Without being bound by any theory, it is believed that the increase in DHAD activity as observed by the present inventors results from the enhanced formation of FeS clusters as mediated by the altered regulation, expression, and/or activity of NFS1 and/or ISD11. Thus, in various embodiments described herein, the

28

364561 yl/CO present invention provides recombinant microorganisms with increased DHAD activity as a result of alterations in NFS1 and/or ISD11 regulation, expression, and/or activity, In one embodiment, the alteration in NFS1 and/or ISD11 regulation, expression, and/or activity increases the activity of a cytosolicaliy-localized DHAD. In another embodiment, the alteration in NFS1 and/or ISD11 regulation, expression, and/or activity increases the activity of a mitochondrially-localized DHAD.

[00120] While particularly useful for the biosynthesis of isobutanoi, the altered regulation, expression, and/or activity of NFS1 and/or ISD11 is also beneficial to any other fermentation process in which increased DHAD activity is desirable, including, but not limited to, the biosynthesis of isoieucine, valine, leucine, pantothenic acid (vitamin B5), 2-methy!-1 -butanol, and 3-methyi-1 -butanol.

[00121] As described herein, the present inventors have observed increased isobutanoi titers, productivity, and yields in recombinant microorganisms exhibiting increased expression of NFS1 and/or ISD11. Without being bound by any theory, it is believed that the increases in isobutanoi titer, productivity, and yield are due to the observed increases in DHAD activity. Thus, in one embodiment, the present invention provides a recombinant microorganism for producing isobutanoi, wherein said recombinant microorganism comprises an isobutanoi producing metabolic pathway, and wherein the expression of NFS1 or a homolog thereof is increased. In another embodiment, the present invention provides a recombinant microorganism for producing isobutanoi, wherein said recombinant microorganism comprises an isobutanoi producing metabolic pathway, and wherein the expression of ISD11 or a homolog thereof is increased. In yet another embodiment, the present invention provides a recombinant microorganism for producing isobutanoi, wherein said recombinant microorganism comprises an isobutanoi producing metabolic pathway, and wherein the expression of NFS1 or a homolog thereof and ISD11 or a homolog thereof is increased. As would be understood in the art, naturally occurring homo!ogs of NFS1 and ISD11 in yeast other than S. cerevisiae can similarly be overexpressed using the methods of the present invention. NFS1 and ISD11 homologs and methods of identifying such NFS1 and ISD11 homologs are described herein. Thus, in some embodiments, nucleic acids having an identity to NFS1 and/or ISD11 of at least about 50%, of at least about 80%, of at least about 70%, at least about 80%, or at least about 90% can be used for a similar purpose.

[00122] In one embodiment, the present invention provides a recombinant microorganism for producing isobutanoi, wherein said recombinant microorganism

29

364561 yl/CO comprises an isobutano! producing metabolic pathway, and wherein the activity of Nfs1 or a homolog thereof is increased. In another embodiment, the present invention provides a recombinant microorganism for producing isobutano!, wherein said recombinant microorganism comprises an isobutano! producing metabolic pathway, and wherein the activity of Isd1 1 or a homolog thereof is increased. In yet another embodiment, the present invention provides a recombinant microorganism for producing isobutano!, wherein said recombinant microorganism comprises an isobutano! producing metabolic pathway, and wherein the activity of Nfs1 or a homolog thereof and !sd1 1 or a homolog thereof is increased.

[00123] In alternative embodiments, proteins having an identity to Nfs1 and/or Isd1 1 of at least about 50%, of at least about 60%, of at least about 70%, at least about 80%, or at least about 90% can be used for a similar purpose.

[00124] In various embodiments described herein, the recombinant microorganism comprises an isobutanol producing metabolic pathway. In one embodiment, the isobutano! producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutano!. In another embodiment, the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. in yet another embodiment, the isobutanol producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that cata!yze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, all of the isobutano! producing metabolic pathway steps in the conversion of pyruvate to isobutano! are converted by exogenously encoded enzymes.

[00125] In one embodiment, one or more of the isobutano! pathway genes encodes an enzyme that is localized to the cytosol. In one embodiment, the recombinant microorganisms comprise an isobutano! producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutano! producing metabolic pathway with at least two isobutano! pathway enzymes localized in the

30

364561 yl/CO cytoso!. In yet another embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least three isobutanoi pathway enzymes localized in the cytosoi. in yet another embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least four isobutanoi pathway enzymes localized in the cytosoi. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with five isobutanoi pathway enzymes localized in the cytosoi. in yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with all isobutanoi pathway enzymes localized in the cytosoi. In a further exemplary embodiment, at least one of the pathway enzymes localized to the cytosoi is a cytosolicaily active DHAD enzyme as disclosed herein.

[00126] In various embodiments described herein, the isobutanoi pathway genes may encode enzyme(s) selected from the group consisting of acetolactate synthase (ALS), ketoi-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2- keto-acid decarboxylase, e.g., keto-isovaierate decarboxylase (KIVD), and alcohol dehydrogenase (ADH).

Enhancing DHAD Activity by Increasing Cysteine Availability in the Yeast Cell

[00127] Because Nfs1 and Isd1 1 catalyze an early step of FeS duster biogenesis in the yeast mitochondria by mobilizing sulfur into the FeS biogenesis pathway from cysteine, overexpression of NFS1 and ISD11 as described herein can be combined with methods for increasing cysteine availability in the ceil to augment the increase in FeS protein activity in the yeast cells. Specifically, increasing the expression of the genes MET3, MET14, MET16, MET10, MET5, MET1, MET8, MET2, MET17, HOM3, HOM2, HOM6, CYS3, CYS4, SUL1 and SUL2 _S alone or in combinations, to increase cysteine biosynthesis by the cell and/or increasing the expression of the transport protein-encoding genes YCT1, MUP1, GAP1, AGP1, GNP1, BAP1, BAP2, BAP3, TAT1 and LA 72, alone or in combinations, to increase uptake of exogenous cysteine by the cell will increase the amount and availability of cysteine for use by Nfs1 and Isd1 1 in yeast overexpressing NFS1 and ISD11. Accordingly, recombinant microorganisms engineered to overexpress MET3, MET14, MET16, MET10, MET5, MET1, MET8, MET2, MET17, HOM3, HOM2, HOM6, CYS3, CYS4, SUL1 SUL2, YCT1, MUP1, GAP1, AGP1, GNP1, BAP1, BAP2, BAP3, TAT1 and L 72, alone or in combinations, are within the scope of the present invention. Such modifications

31

364561 yl/CO can be used to further increase DHAD activity and enhance the titers, productivity, and yields in strains comprising DHAD as part of an DHAD-requiring biosynthetic pathway, e.g., an isobutanol producing metabolic pathway.

[00128] Additionally, the overexpression of heterologous serine o- acetyltransferases, including, but not limited to, the Beta vulgaris serine o- acetyltransferase ("Bv_SAT') (Mulet et a/., 2004, Yeast 21 : 303-312), is another way to increase the level and availability of cysteine within yeast ceils for the production of FeS cluster-containing proteins in yeast overexpressing NFS1 and ISD11. Accordingly, recombinant microorganisms engineered to overexpress a heterologous serine o-acetyltransferase are also within the scope of the present invention. In one embodiment, the heterologous serine o-acetyltransferase is encoded by Bv__SAT. As described herein, the expression of a heterologous serine o-acetyltransferase can be used to further increase DHAD activity and enhance the titers, productivity, and yields in strains comprising DHAD as part of an DHAD-requiring biosynthetic pathway, e.g., an isobutanol producing metabolic pathway.

[00129] Furthermore, the addition of increased exogenous cysteine to yeast ceils, separately from or in addition to increased expression of the transport protein- encoding genes listed above, may also be used to increase the level and availability of cysteine within yeast cells for the production of FeS cluster-containing proteins in yeast overexpressing NFS1 and ISD11, and is therefore also within the scope of the present invention.

Enhancing DHAD Activity by Altering Aft1/Aft2 Activity and/or Expression

[00130] The present inventors have also found that altering the expression of the AFT1 and/or AFT2 genes of S. cerevissae surprisingly increases DHAD activity and contributes to increased isobutanol titers, productivity, and yield in strains comprising DHAD as part of an isobutanoi-producing metabolic pathway. The observed increases in DHAD activity resulting from the increased expression of AFT1 and/or AFT2 therefore has broad applicability to any DHAD-requiring biosynthetic pathway, as DHAD activity is often a rate-limiting component of such pathways.

[00131] Accordingly, one aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more Aft proteins or homologs thereof.

[00132] Without being bound by any theory, it is believed that altered expression of

32

364561 yl/CO an AFT gene {e.g. the AFT1 and/or AFT2 genes) enhances cellular iron availability, which leads to an improvement in the activity of the iron-sulfur (FeS) cluster- containing protein, DHAD. The observation that increased expression of the AFT genes improves DHAD activity is surprising, particularly in light of recently published findings by !hrig et al. (2010, Eukaryotic Cell 9: 460-471 ). Notably, Ihrig et al. observed that the increased expression of Aft1 in S. cerevisiae had little to no effect on the activity of another FeS cluster-containing protein, Leu1 (isopropyimaiate isomerase of the leucine biosynthesis pathway). In contrast to observations made by Ihrig et al, with respect to the FeS protein, Leu1 , the present inventors unexpectedly observed that increased expression of Aft1 and/or Aft2 resulted in a significant increase in the activity of DHAD, also an iron-sulfur (FeS) duster- containing protein. Moreover, in strains comprising DHAD as part of an isobutanol- producing metabolic pathway, the increased expression of Aft1 produced significant increases in isobutano! titer, productivity, and yield.

[00133] In S. cerevisiae, AFT1 and AFT2 encode for the transcription factors, Aft1 and Aft2 ("activator of ferrous transport"), respectively. It is hypothesized that Aft1 and Aft2 activate gene expression when iron is scarce in wild-type S. cerevisiae. Consequently, strains lacking both Aft and Aft2 exhibit reduced expression of the iron regu!on. As with many other paralogous genes, AFT1 and AFT2 code for proteins that have significant regions of identity and overlapping functions. The DNA-binding domain of each protein is in a highly conserved N-termina! region, and a conserved cysteine-to-phenylalanine mutation in either protein generates a factor that activates the high expression of the iron reguion irrespective of iron concentrations.

[00134] In yeast, homeostatic regulation of iron uptake occurs (Eide et al., 1992, J. Biol Chem. 287: 20774-81 ). Iron deprivation induces activity of a high affinity iron uptake system. This induction is mediated by increased transcript levels for genes involved in the iron uptake system, and AFT1 is hypothesized to play a critical role in this process (Yamaguchi-iwai et al., 1995, The EMBO Journal 14: 1231 -9). Yamaguchi-!wai et al. observed that mutant strains lacking AFT1, due to gene deletion, are unable to induce the high-affinity iron uptake system. On the other hand, mutant strains carrying the AFT1 ^UP allele exhibit a gain-of-function phenotype in which iron uptake cannot be repressed by available iron in the environment. The AFT1 ^UP and AFT2 ^UP alleles described above act as gain of function point mutations. AFT1 ^U is due to the mutation Cys ^{2 1} Phe (Rutherford et al., 2005, Journal of

33

364561 yl/CO Biological Chemistry 281 : 10135-40). AFT2" ^' is due to the mutation Cys Phe (Rutherford et ai., 2001 , PNAS 98: 14322-27).

[00135] There are clear phenotyplc differences in strains that separately lack AFT1 or AFT2. An a f? null strain exhibits low ferrous iron uptake and grows poorly under low-iron conditions or on a respiratory carbon source. No phenotype has been attributed to an aft2 null strain. An afti afi2 double null strain is, however, more sensitive to low-iron growth than a single afti null strain, which is consistent with the functional similarity of these factors. The partial redundancy of these factors allows AFT2 to complement an afti null strain when it is overexpressed from a p!asmid. The properties of Afti and Aft2 that distinguish them from each other have not been fully elucidated. Both factors mediate gene regulation via an iron-responsive element that contains the core sequence 5'-CACCC-3 ^! . Without being bound to any theory, it is likely that sequences adjacent to this element influence the ability of each factor to mediate regulation via a particular iron-responsive element. The differential regulation of individual genes by Afti and Aft2 results in each factor generating a distinct global transcriptional profile (Table 1 ) (Rutherford et al., 2004, Eukaryotic Cell 3: 1 -13; Conde e Silva et ai, 2009, Genetics 183: 93-106).

Table 1. Genes Regulated by Metal-Responsive Transcription Factors.

[00136] In S. cerevisiae, the Afti regulon consists of many genes that are involved in the acquisition, compartmentalization, and utilization of iron. These include genes involved in iron uptake (FET3, FTR1, and FRE1, FRE2), siderophore uptake {ARN1-

34

364561 vl/CO 4 and FIT1-3), iron transport across the vacuole membrane (FTH1), and iron-sulfur cluster formation (ISU1 and ISU2). Aft1 binds to a conserved promoter sequence in an iron-dependent manner and activates transcription under low-iron conditions. The Aft2 regulator controls the expression of several distinct genes (Table 2) (Rutherford et al., 2004, Eukaryotic Cell 3: 1 -13). The initial step in iron acquisition requires reduction of ferric iron chelates in the environment by externally directed reductases encoded by the FRE1 and FRE2 genes, thereby generating the ferrous iron substrate for the transport process (Dancis et a/., 1992, PNAS 89: 3889-73; Georgatsou and Aiexandraki, 1994, Moi. Cell. Biol, 14: 3085-73). FET3 encodes a multi-copper oxidase (Askwith et al., 1994, Cell 78: 403-10; De Silva et al,, 1995, J. Biol. Chem. 270: 1098-1 101 ) that forms a molecular complex with the iron permease encoded by FTR1. This complex, located in the yeast plasma membrane, mediates the high-affinity transport of iron into the ceil (Stearman et a/., 1998, Science 271 : 1552-7). AFT genes may be found in yeast strains other than S. cerevisiae. For example, in K. iactis, a homo!og of the S. cerevisiae AFT1 has been found and designated Ki AFT (Conde e Silva et al., 2009, Genetics 183: 93-108). In this fungus, Ki__Aft has been found to activate transcription of genes regulated by Aft1 in S. cerevisiae. Thus, altering the regulation, activity, and/or expression of AFT honioiogs in fungal strains other than S. cerevisiae, is also within the scope of this invention. A person skilled in the art will be able to utilize publicly available sequences to construct relevant recombinant microorganisms with altered expression of AFT homoiogs. A listing of a representative number of AFT homoiogs known in the art and useful in the construction of recombinant microorganisms engineered for increased DHAD activity are listed Table 2. One skilled in the art, equipped with this disclosure, will appreciate other suitable homoiogs for the generation of recombinant microorganisms with increased DHAD activity. Sequences of AFT genes found in sub-species or variants of a given species may not be identical (See, e.g., > 98% identity amongst S. cerevisiae AFT1 genes of SEQ ID NOs: 1 , 208, 210, 212, 214, 216, 218, 220, 222, and 224). While it is preferred to overexpress an AFT gene native to the subspecies or variant, AFT genes may be interchangeably expressed across subspecies or variants of the same species.

Table 2. Representative Aft Homoiogs of Yeast Origin

35

364561 vl/CO (SEQ ID NO) (SEQ ID NO)

Saccharomyces cerevisiae S288c (AFT1) 1 2

Saccharomyces cerevisiae S288c (AFT2) 3 4

Candida glabrata (AFT1) 5 6

Candida glabrata (AFT2) 7 8

Zygosaccharomyces rouxii (AFT) 9 10

Ashbya gossypii (AFT) 1 1 12

Kiuyveromyces lactis (AFT) 13 14

Vanderwaltozyma polyspora (AFT) 15 16

Lachancea thermotoierans (AFT) 17 18

Debaromyces hanseii (AFT) 19 20

Saccharomyces bayanus ^* 21 22

Saccharomyces castelii ^* 23 24

Kiuyveromyces wa!tii ^* 25 26

Saccharomyces kluyveri ^* 27 28

Kiuyveromyces marxianus 29 30 issatchenkia orientaiis (AFT1-1) 31 32 issatchenkia orientaiis (AFT 1-2) 33 34

Saccharomyces bayanus (AFT2) 35 36

Saccharomyces castelii (AFT2) 37 38

S. cerevisiae W303 (AFT1) 208 209

S. cerevisiae DBVPG1106 (AFT1) 210 21 1

S. cerevisiae NCYC361 (AFT1) 212 213

S. cerevisiae Y55 (AFT1) 214 215

S. cerevisiae YJM981 (AFT1) 216 217

S. cerevisiae RM11 1A (AFT1) 218 219

S. cerevisiae UWOPS87_2421 (AFT1) 220 221

S. cerevisiae SK1 (AFT1) 222 22o

S, cerevisiae YPS606 (AFT1) 224 225

^* Byrne K.P,, Wolfe, K.H. (2005) The Yeast Gene Order Browser: combining cu rated homology and syntenic context reveals gene fate in polyploid species. Genome Research, 15(10):1456-61

[00137] Without being bound by any theory, it is believed that increasing the expression of the gene AFT1 or a homolog thereof will modulate the amount and availability of iron in the host cell. Since Aft1 activates the expression of target genes in response to changes in iron availability, overexpression of AFT1 increases the machinery to import more iron into the cytoso! and/or mitochondria. A person skilled in the art, equipped with this disclosure, will appreciate suitable methods for increasing the expression (i.e. overexpressing) AFT1. For instance, in one embodiment, AFT1 or homolog thereof may be overexpressed from a plasmid. In

36

364561 vl/CO another embodiment, one or more copies of the AFT1 gene or a homolog thereof is inserted into the chromosome under the control of a constitutive promoter. In addition, a skilled person in the art, equipped with this disclosure, will recognize that the amount of AFT1 overexpressed may vary from one yeast to the next. For example, the optimal level of overexpression may be one, two, three, four or more copies in a given yeast.

[00138] In additional embodiments, the native Aft1 or homolog thereof may be replaced with a mutant version that is constitutively active. In one embodiment, the native Aft1 is replaced with a mutant version that comprises a modification or mutation at a position corresponding to amino acid cysteine 291 of the S. cerevisiae Aft1 (SEG ID NO: 2). in an exemplary embodiment, the cysteine 291 residue of the native S. cerevisiae Aft1 (SEQ ID NO: 2) or homolog thereof is replaced with a phenylalanine residue.

[00139] As will be understood by one of ordinary skill in the art, modified Aft1 proteins and homologs thereof may be obtained by recombinant or genetic engineering techniques that are routine and well-known in the art. For example, mutant Aft1 proteins and homologs thereof, can be obtained by mutating the gene or genes encoding Aft1 or the homologs of interest by site-directed mutagenesis. Such mutations may include point mutations, deletion mutations and insertional mutations. For example, one or more point mutations (e.g., substitution of one or more amino acids with one or more different amino acids) may be used to construct mutant Aft1 proteins of the invention. The corresponding cysteine position of Aft1 homologs may be readily identified by one skilled in the art. Thus, given the defined region and the examples described in the present application, one with skill in the art can make one or a number of modifications which would result in the constitutive expression of Aft1 .

[00140] Without being bound by any theory, it is believed that increasing the expression of the gene AFT2 or a homolog thereof will modulate the amount and availability of iron in the host ceil. AFT2 overexpression is predicted to result in increased expression of the machinery to import more iron into the cytosoi and/or mitochondria. A person skilled in the art, equipped with this disclosure, will appreciate suitable methods for increasing the expression (i.e. overexpression) of AFT2. For instance, in one embodiment, AFT2 or homolog thereof may be overexpressed from a p!asmid. In another embodiment, one or more copies of the AFT2 gene or a homolog thereof is inserted into the chromosome under the control

37

364561 yl/CO of a constitutive promoter. In addition, a skilled person in the art, equipped with this disclosure, will recognize that the amount of AFT2 overexpressed may vary from one yeast to the next. For example, the optima! level of overexpression may be one, two, three, four or more copies in a given yeast. Moreover, the expression level may be tuned by using a promoter that achieves the optima! expression level in a given yeast

[00141] In another embodiment, the native Aft2 or homolog thereof may be replaced with a mutant version that is constitutively active. In one embodiment, the native Aft2 is replaced with a mutant version that comprises a modification or mutation at a position corresponding to amino acid cysteine 187 of the S. cerevisiae Aft2 (SEQ ID NO: 4). in an exemplary embodiment, the cysteine 187 residue of the native S. cerevisiae Aft2 (SEQ ID NO: 4) or homolog thereof is replaced with a phenylalanine residue.

[00142] As will be understood by one of ordinary skill in the art, modified Aft2 proteins and homologs thereof may be obtained by recombinant or genetic engineering techniques that are routine and well-known in the art. For example, mutant Aft2 proteins and homologs thereof, can be obtained by mutating the gene or genes encoding Aft2 or the homologs of interest by site-directed. Such mutations may include point mutations, deletion mutations and insertiona! mutations. For example, one or more point mutations (e.g., substitution of one or more amino acids with one or more different amino acids) may be used to construct mutant Aft2 proteins of the invention. The corresponding cysteine position of Aft2 homologs may be readily identified by one skilled in the art. Thus, given the defined region and the examples described in the present application, one with skill in the art can make one or a number of modifications which would result in the constitutive expression of Aft2.

[00143] In various exemplary embodiments, increasing the expression of both AFT1 and/or AFT2 will increase DHAD activity and the production of beneficial metabolites from DHAD-requiring biosynthetic pathways.

[00144] Embodiments in which the regulation, activity, and/or expression of AFT1 and/or AFT2 are altered can also be combined with increases in the extracellular iron concentration to provide increased iron in the cytosoi and/or mitochondria of the cell. Increase in iron in either the cytosoi or the mitochondria by this method appears to make iron more available for the FeS cluster-containing protein, DHAD. Without being bound by any theory, it is believed that such an increase in iron leads to a

38

364561 yl/CO corresponding increase in DHAD activity.

[00145] As described herein, the increased activity oi DHAD in a recombinant microorganism is a favorable characteristic for the production of beneficiai metabolites including isobutanoi, 3-methyi-l -butanol, 2-methyi-l -butanol, valine, isoleucine, leucine, and pantothenic acid derived from DHAD-requiring biosynthetic pathways. Without being bound by any theory, it is believed that the increase in DHAD activity as observed by the present inventors results from enhanced cellular iron levels as mediated by the altered regulation, expression, and/or activity of AFT1 and/or AFT2. Thus, in various embodiments described herein, the present invention provides recombinant microorganisms with increased DHAD activity as a result of alterations in AFT1 and/or AFT2 regulation, expression, and/or activity. In one embodiment, the alteration in AFT1 and/or AFT2 regulation, expression, and/or activity increases the activity of a cytosolicaily-iocalized DHAD. In another embodiment, the alteration in AFT1 and/or AFT2 regulation, expression, and/or activity increases the activity of a itochondrially-localized DHAD.

[00146] While particularly useful for the biosynthesis of isobutanoi, the altered regulation, expression, and/or activity of AFT1 and/or AFT2 is also beneficial to any other fermentation process in which increased DHAD activity is desirable, including, but not limited to, the biosynthesis of isoleucine, valine, leucine, pantothenic acid (vitamin B5), 2-methy!-1 -butanol, and 3-methyi-1 -butanol.

[00147] As described herein, the present inventors have observed increased isobutanoi titers, productivity, and yields in recombinant microorganisms exhibiting increased expression o AFT 1 and/or AFT2. Without being bound by any theory, it is believed that the increases in isobutanoi titer, productivity, and yield are due to the observed increases in DHAD activity. Thus, in one embodiment, the present invention provides a recombinant microorganism for producing isobutanoi, wherein said recombinant microorganism comprises an isobutanoi producing metabolic pathway, and wherein the expression of AFT1 or a homolog thereof is increased. In another embodiment, the present invention provides a recombinant microorganism for producing isobutanoi, wherein said recombinant microorganism comprises an isobutanoi producing metabolic pathway, and wherein the expression of AFT2 or a homolog thereof is increased. In yet another embodiment, the present invention provides a recombinant microorganism for producing isobutanoi, wherein said recombinant microorganism comprises an isobutanoi producing metabolic pathway, and wherein the expression of AFT 1 and AFT2 or homoiogs thereof is increased.

39

364561 yl/CO [00148] In alternative embodiments, nucleic acids having a homology to AFT1 and/or AFT2 of at least about 50%, of at least about 60%, of at least about 70%, at least about 80%, or at least about 90% similarity can be used for a similar purpose.

[00149] In one embodiment, the present invention provides a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, and wherein the activity of Aft1 or a homoiog thereof is increased. In another embodiment, the present invention provides a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, and wherein the activity of Aft2 or a homoiog thereof is increased. In yet another embodiment, the present invention provides a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, and wherein the activity of Aft1 and Aft2 or homo!ogs thereof is increased.

[00150] In alternative embodiments, proteins having a homology to Aft1 and/or Aft2 of at least about 50%, of at least about 60%, of at least about 70%, at least about 80%, or at least about 90% similarity can be used for a similar purpose.

[00151] In one embodiment, the isobutanol producing metabolic pathway comprises at least one exogenous gene that catalyzes a step in the conversion of pyruvate to isobutanol. In another embodiment, the isobutanol producing metabolic pathway comprises at least two exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least three exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least four exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at five exogenous genes that catalyze steps in the conversion of pyruvate to isobutanol.

[00152] In one embodiment, one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol. In one embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an

40

364561 yl/CO isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least four isobutanol pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with five isobutanol pathway enzymes localized in the cytosol. In a further exemplary embodiment, at least one of the pathway enzymes localized to the cytosol is a cytosoiically active DHAD enzyme as disclosed herein.

[00153] In various embodiments described herein, the isobutanol pathway genes may encode enzyme(s) selected from the group consisting of acetolactate synthase (ALS), ketoi-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2- keto-acid decarboxylase, e.g., keto-isovalerate decarboxylase (KIVD), and alcohol dehydrogenase (ADH).

[00154] As described above, the transcription factors Aft1 and Aft2 regulate genes involved in the acquisition, compartmentaiization, and utilization of iron. Thus, in additional aspects, the present invention provides methods of increasing DHAD activity and the production of beneficial metabolites produced from DHAD-requiring biosynthetic pathways as a result of alterations in the regulation, expression, and/or activity of genes regulated by Aft1 and Aft2. In one embodiment, the gene(s) regulated by Aft1 and Aft2 is selected from the group consisting of FET3, FET4, FET5, FTR1, FTH1, SMF3, MRS4, CCC2, COT1, ATX1, FRE1, FRE2, FRE3, FRE4, FRE5, FRE6, FIT1, FIT2, FIT3, ARN1, ARN2, ARN3, ARN4, ISU1, ISU2, TIS11, HMX1, AKR1, PCL5, YOR387C, YHL035C, YMRQ34C, !CY2, PRY1, YDL124W, BNA2, ECM4, LAP4, YOL083W, YGR146C, 8/05, YDR271C, OYE3, CTH1, CTH2, MRS3, MRS4, HSP26, YAP2, VMR1, ECL1, OSW1, NFT1, ARA2, TAF1/TAF130/TAF145, YOR225W, YKR104W, YBR012C, and YMR041C or a homoiog thereof. While particularly useful for the biosynthesis of isobutanol, the altered regulation, expression, and/or activity of genes regulated by Aft1 and Aft2 is also beneficial to any other fermentation process in which increased DHAD activity is desirable, including, but not limited to, the biosynthesis of isoleucine, valine, leucine, pantothenic acid (vitamin B5), 1 -butanoi, 2-methyl-1 -butanol, and 3-methyl-1 - butanol.

[00155] In one embodiment, ail genes demonstrated to increase DHAD activity and/or the production of a metabolite from a DHAD-requiring biosynthetic pathway are overexpressed. Where none of the AFT reguion genes expressed alone are

41

364561 yl/CO effective in increasing DHAD activity and/or the production of a metabolite from a DHAD-requiring biosynthetic pathway, then 1 , 2, 3, 4, 5, or more of the genes in the AFT regulon are overexpressed together.

[00156] As described herein, the present inventors have observed increased isobutanol titers, productivity, and yields in recombinant microorganisms exhibiting increased expression of the transcription factors AFT1 and/or AFT2 _S which regulate the expression of genes involved in the acquisition, compartmentaiization, and utilization of iron. Thus, in one embodiment, the present invention provides a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, and wherein the expression and/or activity of one or more genes selected from the group consisting of FET3, FET4, FET5, FTR1, FTH1, 8MF3, MRS4, CCC2, COT1, ATX1, FREl FRE2, FRE3, FRE4, PRE 5, FRE6, FIT1, FIT2, FIT3, ARN1, ARN2, ARN3, ARN4, !SU1, ISU2, TIS11, HMX1, AKR1, PCL5, YOR387C, YHL035C, YMRQ34C, ICY2, PRY1, YDL124W, BNA2, ECM4, LAP4, YGL083W, YGR146C, Bi05, YDR271C, OYE3, CTH1, CTH2, MR83, MRS4, HSP26, YAP2, VMR1, ECU, OSW1, NFT1, ARA2, TAF1/TAF130/TAF145, YOR225W, YKR104W, YBR012C, and YMR041C or a homolog thereof is increased.

Enhancing DHAD Activity by Increased GRX3/GRX4 Activity and/or Expression

[00157] As described herein, increasing the expression of the genes GRX3 and/or

GRX4 will generally modulate the amount and availability of iron in the yeast cytosoi or mitochondria. Accordingly, one aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said microorganism has been engineered to overexpress a polynucleotide encoding Grx3 and/or Grx4 or a homolog thereof. In one embodiment, the polynucleotide encoding the Grx protein or homolog thereof is native to the recombinant microorganism. In another embodiment, the polynucleotide encoding the Grx protein or homolog thereof is heterologous to the recombinant microorganism.

[00158] Grx3 and Grx4 are monothiol glutaredoxins that have been shown to be involved in cellular Fe content modulation and delivery in yeast. Glutaredoxins are glutathione-dependent thiol-disulfide oxidoreductases that function in maintaining the cellular redox homeostasis. S. cerevisiae has two dithioi glutaredoxins (Grx1 and Grx2) and three monothiol glutaredoxins (Grx3, Grx4, and Grx5). The monothiol

42

364561 vl/CO glutarecloxins are believed to reduce mixed disulfides formed between a protein and glutathione in a process known as deglutathionyiation. In contrast, dithioi glutaredoxins can participate in deglutathionyiation as well as in the direct reduction of disulfides. Grx5, the most studied monothiol g!utaredoxin, is localized to the mitochondrial matrix, where it participates in the maturation of Fe-S clusters. Grx3 and Grx4 are predominantly localized to the nucleus. These proteins can substitute for Grx5 when overexpressed and targeted to the mitochondrial matrix; no information on their natural function has been reported. In addition to the reported interaction between Grx3 and Aft1 , iron inhibition of Aft1 requires glutathione, it has been shown that iron sensing is dependent on the presence of the redundant Grx3 and Grx4 proteins. One report indicated that removal of both Grx3 and Grx4 resulted in constitutive expression of the genes regulated by Aft1/Aft2. This result suggested that the ceils accumulated Fe at levels greater than normal.

[00159] In one embodiment, Grx3 is overexpressed from a plasmid or by inserting multiple copies of the gene into the chromosome under the control of a constitutive promoter. In another embodiment, Grx4 is overexpressed from a plasmid or by inserting multiple copies of the gene into the chromosome under the controi of a constitutive promoter. In another embodiment, Grx3 and Grx4 are overexpressed from a plasmid or by inserting multiple copies of the gene into the chromosome under the control of a constitutive promoter. In another embodiment, Grx3, Grx4, or Grx3 and Grx4 are deleted or attenuated. In another embodiment, Grx3 and Aft1 are overexpressed from a plasmid or by inserting multiple copies of the gene into the chromosome under the control of a constitutive promoter. In another embodiment, Grx4 and Aft1 are overexpressed from a plasmid or by inserting multiple copies of the gene into the chromosome under the controi of a constitutive promoter. In another embodiment, Grx3 and Aft2 are overexpressed from a plasmid or by inserting multiple copies of the gene into the chromosome under the control of a constitutive promoter. In another embodiment, Grx4 and Aft2 are overexpressed from a plasmid or by inserting multiple copies of the gene into the chromosome under the control of a constitutive promoter. These embodiments can also be combined with increases in the extracellular iron concentration to provide increased iron in the cytosol or mitochondria of the cell. One or both of: Aft1 , Aft2 is overexpressed either alone or in combination with: Grx3 or Grx4. Such overexpression can be accomplished by plasmid or by inserting multiple copies of the gene into the chromosome under the controi of a constitutive promoter.

43

364561 yl/CO [00160] As described herein, the increased activity of DHAD in a recombinant microorganism is a favorable characteristic for the production of beneficial metabolites including isobutanol, 3-methy!-1 -butanol, 2-methy!-1 -butanol, valine, isoleucine, leucine, and pantothenic acid from DHAD-requiring metabolic pathways. Thus, in various embodiments described herein, the present invention provides recombinant microorganisms with increased DHAD activity as a result of alterations in GRX3 and/or GRX4 regulation, expression, and/or activity. In one embodiment, the alteration in GRX3 and/or GRX4 regulation, expression, and/or activity increases the activity of a cytosolicaliy-localized DHAD. In another embodiment, the alteration in GRX3 and/or GRX4 regulation, expression, and/or activity increases the activity of a mitochondrially-localized DHAD.

[00161] While particularly useful for the biosynthesis of isobutanol, the altered regulation, expression, and/or activity of GRX3 and/or GRX4 is also beneficial to any other fermentation process in which increased DHAD activity is desirable, including, but not limited to, the biosynthesis of isoleucine, valine, leucine, pantothenic acid (vitamin B5), 1 -butanol, 2~methyl-1 ~butanol, and 3-methyi~1 -butanoi,

[00162] In one embodiment, the present invention provides a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, and wherein the expression of GRX3 or a homoiog thereof is increased. In another embodiment, the present invention provides a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, and wherein the expression of GRX4 or a homoiog thereof is increased. In yet another embodiment, the present invention provides a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, and wherein the expression of GRX3 and GRX4 or homologs thereof is increased.

[00163] In alternative embodiments, nucleic acids having a homology to GRX3 and/or GRX4 of at least about 50%, of at least about 80%, of at least about 70%, at least about 80%, or at least about 90% similarity can be used for a similar purpose.

[00164] In one embodiment, the present invention provides a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, and wherein the activity of Grx3 or a homoiog thereof is increased, !n another embodiment, the present invention provides a recombinant microorganism for producing isobutanol, wherein

44

364561 yl/CO said recombinant microorganism comprises an isobutanol producing metabolic pathway, and wherein the activity of Grx4 or a homolog thereof is increased. In yet another embodiment, the present invention provides a recombinant microorganism for producing isobutanol, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, and wherein the activity of Grx3 and Grx4 or homologs thereof is increased.

[00165] In alternative embodiments, proteins having a homology to Grx3 and/or Grx4 of at least about 50%, of at least about 80%, of at least about 70%, at least about 80%, or at least about 90% similarity can be used for a similar purpose.

Altering the Iron-Sulfur Cluster Domain and/or Redox Active Domain

[00166] In general, the yeast cytosol demonstrates a different redox potential than a bacterial cell, as well as the yeast mitochondria. As a result, isobutanol pathway enzymes such as DHAD which exhibit an iron sulfur (FeS) domain and/or redox active domain, may require the redox potential of the native environments to be folded or expressed in a functional form. Expressing the protein in the yeast cytosol, which can harbor unfavorable redox potential, has the propensity to result in an inactive protein, even if the protein is expressed. The present inventors have identified a number of different strategies to overcome this problem, which can arise when an isobutanol pathway enzyme such as DHAD which is suited to a particular environment with a specific redox potential is expressed in the yeast cytosol.

[00167] In one embodiment, the present invention provides DHAD enzymes that exhibit a properly folded iron-sulfur cluster domain and/or redox active domain in the cytosol. Such DHAD enzymes may either be native or heterologous DHAD homologs or functional analogs or comprise a mutated or modified iron-sulfur cluster domain and/or redox active domain, allowing for a DHAD enzyme to be expressed in the yeast cytosol in a functional form. Thus, if an enzyme in the isobutanol production pathway was identified that was fully soluble and active in the cytosol of said recombinant microorganism, such enzyme can be used without addition of chaperone proteins not already present in the cytosol or without increased expression of chaperone proteins already present in the cytosol. However, some

DHAD proteins may need the assistance of additional chaperones or increased chaperone levels to exhibit optimal cytosol ic activity.

[00168] Therefore, in various embodiments described herein, the recombinant microorganisms may further comprise a nucleic acid encoding a chaperone protein,

45

364561 vl/CO wherein said chaperone protein assists the folding of a protein exhibiting cytoso!ic activity. Addition of the chaperone protein can lead to improved activity, solubility, and/or correct folding of the DHAD enzyme. In one embodiment, the chaperone may be a native protein. In another embodiment, the chaperone protein may be an exogenous protein. In some embodiments, the chaperone protein may be selected from the group consisting of: endoplasmic reticulum oxidoreductin 1 (Ero1 , accession no. NP 013578.1 ), including variants of Ero1 that have been suitably altered to reduce or prevent its normal localization to the endoplasmic reticulum; thioredoxins (which includes Trx1 , accession no. NP 013144.1 ; and Trx2, accession no. NP__01 1725.1 ), thioredoxin reductase (Trr1 , accession no. NP_010640.1 ); glutaredoxins (which includes Grx1 , accession no. NPJ309895.1 ; Grx2, accession no. NP_010801 .1 ; Grx3, accession no. NP__010383.1 ; Grx4, accession no. NP_01 101 .1 ; GrxS, accession no. NP_015266.1 ; Grx6, accession no. NP_010274.1 ; Grx7, accession no. NP_009570.1 ; GrxS, accession no. NP_013468.1 ); glutathione reductase Girl (accession no. NP__015234.1 ); Jac1 (accession no. NP__01 1497.1 ), including variants of Jac1 that have been suitably altered to reduce or prevent its normal mitochondrial localization; Hsp60 and Hsp10 proteins (e.g., yeast Hsp 60 and Hsp10 proteins, or other eukaryotic Hsp60 and Hsp10 homoiogs), bacterial chaperonin homoiogs (e.g., GroEL and GroES proteins from Lactococcus lactis); homoiogs or active variants thereof, and combinations thereof.

[00169] As described herein, it is preferred that the DHAD enzymes are properly assembled and folded, thus allowing for said DHADs to exhibit maximal activity in the cytosol. In yeast, the DHAD Ilv3 is involved in biosynthesis of the amino acids leucine, isoleucine and valine. Iiv3 is typically localized to the mitochondria, where the chaperonin proteins Hsp60 and Hsp10 aid in the proper folding of the protein (Dubaquie et. al. The EMBO Journal 1998 17: 5868-5876). In wild-type yeast cells, Iiv3 is found in the soluble fraction of ceil lysates. In extracts from an hsp60 temperature-sensitive mutant, at the non-permissive temperature, there is no detectable soluble Ilv3. All of the protein is found in the insoluble fraction, in a presumably inactivated state. In an hspIO temperature-sensitive mutant, at the non- permissive temperature, about half of the Ilv3 is found in the insoluble portion, indicating that HspI O is also important for proper folding of Ilv3, but that Hsp60 is required. (Dubaquie et. al. The EMBO Journal 1998 17: 5868-5876).

[00170] Thus, in one embodiment of the present invention, wherein the yeast DHAD encoded by ILV3 gene is used in the cytosol of a isobutanoi-producing

46

364561 yl/CO recombinant microorganism (e.g., a yeast microorganism), Hsp60 and/or Hsp10 from the same yeast, homologs thereof from other microorganisms, or active variants thereof can be overexpressed in said microorganism to increase the activity, solubility, and/or correct folding of DHAD encoded by ILV3 gene to increase the productivity, titer, and/or yield of isobutanol produced. Alternatively, if said microorganism is a yeast and it naturally expresses chaperonin proteins homologous to Hsp60 and/or Hsp10 in its cytosol, DHAD encoded by ILV3 can be expressed in said yeast without the overexpression of the Hsp60 and/or the Hsp10 proteins. In another embodiment, wherein the DHAD derived from an organism other than yeast is used for isobutanol production, chaperonin homologs, or active variants thereof derived from said non-yeast organism or related non-yeast organism can be overexpressed together with the DHAD derived from said non-yeast organism. In one embodiment, said non-yeast organism is an eukaryotic organism. In another embodiment, said non-yeast organism is a prokaryotic organism. In a further embodiment, said non-yeast organism is a bacterium (e.g., E. coll., or Lactococcus lactis). For example, the Lactococcus lactis GroEL and GroES chaperonin proteins are expressed in the yeast cytosol in conjunction with the IlvD from Lactococcus lactis. Overexpression of these genes may be accomplished by methods as described herein.

[00171] Also disclosed herein are recombinant microorganisms comprising one or more genes encoding an iron-sulfur cluster assembly protein. Iron-sulfur cluster assembly for insertion into yeast apo-iron-suifur proteins begins in yeast mitochondria. To assemble in yeast the active iron-sulfur proteins containing the cluster, either the apo-iron-suifur protein is imported into the mitochondria from the cytosol and the iron-sulfur cluster is inserted into the protein and the active protein remains localized in the mitochondria; or the iron-sulfur clusters or precursors thereof are exported from the mitochondria to the cytosol and the active protein is assembled in the cytosol or other cellular compartments.

[00172] Targeting of yeast mitochondrial iron-sulfur proteins or non-yeast iron- sulfur proteins to the yeast cytosol can result in such proteins not being properly assembled with their iron-sulfur clusters. This present invention overcomes this problem by co-expression and cytosolic targeting in yeast of proteins for iron-sulfur cluster assembly and cluster insertion into apo-iron-suifur proteins, including iron- sulfur cluster assembly and insertion proteins from organisms other than yeast, together with the apo-iron-suifur protein to provide assembly of active iron-sulfur

47

364561 yl/CO proteins in the yeast cytosol.

[00173] In some embodiments, the present invention provides methods of using Fe-S cluster containing protein in the eukaryotic cytosol for improved isobutanoi production in a microorganism, comprising overexpression of a Fe-S cluster- containing protein in the isobutanoi production pathway in an microorganism. In a preferred embodiment, said microorganism is a yeast microorganism. In one embodiment, said Fe-S cluster-containing protein is a endogenous protein. In another embodiment, said Fe-S duster-containing protein is an exogenous protein, !n one embodiment, said Fe-S cluster-containing protein is derived from a eukaryotic organism. In another embodiment, said Fe-S duster-containing protein is derived from a prokaryotic organism. In one embodiment, said Fe-S cluster-containing protein is DHAD. In one embodiment, said Fe-S cluster is a 2Fe-2S cluster. In another embodiment, said Fe-S cluster is a 4Fe-4S cluster.

[00174] All known DHAD enzymes contain an iron sulfur cluster, which is assembled in vivo by a multi-component pathway. DHADs contain one of at least two types of iron sulfur clusters, a 2Fe-2S cluster as typified by the spinach enzyme (Flint and Emptage, JSC 1988 283(8): 3558) or a 4Fe-4S duster as typified by the £. coii enzyme (Flint ef. a/., JSC 1993 268(20): 14732). In eukaryotic cells, iron-sulfur duster proteins can be found in either the cytosol or, more commonly, in the mitochondria. Within the mitochondria, a set of proteins, collectively similar to the ISC and/or SUF systems of £. coii, are present and participate in the assembly, maturation, and proper insertion of Fe-S clusters into mitochondrial target proteins. (Lill and Muhlenhoff, 2008, Annu. Rev, Biochem., 77:869-700). In addition, a cytosolic iron sulfur assembly system is present and is collectively termed the CIA machinery. The CIA system promotes proper Fe-S duster maturation and loading into cytosoiically-localized iron sulfur proteins such as Leu1 . Importantly, function of the CIA system is dependent on a critical (but still uncharacterized) factor exported from the mitochondria. In the yeast S.cerevisiae, the native DHAD, encoded by ILV3, is a mitochondrial!y-localized protein, where it is presumably properly recognized and activated by Fe-S cluster insertion by the endogenous machinery. Accordingly, ectopic expression of a DHAD in the yeast cytosol might be not expected to be functional due to its presence in a non-native compartment and the concomitant lack of appropriate Fe-S cluster assembly machinery.

[00175] The E, coii DHAD (encoded by HvD) is sensitive to oxygen, becoming quickly inactivated when isolated under aerobic conditions (Flint ef. a/., JSC 1993

48

364561 yl/CO 268(20): 14732; Brown et. al. Archives Biochem. Biophysics 1995 319(1 ): 10), It is thought that this oxygen sensitivity is due to the presence of a labile 4Fe-4S cluster, which is unstable in the presence of oxygen and reactive oxygen species, such as oxygen radicals and hydrogen peroxide. In yeast and other eukaryotes, the mitochondrial environment is reducing, i.e. it is a low oxygen environment, in contrast to the more oxygen-rich environment of the cytosol. The redox state of the cytosoi is thus expected to be a problem for expressing mitochondrialiy localized DHADs, which are natively located in the mitochondria, or in expressing DHADs from many bacterial species which typically have an intracellular reducing environment. The spinach DHAD has been shown to be more oxygen resistant than the £. coil enzyme in in vitro assays (Flint and Emptage, JBC 1988 263(8):3558), which may be due to its endogenous localization to the plastid, where it would normally encounter a relatively high-oxygen environment. It has been suggested that DHADs with 2Fe-2S clusters are inherently more resistant to oxidative damage and they are therefore an attractive possibility for inclusion in the cytosolicaliy localized isobutanoi pathway.

[00176] An additional complication to the oxygen sensitivity of DHADs is that the iron sulfur clusters must be properly assembled and inserted into the enzyme such that an active enzyme results. There are several types of machinery that produce iron sulfur clusters and properly assemble them into proteins, including the NIF system found in bacteria and in some eukaryotes, the ISC system found in bacteria and mitochondria, the SUF system found in bacteria and plastids, and the CIA system found in the cytosol of eukaryotes.

[00177] Thus, the methods of using Fe-S cluster in the eukaryotic cytosoi for improved enzymatic activity in isobutanoi production pathway as described above may further comprise the co-expression a heterologous Fe-S cluster-containing DHAD with the NIF assembly system in the yeast cytosoi to aid in assembling said heterologous DHADs. The N!F system found in the parasite Entamoeba histolytica has been shown to complement the double deletion of the £. coli !SC and SUF assembly systems (Aii et. al. JBC 2004 279(16): 18883) . The critical components of the Entamoeba assembly system comprise only two genes, NifS and NifU. In one embodiment, these two components are overexpressed in the yeast cytosol to increase activity and/or stability of cytosoiic DHADs. In one embodiment, the NIF system is the E. hisotlytica NIF system; in another embodiment, the NIF system is from other organisms (e.g. Lactococcus lactis). An advantage of using the £. hisotlytica assembly system is that it has already been demonstrated to work in a

49

364561 yl/CO heterologous organism, E. coli.

[00178] A 2Fe-2S duster-containing DHAD can be used in the present invention. In one embodiment, the 2Fe-2S cluster DHADs includes all known 2Fe-2S duster dehydratase enzymes identified biochemically. In another embodiment, the 2Fe-2S cluster DHADs include those predicted to be 2Fe-2S cluster dehydratases containing some version of the consensus motif for 2Fe-2S cluster proteins, e.g., the motif CX ₄ CX ₂ CX-30C (SEQ ID NO: 39, Liii and fVluh!enhoff, 2008, Annu. Rev. Biochem., 77:869-700). For example, based on the extremely highly conserved DHAD gene sequences shared amongst plant species, the inventors have synthesized a likely 2Fe-2S DHAD from Arabidopsis (and rice, Oryza sativa japonica) which can be used to improve isobutanoi production in vivo in the cytosolic isobutanoi pathway.

[00179] Alternatively, a DHAD may be determined to be a 2Fe-2S protein or a 4Fe- 48 protein based on a phylogenetic tree, such as Figure 3. Sequences not present on the example phylogenetic tree disclosed here could be added to the tree by one skilled in the art. Furthermore, once a new sequence was added to the DHAD phylogenetic tree, one skilled in the art may be able to determine if it is a 2Fe~2S or a 4Fe-4S cluster containing protein based on the phylogenetic relationship to known 2Fe-2S or a 4Fe-4S cluster containing DHADs.

[00180] In another embodiment, a 4Fe-4S cluster-containing DHAD could substitute for the 2Fe-2S duster-containing DHAD in the cytosoi. In one embodiment, said 4Fe-4S cluster DHAD is engineered to be oxygen resistant, and therefore more active in the cytosoi of cells grown under aerobic conditions.

[00181] In one embodiment of this invention, the apo-iron-sulfur protein DHAD enzyme encoded by the £. coii ilvD gene is expressed in yeast together with £. coii iron-sulfur cluster assembly and insertion genes comprising either the cyaY, iscS, iscU, iscA, hscB _s hscA, fdx and isuX genes or the suf A, sufB, sufC, sufD, sufS and sufE genes. This strategy allows for both the apo-iron-sulfur protein (DHAD) and the iron-sulfur cluster assembly and insertion components (the products of the isc or suf genes) to come from the same organism, causing assembly of the active DHAD iron- sulfur protein in the yeast cytosoi. As a modification of this embodiment, for those £. coii iron-sulfur cluster assembly and insertion components that localize to or are predicted to localize to the yeast mitochondria upon expression in yeast, the genes for these components are engineered to eliminate such targeting signals to ensure localization of the components in the yeast cytoplasm. Thus, in some embodiments, one or more genes encoding an iron-sulfur cluster assembly protein may be mutated

50

364561 yl/CO or modified to remove a signal peptide, whereby localization of the product of said one or more genes to the mitochondria is prevented. In certain embodiments, it may be preferable to overexpress one or more genes encoding an iron-sulfur cluster assembly protein.

[00182] In additional embodiments, iron-sulfur cluster assembly and insertion components from other than E. coll can be co-expressed with the E. coll DHAD protein to provide assembly of the active DHAD iron-sulfur cluster protein. Such iron- sulfur cluster assembly and insertion components from other organisms can consist of the products of the Helicobacter pylori nifS and nifU genes or the Entamoeba histolytica nifS and nifU genes. As a modification of this embodiment, for those non- E. coli iron-sulfur cluster assembly and insertion components that localize to or are predicted to localize to the yeast mitochondria upon expression in yeast, the genes for these components can be engineered to eliminate such targeting signals to ensure localization of the components in the yeast cytoplasm.

[00183] As a further modification of this embodiment, in addition to co-expression of these proteins in aerobically-grown yeast, these proteins may be co-expressed in anaerobicaliy-grown yeast to lower the redox state of the yeast cytoplasm to improve assembly of the active iron-sulfur protein.

[00184] In another embodiment, the above iron-sulfur cluster assembly and insertion components can be co-expressed with DHAD apo-iron-suifur enzymes other than the £. coil IlvD gene product to generate active DHAD enzymes in the yeast cytoplasm. As a modification of this embodiment, for those DHAD enzymes that localize to or are predicted to localize to the yeast mitochondria upon expression in yeast, then the genes for these enzymes can be engineered to eliminate such targeting signals to ensure localization of the enzymes in the yeast cytoplasm.

[00185] In additional embodiments, the above methods used to generate active DHAD enzymes localized to yeast cytoplasm may be combined with methods to generate active acetoiactate synthase, KARI, KIVD and ADH enzymes in the same yeast for the production of isobutanoi by yeast.

[00186] In another embodiment, production of active iron-sulfur proteins other than DHAD enzymes in yeast cytoplasm can be accomplished by co-expression with iron- sulfur cluster assembly and insertion proteins from organisms other than yeast, with proper targeting of the proteins to the yeast cytoplasm if necessary and expression in anaerobically growing yeast if needed to improve assembly of the active proteins.

51

364561 vl/CO [00187] In another embodiment, the iron-sulfur cluster assembly protein encoding genes may be derived from eukaryotic organisms, including, but not limited to yeasts and plants. In one embodiment, the iron-sulfur duster protein encoding genes are derived from a yeast organism, including, but not limited to S. cerevisiae. In specific embodiments, the yeast-derived genes encoding iron-sulfur cluster assembly proteins are selected from the group consisting of Cfd1 (accession no. NP 012263.1 ), Nbp35 (accession no. NP 01 1424.1 ), Nar1 (accession no. NP__014159.1 ), Cia1 (accession no. NP__010553.1 ), and homologs or variants thereof. In a further embodiment, the iron-sulfur cluster assembly protein encoding genes may be derived from plant nuclear genes which encode proteins translocated to chloroplasts or plant genes found in the chloropiast genome itself.

[00188] In certain embodiments described herein, it may be desirable to reduce or eliminate the activity and/or proteins levels of one or more iron-sulfur cluster containing cytosoiic proteins. This modification increases the capacity of a yeast to incorporate [Fe-S] clusters into cytosolically expressed proteins wherein said proteins can be native proteins that are expressed in a non-native compartment or heterologous proteins. This is achieved by deletion of a highly expressed native cytoplasmic [Fe-S]-dependent protein. More specifically, the gene LEU1 is deleted coding for the 3-isopropy!ma!ate dehydratase which catalyses the conversion of 3- isopropyimaiate into 2-isopropyimaleate as part of the leucine biosynthetic pathway in yeast. Leul p contains an 4Fe-4S cluster which takes part in the catalysis of the dehydratase. Some DHAD enzymes also contain a 4Fe-4S cluster involved in its dehydratase activity. Therefore, although the two enzymes have different substrate preferences the process of incorporation of the Fe-S cluster is generally similar for the two proteins. Given that Leul p is present in yeast at 10000 molecules per ceil (Ghaemmaghami S. et al. Nature 2003 425: 737), deletion of LEU1 therefore ensures that the ceil has enough spare capacity to incorporate [Fe-S] clusters into at least 10000 molecules of cytosolically expressed DHAD. Taking into account the specific activity of DHAD (£. coli DHAD is reported to have a specific activity of 63 U/mg (Flint, D.H. et al., JBC 1993 268: 14732), the LEU1 deletion yeast strain would generally exhibit an increased capacity for DHAD activity in the cytosol as measured in cell iysate.

[00189] In alternative embodiments, it may be desirable to further overexpress an additional enzyme that converts 2,3-dihydroxyisovalerate to ketoisovaierate in the cytosol. In a specific embodiment, the enzyme may be selected from the group

52

364561 yl/CO consisting of 3-isopropylrnalate dehydratase (Leul p) and imidazoleglycerol- phosphate dehydrogenase (His3p) or other dehydratases iis ed in Table 3.

Table 3. Dehydratases with putative activity towards 2,3-dihydroxyisovalerate.

[00190] Because in some embodiments, DHAD activity may be limited in the cytosol, alternative dehydratases that convert dihydroxyisova!erate (DHIV) to 2- ketoisovalerate (KIV) and are physiologically localized to the yeast cytosol may be utilized. Leul p and His3p and other enzymes encoded by genes iisted in Table 3 are dehydratases that potentially may exhibit affinity for DHIV. Leul p is an Fe-S binding protein that is involved in leucine biosynthesis and is also normally localized to the cytosol. His3p is involved in histidine biosynthesis and is similar to Leul p, it is generally localized to the cytosol or predicted to be localized to the cytosol. This modification overcomes the problem of a DHAD that is limiting isobutanoi production in the cytosol of yeast. The use of an alternative dehydratase that has activity in the cytosol with a low activity towards DHIV may thus be used in place of the DHAD in the isobutanoi pathway. As described herein, such enzyme may be further engineered to increase activity with DHIV.

Increased Mitochondrial Export of Essential Components for Iron Sulfur Protein Assem bly in the Cytosol

[00191] As noted herein, the third step in an exemplary isobutanoi biosynthetic pathway is the conversion of dihydroxyisovalerate (DHIV) to ketoisovaierate (KIV) by a dihydroxyacid dehydratase (DHAD). DHADs often require iron sulfur clusters for activity, and the native yeast DHAD acquires its iron sulfur cluster via the

53

364561 vl/CO mitochondrial ISC machinery, remaining within the mitochondria as an active enzyme. However, isobutanol production by the engineered pathway requires DHAD to be functionally expressed within the cytosol, and such a DHAD presumably requires iron sulfur clusters to be added in the cytosol. One of the inventions disclosed herein addresses possible genetic or chemical approaches to increase the functional activity of cytosol DHADs. The present invention provides ways to increase the export of an essential compound that is generated in mitochondria, thereby increasing the amount of the compound available for use by the cytosolic iron sulfur assembly machinery (e.g. CIA) to effectively increase the functional expression of cytosolic DHADs.

Overexpressing Mitochondrial Iron Sulfur Cluster (ISC) Machinery

[00192] The compound generated within the mitochondrial matrix that is essential for iron sulfur protein assembly in the cytosol is subsequently exported through the

ABC transporter, Atm1 , and is chaperoned across the intermembrane space of the mitochondria to the cytosol by Erv1 (reviewed in Liil and Muhlenhoff, 2008, Annu,

Rev. Biochem., 77:889-700). Sc_ BAT 1 was identified as a third putative component of the mitochondrial export machinery required for the export of an unknown compound essential for cytosolic iron-sulfur cluster biosynthesis from the mitochondrial matrix to the cytosol by a genetic selection of suppressors of a

Sc_atm1 temperature sensitive allele (Kispa! ef a/, 1998, JSC, 271 :24458-24484). It is also suggested that a further strong indication for a direct functional relationship between Atml p and Batl p is the leucine auxotrophy associated with the deletion of the A TM1 gene.

[00193] To facilitate export of the essential compound, the present invention provides in an embodiment recombinant microorganisms that have been engineered to overexpress one or more mitochondrial export proteins. In various embodiments described herein, the mitochondrial export protein may be selected from the group consisting of the S. cerevisiae ATM1, the S. cerevisiae ERV1, and the S. cerevisiae BAT1, or homologs thereof. Such manipulations can increase the export of the essential compound out of the mitochondria to increase the amount available for use by the cytosolic iron sulfur assembly machinery (e.g. CIA) to effectively increase the functional expression of cytosolic DHADs.

Increasing Inner Mitochondrial Membrane Electrical Potential

[00194] In one embodiment, the present invention provides recombinant

54

364561 yl/CO microorganisms that have further been engineered to increase inner mitochondrial membrane potential, ΔΜ½. As described herein, although yeast cells require a function mitochondrial compartment, they are viable without the mitochondrial genome (mtDNA). However, loss of mtDNA has been linked to destabilization of the nuclear genome (Veatch et ai, 2009, Cell, 1 37(7): 1 179-1 1 81 ). Nuclear genome stability was restored in yeast lacking mtDNA when a suppressor mutation {ATP1- 111) was introduced (Veatch et a!., 2009, Cell, 1 37(7): 1 179-1 181 , Francis et ai,, 2007, J. Bioenerg. Biomembr. 39(2): 149-157). The mutation has been shown to increase ATP hydrolysis activity of the mitochondrial ATP synthase, and similar mutations in the ATP synthase complex have also been shown to increase the electrical potential across the inner membrane of mitochondria, ΔΨ _Μ , in ceils lacking mtDNA (Smith et ai, 2005, Euk Cell, 4(12):2057-2085; Kominsky et ai, 2002, Genetics, 162:1595-1804). Generation of ΔΨ _Μ is required for efficient import of proteins into the mitochondrial matrix, including those involved in assembly and export of a complex required for the assembly of iron sulfur clusters into proteins in the cytosol. The link between ΔΨΜ and iron sulfur cluster assembly in the cytosol is supported by microarray data that indicate that the transcriptional profile of cells lacking mtDNA (decreased ΔΨ _Μ ) is similar to yeast grown under iron depletion conditions (Veatch et ai, 2009, Cell, 137(7):1 179-1 181 ). Introduction of the ATP1- 111 suppressor mutation restores the transcriptional profile to one resembling a wild- type cell's transcriptional profile (Veatch et al., 2009, Cell, 1 37(7): 1 1 79-1 181 ). Taken together, these data indicate that ΔΨ _Μ must be sufficient to support assembly of cytosolic iron sulfur proteins, particularly those involved in nuclear genome stability (Veatch et al., Ce// 2009, 137(7):1247-1258).

[00195] Thus, the present invention aims to generate the highest possible ΔΨ _Μ in a yeast with an intact mitochondrial genome, allowing for the maximization the export of the complex required for assembly of cytosolic iron sulfur proteins, which can in turn increase the amount available for use by the cytosolic iron sulfur assembly machinery (e.g. CIA) to effectively increase the functional expression of cytosolic DHADs. ΔΨΜ can be maximized several different ways, including, but not limited to: (1 ) Introducing mutations in the mitochondrial ATP synthase complex that increase ATP hydrolysis activity, or active variants thereof; (2) Overexpressing an ATP/ADP carrier protein that leads to an increase ATP ^4" import into the mitochondrial matrix in exchange for ADP ^3" , contributing to generation of ΔΨ _Μ ; (3) Removal and/or overexpression of additional gene(s) involved in generation of ΔΨ _Μ ; and (4) Addition

55

364561 yl/CO of chemical reagents that lead to an increase in ΔΨ _Μ .

[00196] In various embodiments described herein, the recombinant microorganism may comprise a mutation in the mitochondrial ATP synthase complex that increases ATP hydrolysis activity. In one embodiment, said mutant mitochondrial is an ATP synthase which can increase ATP hydrolysis activity is from a eukaryotic organism (e.g., a yeast ATP1 , ATP2, ATP3). In another embodiment, said mutant mitochondrial ATP synthase is from a prokaryotic organism (e.g., bacteria). Non- limiting examples of said mutant mitochondrial ATP synthase include, mutant ATPase from the ATP1-111 strain in Francis ef a/., J Bioenerg Biomembr, 2007, 39(2):127-144), a mutant ATPase from the atp2-227 strain in Smith et a/., 2005, Euk Ceil, 4(12):2057-2085, or a mutant ATPase from the yme1 strain in Kominsky et a/., 2002, Genetics, 162:1595-1804). In another embodiment, active variants, or homoiogs of the mutant mitochondrial ATP synthases described above can be applied. In one embodiment, an ATP synthase having a homology to any of ATP1 , ATP2, and ATP3 of at least about 70%, at least about 80%, or at least about 90% similarity can be used for a similar purpose.

[00197] In one embodiment, the inner mitochondrial membrane electrical potential can be increased by overexpressing an ATP/ADP carrier protein. Overexpression of the ATP/ADP carrier protein increases ATP ^4" import into the mitochondrial matrix in exchange for ADP ^3" . Non-limiting examples of ATP/ADP carrier proteins include the S. cerew ^' s/ae_AAC1 or the S. cerevisiae_AAC3, and active variants or homoiogs thereof. In one embodiment, an ATP/ADP carrier protein having a homology to either the S. cerevisiae__AAC\ or S. cerevisiae_PAC3 of at least about 70%, at least about 80%, or at least about 90% similarity can be used for a similar purpose.

[00198] In another embodiment, the inner mitochondrial membrane electrical potential can be increased by removal and/or overexpression of additional gene(s) involved in the generation of ΔΨ _Μ . A person skilled in the art will be familiar with proteins encoded by such genes. Non-limiting examples include the protein complexes in the mitochondrial electron transport chain which are responsible for establishing f-T ions gradient. For examples, complexes on the inner membrane of mitochondria that are involved in conversion of NADH to NAD ^÷ (Complex I, NADH dehydrogenase), succinate to fumarate (Complex II, cytochrome 6ci complex), and oxygen to water (Complex IV, cytochrome c oxidase), which are responsible for the transfer of H ⁺ ions. In another embodiment, enzymes in the citric acid cycle in the matrix of mitochondria can be overexpressed to increase NADH and succinate

56

364561 yl/CO production, such that more H ^÷ ions are avaiiab!e. These enzymes include, citrate synthase, aconitase, isocitrate dehydrogenase, a-Ketogiutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and ma!ate dehydrogenase.

[00199] In yet another embodiment, the inner mitochondrial membrane electrical potential can be increased by the addition of chemical reagents that lead to an increase in ΔΨ _Μ . in one embodiment, said chemical reagents are substrates in the citric acid cycle in the matrix of mitochondria, wherein when added into the culture, more NADH and succinate can be produced which in turn increase ΔΨ _Μ in the mitochondria. Non-limiting examples of said substrates include, oxaioacefate, acetyl CoA.citrate, cis-Aconitate, isocitrate, oxaiosuccinate, a-Ketoglutarate, succinyi-CoA, succinate, fumarate and L-Maiate.

Enhancing Cytosolic DHADs Activity by Increasing Cytosol Sulfur Levels

[00200] Also provided herein are methods of increasing the levels of sulfur- containing compounds within yeast cells, including the amino acid cysteine, such that this sulfur is more available for the production of iron-sulfur cluster-containing proteins in the yeast cytosol or mitochondria. Specifically, by increasing the concentration of sulfur-containing compounds in the ceil such, the activity of a functional DHAD is enhanced in the yeast cytosol or mitochondria.

[00201] Accordingly, the present invention provides in an embodiment recombinant microorganisms that have been engineered to overexpress one or more genes to increase biosynthesis of cysteine or uptake of exogenous cysteine by the cell in order to increase the amount and availability of sulfur-containing compounds for the production of active iron-sulfur cluster-containing proteins in the yeast cytosol or mitochondria. In one embodiment, the recombinant microorganisms have been engineered to increase the expression of one or more proteins to increase cysteine biosynthesis by the cell, including, but not limited to MET3, MET14, MET16, MET10, MET5, MET1, MET8, MET2, MET11, HOM3, HOM2, HOM6, CYS3, CYS4, 8UL1, SUL2, active variants thereof, homologs thereof, and combination thereof, to increase cysteine biosynthesis by the ceil. In another embodiment, the recombinant microorganisms have been engineered to increase the expression of one or more transport proteins, including, but not limited to YCT1, MUP1, GAP1, AGP1, GNP1, BAP1, BAP2, TAT1, active variants thereof, homologs thereof, and combination thereof.

57

364561 vl/CO [00202] As noted above, increasing uptake of exogenous cysteine by the ceil will increase the amount and availability of sulfur-containing compounds for the production of active iron-sulfur cluster containing proteins in the cytosoi or mitochondria of the cell. Addition of increased exogenous cysteine to yeast cells, separately from or in addition to increased expression of the transport protein- encoding genes as described above, can also increase the level and availability of sulfur-containing compounds within the cell such that the sulfur is more available for the production of iron-sulfur cluster-containing proteins in the ceil cytosoi or mitochondria.

[00203] Sulfur is a necessary element for the biogenesis of iron-sulfur cluster (FeS ciuster)-containing protein in vivo. Sulfur is a component of the FeS clusters that are incorporated into such proteins and is also a component of compounds such as glutathiones, which are essential for FeS cluster biogenesis in many organisms as well as being involved in cellular redox homeostasis. The direct source of the sulfur for these processes in many organisms is the amino acid cysteine. The sulfur from cysteine is mobilized into FeS clusters during FeS duster biogenesis using cysteine desulfurase proteins identified in many organisms such as IscS, SufS (together with SufE), NifS and Nfs1 (together with isd1 1 ). Additionally, glutathione biosynthesis requires cysteine.

[00204] Increased expression of Fe-S cluster-containing proteins in organisms such as the budding yeast S. cerevisiae results in an increased demand for sulfur, in the form of cysteine, in the ceil. Such an increased demand for cysteine may possibly be met by natural induction of the endogenous cysteine biosynthetic pathway but maximal natural induction of this pathway may be insufficient to provide enough cysteine for the proper assemble and maintenance of increased levels of FeS cluster-containing proteins in the ceil. Such ceils with an increased demand for cysteine may also induce cysteine and/or sulfate transport pathways to bring in exogenous cysteine for or sulfate, which is the sulfur donor for cysteine biosynthesis. However, maximal natural induction of these transport systems may also be insufficient to meet the sulfur requirement of such cells.

[00205] Assembly of active FeS cluster-containing proteins in the native yeast cytosoi requires the production and export to the cytosoi by the mitochondria of an unidentified sulfur-containing compound derived from the mitochondrial FeS cluster biogenesis pathway and the amino acid cysteine and requiring glutathione for export. Overexpression of an FeS cluster-containing protein in the yeast cytosoi or the

58

364561 yl/CO localization of a previously non-cytoso!ic FeS cluster-containing protein to the yeast cytosol may result in the decreased availability of this unidentified sulfur-containing compound in the yeast cytosoi and low activity of the cytosolic FeS cluster- containing protein or proteins. Increased availability of cysteine to the cell may prevent this limitation by providing increased sulfur for the biosynthesis of this compound and sufficient glutathione for its export from the mitochondria.

[00206] Sulfur for the assembly of FeS cluster-containing proteins expressed in the yeast cytosoi may also be provided by localization of cysteine desuifurase proteins to the yeast cytosoi. Expression of such proteins in the yeast cytosoi may result in an increased demand for cysteine by such cells, especially in the cytosoi. Additionally, damage to the FeS cluster of FeS cluster-containing proteins expressed in the yeast cytosoi, due to the oxic nature of the yeast cytosoi or due to reactive oxygen or nitrogen species, may require additional sulfur derived from cysteine for repair or regeneration of the damaged clusters. As well, additional sulfur derived from cysteine may modulate the redox balance of the yeast cytosoi through the production of increased levels of compounds such as glutathione which may positively affect the assembly or activity of FeS cluster-containing proteins in the yeast cytosoi.

[00207] Increased cellular sulfur in the form of cysteine can be provided by increasing the biosynthesis of cysteine in the cell or by increasing cellular uptake of exogenous cysteine. Increasing the cellular level of cysteine in these ways is expected to increase the level of other sulfur-containing compounds in the cell that derive their sulfur from cysteine or the cysteine biosynthesis pathway. Cysteine biosynthesis in S. cerevisiae involves the uptake of exogenous sulfate by transport proteins encoded by the SUL1 and/or SUL2 genes and the action of the proteins encoded by the MET3, MET14, MET16, MET10, MET5, MET1, MET8, MET2, MET17, HOM3, HOM2, HOM6, CYS4 and CYS4 genes. Exogenous cysteine is taken up into S. cerevisiae by the high-affinity transport system encoded by the yCTf gene but also by the broader-specificity transport proteins encoded by the MUP1, GAP1, AGP1, GNP1, BAP1, BAP2, TAT1 and TAT2 genes.

[00208] Thus, in an additional aspect, the invention is directed to methods of increasing the levels of sulfur-containing compounds within the yeast cytosoi and/or mitochondria, such that sulfur is more available for the production of iron-sulfur cluster-containing proteins in the cytosoi or mitochondria. In one embodiment, the levels of sulfur-containing compounds within the yeast cytosoi and/or mitochondria are increased. In another embodiment, an increase in sulfur-containing compounds

59

364561 yl/CO in the yeast cytosol or mitochondria leads to an increase in activity of a cytosolicaily expressed FeS cluster-containing protein DHAD, which catalyzes the reaction of 2,3- dihydroxyisovaierate to 2-ketoisova!erate. In another embodiment, an increase in sulfur-containing compounds in the yeast cytosol or mitochondria leads to an increase in activity of a cytosolicaily expressed DHAD. !n another embodiment, an increase in sulfur-containing compounds in the yeast cytosol and/or mitochondria leads to an increase in activity of a cytosolicaily expressed DHAD and a subsequent increase in the productivity, titer, and/or yield of isobutanol produced by the DHAD- containing strain. !n another embodiment, an increase in sulfur-containing compounds in the yeast cytosol or mitochondria leads to an increase in activity of a mitochondrialiy expressed FeS cluster-containing protein DHAD, which catalyzes the reaction of 2,3-dihydroxyisovalerate to 2-ketoisovalerate. In another embodiment, an increase in sulfur-containing compounds in the yeast cytosol or mitochondria leads to an increase in activity of a mitochondrialiy expressed DHAD. In another embodiment, an increase in sulfur-containing compounds in the yeast cytosol and/or mitochondria leads to an increase in activity of a mitochondrialiy expressed DHAD and a subsequent increase in the productivity, titer, and/or yield of isobutanol produced by the DHAD-containirig strain.

[00209] In another embodiment, the genes YCT1, MUP1, GAP1, AGP1, GNP1, BAP1, BAP2, TAT1, and LA 72, active variants thereof, homologs thereof or combination thereof are overexpressed from a p!asmid or by inserting multiple copies of the gene or genes into the chromosome under the control of a constitutive promoter. This embodiment can also be combined with providing increased extracellular cysteine to the yeast cells to provide increased sulfur-containing compounds in the cytosol and/or mitochondria of the ceils. Overexpression of these genes may be accomplished by methods as described above.

[00210] In another embodiment, providing increased extracellular cysteine to the yeast cells in the absence of any additional engineered expression of transport proteins will provide increased sulfur containing compounds in the cytosol and/or mitochondria of the cells for the improved production of active FeS cluster-containing proteins in the yeast cytosol or mitochondria, which leads to increased isobutanol productivity, titer, and/or yield by the cell.

Enhancing Cytosoiic DHAD Activity by Mitigating Oxidative Species or Oxidative Stress

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364561 yl/CO [00211] The present application also describes methods of protecting enzymes in a DHAD-requiring biosyn hetic pathway (specifically DHAD) in a microorganism to increase the production of beneficial metabolites by mitigating oxidative species or oxidative stress induced damage in the cytosol of said microorganism. Non-limiting examples of oxidative species include, nitric oxide (NO), reactive nitrogen species (RNS), reactive oxygen species (ROS), hydroxy! radical species, organic hydroperoxide, hypochiorous acids, and combinations thereof. As used herein, the phrase "reactive oxygen species" or "ROS" refers to free radicals that contain the oxygen atom. ROS are very small molecules that include oxygen ions and peroxides and can be either inorganic or organic. They are highly reactive due to the presence of unpaired valence shell electrons. During times of environmental stress (e.g. UV or heat exposure) ROS levels can increase dramatically, which can result in significant damage to cell structures. This cumulates into a situation known as oxidative stress. ROS are also generated by exogenous sources such as ionizing radiation.

[00212] Oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. AN forms of life maintain a reducing environment within their ceils. This reducing environment is preserved by enzymes that maintain the reduced state through a constant input of metabolic energy. Disturbances in this normal redox state can cause toxic effects through the production of peroxides and free radicals that damage ail components of the cell, including proteins, lipids, and DNA.

[00213] In chemical terms, oxidative stress is a large rise (becoming less negative) in the cellular reduction potential, or a large decrease in the reducing capacity of the cellular redox couples, such as glutathione. The effects of oxidative stress depend upon the size of these changes, with a cell being able to overcome small perturbations and regain its original state. However, more severe oxidative stress can cause cell death and even moderate oxidation can trigger apoptosis, while more intense stresses may cause necrosis.

[00214] A particularly destructive aspect of oxidative stress is the production of reactive oxygen species, which include free radicals and peroxides, and/or other reactive species. Some of the less reactive of these species (such as superoxide) can be converted by oxidoreduction reactions with transition metals or other redox cycling compounds (including quinones) into more aggressive radical species that can cause extensive cellular damage. The major portion of long term effects is

61

364561 yl/CO inflicted by damage on DNA. Most of these oxygen-derived species are produced at a low level by normal aerobic metabolism and the damage they cause to cells is constantly repaired. However, under the severe levels of oxidative stress that cause necrosis, the damage causes ATP depletion, preventing controlled apoptotic death and causing the cell to simply fall apart. Non-limiting example of oxidants include, superoxide anion (Ό2-, formed in many autoxidation reactions and by the electron transport chain), hydrogen peroxide CH2Q2, formed by disputation of *02- or by direct reduction of O2) , organic hydroperoxide (ROOH, formed by radical reactions with cellular components such as lipids and/or nucieobases), oxygen centered organic radicals (e.g., RG ^s aikoxy and ROO ^» , peroxy radicals, formed in the presence of oxygen by radical addition to double bonds or hydrogen abstraction), hypochlorous acid (HOCi, formed from H2O2 by myeloperoxidase, and peroxynitrife (ONOO-, formed in a rapid reaction between *0 ₂ - and NO*).

[00215] Biological defenses against oxidative damage include protective proteins that remove reactive oxygen species, molecules that sequester metal ions, and enzymes that repair damaged cellular components. Oxidative stress can be defined as a disturbance in the prooxidant-antioxidant balance in favor of prooxidants. One such class of prooxidants are reactive oxygen species, or ROS. ROS are highly reactive species of oxygen, such as superoxide (02- ^" ), hydrogen peroxide {H2O2), and hydroxy! radicals (OH-), produced within the cell, usually as side products of aerobic respiration. By some reports, as much as 2% of the oxygen that enters the respiratory chain is converted to superoxide through a one-electron reduction of oxygen. A small amount of superoxide radical is always released from the enzyme when oxygen is reduced by electron carriers such as flavoproteins or cytochromes. This is because the electrons are transferred to oxygen one at a time. The hydroxy! radical and hydrogen peroxide are derived from the superoxide radical.

[00216] Many microbes possess native enzymes to detoxify these ROS. One example of such a system is superoxide dismutase (SOD) plus catalase. SOD catalyzes a reaction where one superoxide radical transfers Its extra electron to the second radical, which is then reduced to hydrogen peroxide. Catalase catalyzes the transfer of two electrons from one hydrogen peroxide molecule to the second, oxidizing the first to oxygen and reducing the second to two molecules of water. If the hydrogen peroxide is not disposed of, then it can oxidize transition metals, such as free iron(!l) in the Fenton reaction, and form the free hydroxy! radical, OH -. No known mechanisms exists to detoxify hydroxyl radicals, and thus protection from

62

364561 yl/CO toxic forms of oxygen must rely on eliminating superoxide and hydrogen peroxide.

[00217] In yeast, to counteract damage of oxidative stress, there are several antioxidant systems with an apparent functional redundancy. For example, there are detoxifying enzymes such as catalases, cytochrome c peroxidase, glutathione peroxidases, giytaredoxins and peroxi redox! ns, and many isoforms in distinct cellular compartments (Jamieson et al., 1998, Yeast. 14:151 1 -1527; Grant et ai, 2001 , Mol. Microbiol 39:533-541 ; Coliinson et al., 2003, J. Biol. Chem. 278:22492-22497; Park et al., 2000, J. Biol. Chem. 275:5723-5732).

[00218] As described above, an enzyme involved in the isobutanol production pathway, dihydroxyacid dehydratase (DHAD), contains an iron-sulfur (FeS) cluster domain. This iron-sulfur (FeS) cluster domain is sensitive to damage by ROS, which can lead to inactive enzyme. Both 2Fe-2S and 4Fe-4S DHAD enzymes may be susceptible to inactivation by ROS, however direct evidence exists for inactivation of 4Fe-4S cluster containing proteins, such as homoaconitase and isopropyimalate dehydratase in yeast and DHAD and fumarase from E. coli. Therefore, to achieve a functional DHAD expressed in the yeast cytosol in an environment where a substantial amount of ROS may exist from respiration, it may be beneficial to protect the DHAD enzyme from ROS inactivation or oxidative stress through expression of on or more enzymes that reduce or eliminate ROS from the cell.

[00219] To mitigate the potential harmful effects of reactive oxygen species (ROS) or oxidative stress on DHAD in the yeast cytoso!, the present inventors have devised several strategies to protect or repair the DHAD from ROS damage. In various embodiments described herein, the invention provides recombinant microorganisms that have been engineered to express one or more proteins in the cytosol that reduce the concentration of reactive oxygen species (ROS) in said cytosol.

[00220] In one embodiment, enzymes that reduce or eliminate the amount of ROS in the cytosol are expressed and targeted to the yeast cytosol. Specifically, enzymes such as cataiase, superoxide dismutase (SOD), cytochrome c peroxidase, glutathione peroxidases, giytaredoxins, peroxiredoxins, meta!Iothioneins, and methionine su!phoxide reductases, or any isoforms thereof are expressed, such that they lead to reduction in ROS such as hydrogen peroxide, superoxide, peroxide radicals, and other ROS in the yeast cytosol.

[00221] In one embodiment, a cataiase is expressed to reduce the concentration of ROS in the cytosol. In another embodiment, a superoxide dismutase (SOD) is expressed to reduce the concentration of ROS in the cytosol. Usually, microbes that

63

364561 yl/CO grow by aerobic respiration possess one or both of SOD and cata!ase. For example, the bacterium E. coii and the yeast Saccharomyces cerevisiae each possesses at least one native SOD and catalase (e.g., SOD1 or SOD2 from yeast). In E. coii, the genes katG and katE encode cataiase enzymes, and the genes sodA, sodB and sodC encode SodA, SodB, and SodC superoxide dismutase enzymes, respectively. In S. cerevisiae, the genes CTT1 and CTA1 encode cataiase CTT1 and CTA1 enzymes, and the genes SOD1 and SOD2 encode SOD1 and SOD2 superoxide dismutase enzymes. Many other organisms possess cataiase and SOD enzymes and these genes may also be useful for reduction of ROS in the yeast cytosoi. In one embodiment, SOD homologs from species other than E, coii or yeast can be expressed in yeast cytosoi to reduce oxidative stress. In one embodiment, said other species is a plant or a fungus. For example, SOD1 from N. crassa (fungus) may be functionally expressed in the yeast cytosoi. In various embodiments described herein, active variants or homoiogs of the above-described cataiases and SODs can be functionaiiy expressed in the yeast cytosoi. In another embodiment, protein having a homology to any one of the cataiases or SODs described above possessing at least about 70%, at least about 80%, or at least about 90% similarity can be functionally expressed in the yeast cytosoi.

[00222] In one embodiment, the cataiase genes from £. coii are expressed in and targeted to the cytosoi of yeast to reduce the amount of ROS and increase the activity of DHAD also expressed in and targeted to the yeast cytosoi. In another embodiment, the cataiase genes from S. cerevisiae are overexpressed in and targeted to the cytosoi of yeast to reduce the amount of ROS and increase the activity of DHAD also expressed in and targeted to the yeast cytosoi. In one embodiment, the SOD genes from E. coii are expressed in and targeted to the cytosoi of yeast to reduce the amount of ROS and increase the activity of DHAD also expressed in and targeted to the yeast cytosoi. In another embodiment, the SOD genes from S. cerevisiae are expressed in and targeted to the cytosoi of yeast to reduce the amount of ROS and increase the activity of DHAD also expressed in and targeted to the yeast cytosoi. In another embodiment, promoters of native genes are altered, such that the level of SOD or cataiase in the S. cerevisiae cytosoi is increased. In yet another embodiment, expression of SOD or cataiase in the yeast cytosoi is mediated by a piasmid. In yet another embodiment, expression of SOD or cataiase in the yeast cytosoi is mediated by expression of one or more copies of the gene from the chromosome. Other homoiogs of cataiase or SOD may be identified

64

364561 yl/CO by one skilled in the art through tools such as BLAST and sequence alignment. These other homoiogs may be expressed in a similar manner described above to achieve a functional catalase or SOD in the yeast cytosol.

[00223] In another embodiment, a methionine sulphoxide reductase enzyme is expressed to reduce the amount of ROS and protect DHAD from ROS damage and inactivation. In one embodiment, the methionine sulphoxide reductase may be derived from a eukaryotic organism (e.g., a yeast, fungus, or plant). In another embodiment, the methionine sulphoxide reductases may be derived from a prokaryotic organism (e.g., E. coli), The principal enzymatic mechanism for reversing protein oxidation acts on the oxidation product of just one amino acid residue, methionine. This specificity for Met reflects the fact that Met is particularly susceptible to oxidation compared with other amino acids. Methionine sulphoxide reductases (MSRs) are conserved across nearly all organisms from bacteria to humans, and have been the focus of considerable attention in recent years. Two MSR activities have been characterized in the yeast Saccharomyces cerevisiae MsrA (encoded by MXR1 ) reduces the S stereoisomer of methionine sulphoxide (MetO), while MsrB (encoded by the YCL033c ORF), which we term here MXR2) reduces the R stereoisomer of MetO. Consistent with defense against oxidative damage, mutants deficient in MSR activity are hypersensitive to pro-oxidants such as H2O2, paraquat and Cr, while MSR overexpression enhances resistance. Besides methionine residues, iron-sulfur (FeS) clusters are exquisitely ROS-sensitive components of many cellular proteins. It has been reported that MSR activity helps to preserve the function of cellular FeS clusters.

[00224] In one embodiment, the methionine sulphoxide reductase genes from S. cerevisiae are expressed in and targeted to the cytosol of yeast to reduce the amount of ROS and increase the activity of DHAD also expressed in and targeted to the yeast cytosol. Specifically, the S. cerevisiae methionine sulphoxide reductase genes MsrA (encoded by MXR1) and MsrB (encoded by the YCL033c ORF) are expressed in and targeted to the cytosol of yeast to reduce the amount of ROS and increase the activity of DHAD also expressed in and targeted to the yeast cytosol. The resulting methionine sulphoxide reductase expressing strain will generally demonstrate improved isobutano! productivity, titer, and/or yield compared to the parental strain that does not comprise methionine sulphoxide reductase genes that are expressed in and targeted to the cytosol. Methionine sulphoxide reductases from other organisms, such as bacteria, may be identified by sequence homology

65

364561 yl/CO using tools such as BLAST and pairwise sequence alignments by one skilled in the art.

[00225] In yet another embodiment, expression or overexpression of glutathione synthesis enzymes, for example GSH1 , leads to increased glutathione in the cell and protection of the DHAD enzyme in the yeast cytosol. In one embodiment, said enzymes are derived from a bacteria (e.g., E. co//„). In another embodiment, said enzymes are derived from yeast (e.g., S.cerevisiae). In yet another embodiment, said enzymes are derived from a yeast species different from the yeast used for isobutanol production.

[00226] In one embodiment, one or more metailothionein proteins are expressed in the yeast cytosol to mitigate oxidative stress. Metailotbioneins are a family of proteins found in many organisms including yeast and mammals. The biologic function of metailothionein (MT) has been a perplexing topic ever since the discovery of this protein. Many studies have suggested that MT plays a role in the homeostasis of essential metals such as zinc and copper, detoxification of toxic metals such as cadmium, and protection against oxidative stress. MT contains high levels of sulfur. The mutual affinity of sulfur for transition metals makes the binding of these metals to MT thermodynamically stable. Under physiologic conditions, zinc-MT is the predominant form of the metal-binding protein. However, other metals such as copper (Cu) are also bound by MT. Oxidation of the thioiate cluster by a number of mild cellular oxidants causes metal release and formation of MT-disulfide (or thionin if all metals are released from MT, but this is unlikely to occur in vivo), which have been demonstrated in vivo. MT-disulfide can be reduced by glutathione in the presence of selenium catalyst, restoring the capacity of the protein to bind metals like Zn and Cu. This MT redox cycle may play a crucial role in MT biologic function. It may link to the homeostasis of essential metals, detoxification of toxic metals and protection against oxidative stress. In fact, MT has been shown to substitute for superoxide dismutase in yeast cells in the presence of Cu to protect cells and proteins from oxidative stress.

[00227] In one embodiment, said meta!lothuineins are derived from a eukaryotic organism (e.g., a yeast, fungus, or plant). In another embodiment, said metallothuineins are derived from a prokaryotic organism (e.g., £. co!i, Mycobacterium tuberculosis). For example, the metailothionein genes CUP1-1 and CUP1-2 encoding metailothionein CUP1 from S. cerevisiae, active variants thereof, homoiogs thereof, or combination thereof are expressed in and targeted to the

66

364561 yl/CO cytosol of yeast to reduce the amount of ROS and increase the activity of DHAD also expressed in and targeted to the yeast cytosol. In another embodiment, S. cerevisiae metal!othionein genes CUP1-1 and CUP1-2 are expressed in and targeted to the cytosol of yeast to reduce the amount of ROS and increase the activity of DHAD also expressed in and targeted to the yeast cytosol. In another embodiment, Mycobacterium tuberculosis metallothionein gene MymT encoding metaliothionein is expressed in and targeted to the cytosol of yeast to reduce the amount of ROS and increase the activity of DHAD that is also expressed in and targeted to the yeast cytosol. In another embodiment, Synechococcus PCC 7942 metallothionein gene SmtA is expressed in and targeted to the cytosol of yeast to reduce the amount of ROS and increase the activity of DHAD that is also expressed in and targeted to the yeast cytosol. The resulting metallothionein expressing strain has improved isobutanol productivity, titer, and/or yield compared to the parental strain. Metallothioneins from other organisms, such as bacteria, may be identified by sequence homology using tools such as BLAST and pairwise sequence alignments by one skilled in the art.

[00228] In another embodiment, one or more proteins in the thioredoxin system and/or the glutathione/glutaredoxin system, active variants thereof, homologs thereof, or combination thereof are expressed in the yeast cytosol to mitigate oxidative stress. In one embodiment, said proteins in the thioredoxin system and/or the glutathione/glutaredoxin system are derived from a eukaryotic organism (e.g., a yeast, fungus, or plant). In another embodiment, said proteins in the thioredoxin system and/or the glutathione/glutaredoxin system are derived from a prokaryotic organism (e.g., E. coii). The thioredoxin system and the glutathione/glutaredoxin system help maintain the reduced environment of the cell and play significant roles in defending the ceil against oxidative stress. Glutathione is the major protective small molecule against oxidative stress in Saccharomyces cerevisiae. Glutathione, the tripeptide γ-giutamyl-cysteinyl-glycine, makes up the major free thiol pool present in miilimolar concentrations in aerobic ceils. The biosynthesis of glutathione requires γ- glutamyl cysteine synthase (termed Gshl p) glutathione synthase (Gsh2p) and ATP. Glutathione is essential for viability of yeast but not of bacteria such as E, coii. Yeast cells lacking Gshl p (genotype gsh1A) are able to survive in the presence of an external source of glutathione. Deletion of the GSH1 gene encoding the enzyme that catalyzes the first step of glutathione biosynthesis leads to growth arrest, which can be relieved by either glutathione or reducing agents such as dithiothreitol.

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364561 yl/CO Evidence suggests that glutathione, in addition to its protective role against oxidative damage, performs a novel and specific function in the maturation of cytosolic Fe/S proteins. Therefore, increasing the levels of glutathione in the yeast cytosol is predicted to protect or increase the steady-state levels of active FeS cluster containing proteins expressed in the yeast cytosol. Specifically, increasing glutathione within the yeast cytosol may increase the amount of active DHAD enzyme expressed in the yeast cytosol, thereby leading to an increase in the titer, productivity, and/or yield of isobutanol produced from the pathway within which DHAD participates (e.g. the isobutanol pathway in Figures 1 -2).

[00229] Thioredoxins and glutaredoxins are small heat-stable proteins with redox- acfive cysteines that facilitate the reduction of other proteins by catalyzing cysteine thiol-disulfide exchange reactions. The g!utathione/glutaredoxin system consists of glutaredoxin, glutathione (produced by glutathione synthase), glutathione reductase and NADPH (as an electron donor). Thus, to increase the effective levels of available glutathione, one or a combination of each of the following enzymes is functionally overexpressed in the yeast cytosol: glutaredoxin (encoded in S.cerevisiae by GRX2, GRX4 _S GRX8, and GRX7), glutathione reductase (encoded in S.cerevisiae by GLRT); and glutathione synthase (encoded in S.cerevisiae by GSH1 and GSH2). In one embodiment, homologs thereof, active variants thereof, or combination thereof can be expressed in the yeast cytosol to mitigate oxidative stress.

[00230] In another embodiment, the γ-glutamyi cysteine synthase and glutathione synthase genes from S. cerevisiae are expressed in and targeted to the cytosol of yeast to increase the amount of glutathione and increase the activity of DHAD also expressed in and targeted to the yeast cytosol. In another embodiment, S. cerevisiae γ-glutamy! cysteine synthase and glutathione synthase genes Gsh1 and Gsh2 are expressed in and targeted to the cytosol of yeast to increase the amount of glutathione and increase the activity of DHAD also expressed in and targeted to the yeast cytosol. The resulting y-g!utamy! cysteine synthase and glutathione synthase expressing strain has improved isobutanol productivity, titer, and/or yield compared to the parental strain. Homologous genes encoding γ-giutamyl cysteine synthase and glutathione synthase from other organisms, such as other yeast strains, may be identified by sequence homology using tools such as BLAST and pairwise sequence alignments by one skilled in the art.

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364561 vl/CO [00231] Thioredoxins contain two conserved cysteines that exist in either a reduced form as in thioredoxin-{SH) ₂ ) or in an oxidized form as in thioredoxin^) when they form an intramolecular disulfide bridge. Thioredoxins donate electrons from their active center dithiol to protein disulfide bonds (PrQtein-82) that are then reduced to dithiois (Protein-(SH) ₂ ). The resulting oxidized thioredoxin disulfide is reduced directly by thioredoxin reductase with electrons donated by NADPH. Hence the thioredoxin reduction system consists of thioredoxin, thioredoxin reductase, and NADPH. Oxidized glutaredoxins, on the other hand, are reduced by the tripeptide glutathione (gamma-Glu-Cys-GIy, known as GSH) using electrons donated by NADPH. Hence the glutathione/giutaredoxin system consists of glutaredoxin, glutathione, glutathione reductase and NADPH.

[00232] S. cerevisiae contains a cytoplasmic thioredoxin system comprised of the thioredoxins Trxl p and Trx2p and the thioredoxin reductase Trrl p, and a complete mitochondrial thioredoxin system comprised of the thioredoxin Trx3p and the thioredoxin reductase Trr2p. Evidence suggests that the cytoplasmic thioredoxin system may have overlapping function with the glutathione/giutaredoxin system. The mitochondrial thioredoxin system, on the other hand, does not appear to be able to substitute for either the cytoplasmic thioredoxin or glutathione/giutaredoxin systems. Instead, the mitochondrial thioredoxin proteins, thioredoxin (Trx3p) and thioredoxin reductase (Trr2p) have been implicated in the defense against oxidative stress generated during respiratory metabolism.

[00233] Overexpression of the essential cytosoiic functional components of the thioredoxin system is thus predicted to increase the amount of bioavaiiable cytosoiic thioredoxin, resulting in a significant increase in cellular redox buffering potential and concomitant increase in stable, active cytosoiic FeS clusters and DHAD activity. Thus, one or more of the following genes are expressed either singly or in combination, thereby resulting in a functional increase in available thioredoxin: a thioredoxin (encoded in S.cerevisiae by TRX1 and TRX2) and a thioredoxin reductase (encoded in S.cerevisiae by TRR1). Separately, or in combination with the aforementioned genes, the mitochondrial thioredoxin system (encoded by thioredoxin gene TRX3 and thioredoxin reductase gene TRR2) are overexpressed, and, although functional in the mitochondria, provide an added or synergistic effect on FeS cluster assembly or stability, as assayed by increased DHAD activity and/or output of isobutanoi in a fermentation. Overexpression of these genes may be accomplished by methods as described above. In one embodiment, active variants

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364561 yl/CO of any one of the aforementioned thioredoxins or thioredoxin reductases, honiologs thereof, or combination thereof are expressed in the yeast cytosoi to mitigate oxidative stress.

Enhancing Cytosolic DHAD Activity by Mitigating Stress Mediated by Reactive Nitrogen Species (RNS)

[00234] Nitric oxide and reactive nitrogen species are highly reactive, short-lived molecules that can be generated during periods of cellular stress. The exact mechanisms by which these molecules are created, or their downstream targets, is not completely understood and is the subject of intense investigation. However, the functional groups present in many proteins -- for example, FeS clusters are readily attacked and inactivated by NO/RNS. Loss of these labile functional groups usually results in an inactive enzyme.

[00235] Nitric oxide and reactive nitrogen species are highly reactive, short-lived molecules that can be generated during normal cellular function, respiration, and during periods of cellular or redox stress. RNS are produced in eukaryotic cells starting with the reaction of nitric oxide (·ΝΟ) with superoxide (02—) to form peroxynitrite (ONOQ-):

•NO (nitric oxide) + O2— (super oxide)→ ONOO- (peroxynitrite)

[00236] Peroxynitrite itself is a highly reactive species which can directly react with various components of the cell. Alternatively peroxynitrite can react with other molecules to form additional types of RNS including nitrogen dioxide ( ^e NQ ₂ ) and dinitrogen trioxide (N2O3) as well as other types of chemically reactive radicals. Important reactions involving RNS include:

ONOO- + H+→ ONOOH (peroxynitrous acid)→ ^« ΝΟ2 (nitrogen dioxide) + OH

(hydroxy! radical)

ONOO- + C0 ₂ (carbon dioxide)→ ONOOC0 ₂ ^~ (nitrosoperoxycarbonate)

ONOOCO ₂ ^~ → ^s NO ₂ (nitrogen dioxide) + 0=C(O)0- (carbonate radical)

*NO + * 02 is in equilibrium with N2O3 (dinitrogen trioxide)

[00237] NO exhibits other types of interaction that are candidates for mediating aspects of its physiological action. Notably, in a process known as nitrosylation, or

70

364561 vl/CO nitrosation, NO can modify free suifydryi (thiol) groups of cysteines in proteins to produce nitrosothiois, SNOs. Transfer of the NO adduct from one suifydryi to another transnitrosylation) is likely to play a signal transduction role (reviewed in Stamier et a!., 2001 ). Study of this post-translationai modification, which is proposed to be a widespread mediator of signaling, is a relatively new field, and the list of proteins that are modified through nitrosyiation is expanding rapidly. Because NO is highly reactive, transport of an NO signal in tissues can be facilitated through reaction with glutathione and movement of the resulting S-nitrosogiutathione (GSNO), which can subsequently signal by modifying thiol groups on target proteins by transnitrosylation (Lipton et a/,, 2001 ; Foster ef a/., 2003). The discovery of GSNO reductase (GSNOR), which reduces GSNO to restore GSH and to eliminate the NO adduct as NH ⁴⁺ (Jensen ef a/., 1998), revealed the importance of the control of this NO metabolite.

[00238] The exact mechanisms by which the aforementioned molecules are generated, or their downstream targets, are not completely understood and are the subject of intense investigation. However, the functional groups present in many proteins ~ for example, FeS clusters ~ are readily attacked by NO/RNS. The enzyme dihydroxyacid dehydratase (DHAD) contains an iron-sulfur (FeS) cluster cofactor that is sensitive to damage by NO or RNS. As an example of the biological sensitivity of this class of enzyme to attack by NO/RNS, inactivation of the E.coli DHAD (encoded by HvD) and subsequent bacterial cell death resulting from macrophage-generated NO is a major component of the mammalian humoral immune response.

[00239] The present invention provides methods of mitigating the potentially harmful effects of oxidative and nitrosative stress (e.g., NO and/or or RNS) on enzymes involved in the production of isobutanol in the yeast cytosol. Specifically, the enzyme dihydroxyacid dehydratase (DHAD) contains an iron-sulfur (Fe-S) cluster that is sensitive to damage by NO and/or RNS, leading to inactive enzyme. Strategies of mitigating such harmful effects include, but are not limited to, increasing repair of iron-sulfur clusters damaged by oxidative and nitrosative stress conditions; reducing nitric oxide levels by introduction of a nitric oxide reductase (NOR) activity in the cell; reducing the levels of SNO's by overexpression of a GSNO-reductase; or combination thereof.

[00240] Strategies disclosed herein are intended to protect or repair DHAD from NO/RNS damage. Accordingly, in one embodiment, the present invention provides

71

364561 yl/CO recombinant microorganisms that have been engineered to express one or more enzymes in the cytosoi that reduce the concentration of reactive nitrogen species (RNS) and/or nitric oxide in said cytosoi.

[00241] In one embodiment, the present invention provides recombinant microorganisms that have been engineered to express a nitric oxide reductase that reduce the concentration of reactive nitrogen species (RNS) and/or nitric oxide in said cytosoi. To reduce nitric oxide levels in the yeast cytosoi, one or more nitric oxide reductases (NORs) or active variants thereof can be introduced into the ceil by overexpression. Genes present in several microbial species have been shown to encode a nitric oxide reductase activity. For example, in E.coli the gene for a flavorubredoxin, norV, encodes a flavo-diiron NO reductase that is one of the most highly induced genes when E.coli cells are exposed to NO or GSNO. Previous work has identified a gene present in the microbe Fusahum oxysporum as encoding a cytochrome P-450 55A1 (P~450dN!R) that encodes a nitric oxide reductase (Nakahara et al., 1993, J. Biol. Chem. 268:8350-8355). When expressed in a eukaryotic cell, this gene product appears to be cytosolicaiiy localized and exhibits effects consistent with its reducing intracellular NO levels (Dijkers et al, 2009, Molecular Biology of the Cell, 20: 4083-4090). Thus, in one embodiment, homologs of any above-described nitric oxide reductases, active variants thereof, or combinations thereof are expressed in the yeast cytosoi to mitigate nitric oxide.

[00242] In contrast to £, coli and F. oxysporum, S. cerevisiae lacks an endogenous NOR activity (and no homologs of either NOR protein is found in the S. cerevisiae genome). Thus, to provide such an activity, the F. oxysporum NOR gene is synthesized or amplified from genomic DNA, or the E. coli norV gene is amplified from genomic DNA, and either (or both) cloned into a suitable yeast expression vector. Such a vector could either be high copy (e.g., 2micron origin) or low copy (CEN/ARSH), or a single or multiple copies of the gene could be stably integrated into the genome of a host organism, specifically a yeast containing a cytosolic isobutanoi pathway. In each case, methods to clone a gene into a plasmid so that it is expressed at a desired level under the control of a known yeast promoter (including those steps required to transform a host yeast cell) are well known to those skilled in the art. In those cases where the NOR gene is expressed from an episomai plasmid, it can be advantageous to simultaneous overexpress a desired DHAD gene, either from the same or from another plasmid, thereby allowing one to assay the resulting output in DHAD activity. Similar approaches are undertaken to

72

364561 yl/CO express the NOR gene in the presence of a plasmid(s) encoding an isobutanoi production pathway, where the results of NOR expression are manifested in changes in isobutanoi productivity, titer, or yield. It is understood by one skilled in the art that expression of all genes, both NOR and genes encoding the isobutanoi pathway may be integrated into the genome of a host organism in a single or multiple copies of the gene(s), specifically a yeast containing a cytosolic isobutanoi pathway.

[00243] In another embodiment, the present invention provides recombinant microorganisms that have been engineered to express a g!utathione-S-nitrosothiol reductase (GSNO-reductase) that reduces the concentration of reactive nitrogen species (RNS) and/or nitric oxide in said cytosol. To reduce the levels of SNO's, one or more GSNO-reductases or active variants thereof can be introduced into the ceil by overexpression. In S. cerevisiae, the gene SFA1 has been shown to encode a formaldehyde dehydrogenase that possesses GSNO reductase activity (Liu et a/., 2001 , Nature 410:490-494). Sfai p is a member of the class HI alcohol dehydrogenases (EC:1 .1 .1 .284), which are bifunctionai enzymes containing both alcohol dehydrogenase and glutathione-dependent formaldehyde dehydrogenase activities. The glutathione-dependent formaldehyde dehydrogenase activity of Sfa1 p is required for the detoxification of formaldehyde, and the alcohol dehydrogenase activity of Sfai p can catalyze the final reactions in phenylalanine and tryptophan degradation. Sfai p is also able to act as a hydroxymethylfurfurai (HMF) reductase and catabolize HMF, a compound formed in the production of certain biofuels. Sfai p has been localized to the cytoplasm and the mitochondria, and can act on a variety of substrates, including S-hydroxymethylg!utathione, phenyiacetaldehyde, indole acetaidehyde, octanoi, 10-hydroxydecanoic acid, 12-hydroxydodecanoic acid, and S- nitrosogiutathione.

[00244] Sfa1 protein levels are reported as being low-to-moderate from proteome- wide analyses (Ghaemmaghami ef a/., 2003, Nature 425(6959):737-41 ). Thus, in an analogous fashion to the approach described for overexpression of NOR, the gene SFA 1 is overexpressed, thereby decoupling it from its normal regulatory control and permitting significant increase in Sfa1 activity in the cell, which results in measureable increases in DHAD activity and/or fermentation output, as described above. Overexpression of these genes may be accomplished by methods as described above. In one embodiment, homoiogs of SFA1, active variants thereof, or combinations thereof are expressed in the yeast cytosol to mitigate stresses brought on by reactive nitrogen species.

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364561 yl/CO [00245] In additional embodiments, alternative enzymes may be expressed and targeted to the yeast cytosol containing the isobutanoi pathway to mitigate the effects of reactive nitrogen species. Specifically, the enzyme YtfE encoded by E.coli ytfE, homologs thereof, active variants thereof, may be expressed, such that they lead to reduction in NO/RNS in the yeast cytosol and/or a concomitant increase in DHAD function. Such an increase is detected by in vitro assay of DHAD activity, and/or by an increase in productivity, titer, or yield of isobutanoi produced by isobutanoi pathway-containing cells.

[00246] To increase repairment of iron-sulfur clusters, in one embodiment, the gene ytfE from E.coli is expressed in the yeast cytosol which contains a functional isobutanoi pathway and DHAD such that DHAD activity and/or isobutanoi productivity, titer, or yield are increased from the yeast ceils. In £. co!i, the gene ytfE has been shown to play an important role in maintaining active Fe-S clusters. A recent report (Justino et al., (2009). Escherichia coli Di-iron YtfE protein is necessary for the repair of stress-damaged Iron-Sulfur Clusters. JBC 282(14): 10352-10359) showed that AytfE strains have several phenotypes, including enhanced susceptibility to nitrosative stress and are defective in the activity of several iron-sulfur-containing proteins. For example, the damage of the [4Fe-4S] ^2'*' clusters of aconitase B and fumarase A caused by exposure to hydrogen peroxide and nitric oxide stress occurs at higher rates in the absence of ytfE. The ytfE null mutation also abolished the recovery of aconitase and fumarase activities, which is observed in wild-type £. coti once the stress is scavenged. Notably, upon the addition of purified holo-YtfE protein to mutant cell extracts, the enzymatic activities of fumarase and aconitase were fully recovered, and at rates similar to the wild-type strain. Thus, YtfE is critical for the repair of iron-sulfur clusters damaged by oxidative and nitrosative stress conditions, and presents an attractive candidate for overexpression in a host cell that normally lacks this activity, such as S. cerevisiae, where Fe-S cluster proteins are also being overexpressed as part of the isobutanoi pathway.

[00247] To provide such an activity, the E.coli ytfE gene can be amplified from genomic DNA by PGR with appropriate primers, and cloned into a suitable yeast expression vector. Such a vector could either be high copy (e.g., 2micron origin) or low copy (CEN/ARS), or a single or multiple copies of the gene could be stably integrated into the genome of a host organism. In each case, methods to clone a gene into a plasmid so that it is expressed at a desired level under the control of a

74

364561 yl/CO known yeast promoter (including those steps required to transform a host yeast celi) are well known to those skilled in the art. In those cases where the ytfE gene is expressed from an episomal plasmid, it can be advantageous to simultaneous overexpress a desired DHAD gene, either from the same or from another plasmid, thereby allowing one to assay the resulting output in DHAD activity. Similar approaches are undertaken to express the ytfE gene in the presence of a piasmid(s) encoding an isobutanoi production pathway, where the results oft ytfE expression are manifested in changes in isobutanoi productivity, titer, or yield. More specifically, ytfE is expressed in the yeast cytosoi which contains a functional isobutanoi pathway and DHAD such that DHAD activity and/or isobutanoi productivity, titer, or yield are increased from the yeast cells.

[00248] In addition, functional homologs of E.coti ytfE have been identified and characterized. For example, genes from two pathogenic prokaryotes— scdA from Staphylococcus aureus, and dnrN from Neisseria gonorrhoeae, have been shown to have properties similar to that of ytfE (Overton, T.W., et ai (2008). Widespread distribution in pathogenic bacteria of di-iron proteins that repair oxidative and nitrosative damage to iron-sulfur centers. J. Bacteriology 190(6): 2004-2013). Thus, similar approaches to overexpress either of these genes are employed, as described for E.coli ytfE, above. Overexpression of these genes may be accomplished by methods as described above.

The Microorganism in General

[00249] The recombinant microorganisms provided herein can express a plurality of heterologous and/or native target enzymes involved in pathways for the production of beneficial metabolites such as isobutanoi, 3-methyi-1 -butanoi, 2~ methyl-1 -butanoi, valine, isoleucine, leucine, and pantothenic acid from a suitable carbon source.

[00250] Accordingly, "engineered" or "modified" microorganisms are produced via the introduction of genetic material into a host or parental microorganism of choice and/or by modification of the expression of native genes, thereby modifying or altering the cellular physiology and biochemistry of the microorganism. Through the introduction of genetic material and/or the modification of the expression of native genes the parental microorganism acquires new properties, e.g. the ability to produce a new, or greater quantities of, an intracellular metabolite. As described herein, the introduction of genetic material into and/or the modification of the

75

364561 vl/CO expression of native genes in a parental microorganism results in a new or modified ability to produce beneficial metabolites such as isobutanoi, 3-methyl-l -butanoi, 2- methyl-1 -butano!, valine, isoleucine, leucine, and pantothenic acid from a suitable carbon source. The genetic material introduced into and/or the genes modified for expression in 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 one or more metabolites selected from isobutanoi, 3-methy!-1 -butanol, 2~methyl-1 ~ butanoi, valine, isoleucine, leucine, and pantothenic acid and may also include additional elements for the expression and/or regulation of expression of these genes, e.g. promoter sequences.

[00251] In addition to the introduction of a genetic material into a host or parental microorganism, an engineered or modified microorganism can also include alteration, disruption, deletion or knocking-out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism. Through the alteration, disruption, deletion or knocking-out of a gene or polynucleotide the microorganism acquires new or improved properties (e.g., the ability to produce a new metabolite or greater quantities of an intracellular metabolite, improve the flux of a metabolite down a desired pathway, and/or reduce the production of byproducts).

[00252] Recombinant microorganisms provided herein may also 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-ketoisova!erate), or an end product (e.g., isobutanoi) 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.

[00253] 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 mutations and silent mutations. Such modified or mutated

76

364561 vl/CO polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.

[00254] Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to clone and express the polynucleotides encoding such enzymes,

[00255] 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."

[00256] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et a/., 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 £. coli commonly use UAA as the stop codon (Daiphin et a/., 1996, Nucl Acids Res. 24: 218-8). 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.

[00257] 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

77

364561 yl/CO 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.

[00258] In addition, homo!ogs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.

[00259] 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 nonhomologous 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 typicaily 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.

[00260] 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

78

364561 yl/CO of the substitution. Means for making this adjustment are well known to those of skill in the art (See, e.g., Pearson W.R., 1994 _. , Methods in Mol Biol 25: 365-89.

[00261] 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), Giutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[00262] Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See commonly owned and co-pending application US 2009/0226991 . 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. 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 described in commonly owned and co-pending application US 2009/0226991 .

[00263] It is understood that a range of microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of beneficial metabolites from DHAD-requiring biosynthetic pathways. In various embodiments, microorganisms may be selected from yeast microorganisms. Yeast microorganisms for the production of a metabolite such as isobutanol, 3-methyl-1 -butanol, 2-methyl- 1 -butanoi, valine, isoleucine, leucine, and pantothenic acid may be selected based on certain characteristics:

[00264] One characteristic may include the property that the microorganism is selected to convert various carbon sources into beneficial metabolites such as isobutanol, 3-methyi-1 -butanoi, 2-methy!-1 -butanol, valine, isoleucine, leucine, and pantothenic acid. The term "carbon source" generally refers to a substance suitable to be used as a source of carbon for prokaryotic or eukaryotic cell growth. Examples of suitable carbon sources are described in commonly owned and co-pending application US 2009/0228991 . Accordingly, in one embodiment, the recombinant microorganism herein disclosed can convert a variety of carbon sources to products, including but not limited to glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof.

[00265] The recombinant microorganism may thus further include a pathway for the production of isobutanol, 3-methyi-l -butanol, 2-methyi-1 -butanoi, valine,

79

364561 yl/CO isoleucine, leucine, and/or pantothenic acid from five-carbon (pentose) sugars including xylose. Most yeast species metabolize xylose via a complex route, in which xylose is first reduced to xylitol via a xylose reductase (XR) enzyme. The xylitol is then oxidized to xyiuiose via a xyiitoi dehydrogenase (XDH) enzyme. The xylulose is then phosphorylated via a xyiulokinase (XK) enzyme. This pathway operates inefficiently in yeast species because it introduces a redox imbalance in the ceil. The xylose-to-xylitol step uses primarily NADPH as a cofactor (generating NADP+), whereas the xyiitoi-to-xyiulose step uses NAD+ as a cofactor (generating NADH). Other processes must operate to restore the redox imbalance within the cell. This often means that the organism cannot grow anaerobically on xylose or other pentose sugars. Accordingly, a yeast species that can efficiently ferment xylose and other pentose sugars into a desired fermentation product is therefore very desirable.

[00266] Thus, in one aspect, the recombinant microorganism is engineered to express a functional exogenous xylose isomerase. Exogenous xylose isomerases (XI) functional in yeast are known in the art. See, e.g., Rajgarhia et al., US2006/0234364, which is herein incorporated by reference in its entirety. In an embodiment according to this aspect, the exogenous XI gene is operatively linked to promoter and terminator sequences that are functional in the yeast cell. In a preferred embodiment, the recombinant microorganism further has a deletion or disruption of a native gene that encodes for an enzyme (e.g., XR and/or XDH) that catalyzes the conversion of xylose to xylitol. In a further preferred embodiment, the recombinant microorganism also contains a functional, exogenous xyiulokinase (XK) gene operatively linked to promoter and terminator sequences that are functional in the yeast ceil. In one embodiment, the xyiulokinase (XK) gene is overexpressed.

[00267] In one embodiment, the microorganism has reduced or no pyruvate decarboxylase (PDC) activity. PDC catalyzes the decarboxylation of pyruvate to acetaidehyde, which is then reduced to ethanol by ADH via an oxidation of NADH to NADH+. Ethanol production is the main pathway to oxidize the NADH from glycolysis. Deletion of this pathway increases the pyruvate and the reducing equivalents (NADH) available for the DHAD-requiring biosynthetic pathway. Accordingly, deletion of PDC genes can further increase the yield of desired metabolites.

[00268] In another embodiment, the microorganism has reduced or no giycerol-3- phosphate dehydrogenase (GPD) activity. GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glyceroi-3-phosphate (G3P) via the

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364561 yl/CO oxidation of NADH to NAD+. Glycerol is then produced from G3P by G!ycerol-3- phosphatase (GPP). Glycerol production is a secondary pathway to oxidize excess NADH from glycolysis. Reduction or elimination of this pathway would increase the pyruvate and reducing equivalents (NADH) available for the DHAD-requiring biosynthetic pathway. Thus, deletion of GPD genes can further increase the yield of desired metabolites.

[00269] In yet another embodiment, the microorganism has reduced or no PDC activity and reduced or no GPD activity. PDOminus, GPD-minus yeast production strains are described in commonly owned and co-pending publications, US 2009/0228991 and US 201 1/0020889, both of which are herein incorporated by reference in their entireties for all purposes.

[00270] In yet another embodiment, the microorganism has reduced or no 3-keto acid reductase (3-KAR) activity. 3-keto acid reductase catalyzes the conversion of 3-keto acids (e.g., acetolactate) to 3-hydroxyacids (e.g., DH2MB). 3-KAR-minus yeast production strains are described in commonly owned and co-pending U.S. Application Serial No. 201 1/0201090, which is herein incorporated by reference in its entirety for ail purposes.

[00271] In yet another embodiment, the microorganism has reduced or no aldehyde dehydrogenase (ALDH) activity. Aldehyde dehydrogenases catalyze the conversion of aldehydes (e.g., isobutyraldehyde) to acid by-products (e.g., isobutyrate). ALDH-minus yeast production strains are described in commonly owned and co-pending U.S. Application Serial No. 201 1/0201090, which is herein incorporated by reference in its entirety for all purposes.

[00272] In one embodiment, the yeast microorganisms may be selected from the "Saccharomyces Yeast Ciade", as described in commonly owned and co-pending application US 2009/0226991 .

[00273] The term "Saccharomyces sensu stricto" taxonomy group is a cluster of yeast species that are highly related to S. cerevisiae (Rainier! et al., 2003, J. Biosci Bioengin 96: 1 -9). Saccharomyces sensu stricto yeast species include but are not limited to S. cerevisiae, S. cerevisiae, S. kudriavzevii, S, mikatae, S. bayanus, S, uvarum, S. carocanis and hybrids derived from these species (Masneuf et a/., 1998, Yeast 7: 61 -72).

[00274] An ancient whole genome duplication (WGD) event occurred during the evolution of the hemiascomycete yeast and was discovered using comparative genomic tools (Kellis et al., 2004, Nature 428: 617-24; Dujon et a/., 2004, Nature

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364561 yl/CO 430:35-44; Langkjaer et a/., 2003, Nature 428: 848-52; Wolfe et al., 1997, Nature 387: 708-13). Using this major evolutionary event, yeasi can be divided into species that diverged from a common ancestor following the WGD event (termed "post-WGD yeast" herein) and species that diverged from the yeast lineage prior to the WGD event (termed "pre-WGD yeast" herein).

[00275] Accordingly, in one embodiment, the yeast microorganism may be selected from a post-WGD yeast genus, including but not limited to Saccharomyces and Candida. The favored post-WGD yeast species include: S. cerevisiae, S, uvarum, S. bayanus, S. paradoxus, S, castelli, and C. glabrata.

[00276] In another embodiment, the yeast microorganism may be selected from a pre-whole genome duplication (pre-WGD) yeast genus including but not limited to Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Yarrowia and, Schizosaccharomyces. Representative pre-WGD yeast species include: S. kluyveri, K. thermotolerans, K. marxianus, K, waltii, K, lactis, C. tropicalis, P. pastoris, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I. scutulata, D. hansenii, H, anomala, Y, lipolytica, and S. pombe,

[00277] A yeast microorganism may be either Crabtree-negative or Crabtree- positive as described in described in commonly owned and co-pending application US 2009/0226991 . In one embodiment the yeast microorganism may be selected from yeast with a Crabtree-negative phenotype including but not limited to the following genera: Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida. Crabtree-negative species include but are not limited to: K. lactis, K. marxianus, P. anomala, P, stipitis, I, orientalis, I. occidentalis, I. scutulata, H. anomala, and C. utilis. In another embodiment, the yeast microorganism may be selected from a yeast with a Crabtree-positive phenotype, including but not limited to Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia and Schizosaccharomyces. Crabtree-positive yeast species include but are not limited to: S. cerevisiae, S, uvarum, S. bayanus, S. paradoxus, S. castelli, S, kluyveri, K. thermotolerans, C. glabrata, Z. baiiii, Z. rouxii, D. hansenii, P. pasiorius, and S. pombe,

[00278] Another characteristic may include the property that the microorganism is that it is non-fermenting. In other words, it cannot metabolize a carbon source anaerobicaily while the yeast is able to metabolize a carbon source in the presence of oxygen. Nonfermenting yeast refers to both naturally occurring yeasts as well as genetically modified yeast. During anaerobic fermentation with fermentative yeast,

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364561 yl/CO the main pathway to oxidize the NADH from glycolysis is through the production of ethanoL Eihanol is produced by alcohol dehydrogenase (ADH) via the reduction of acetaidehyde, which is generated from pyruvate by pyruvate decarboxylase (PDC). In one embodiment, a fermentative yeast can be engineered to be non-fermentative by the reduction or elimination of the native PDC activity. Thus, most of the pyruvate produced by glycolysis is not consumed by PDC and is available for the isobutanoi pathway. Deletion of this pathway increases the pyruvate and the reducing equivalents available for the biosynthetic pathway. Fermentative pathways contribute to low yield and low productivity of desired metabolites such as isobutanoi. Accordingly, deletion of PDC genes may increase yield and productivity of desired metabolites such as isobutanoi, 3-methyI-1 -butanoi, 2-meihyi-1 -butanoi, valine, isoleucine, leucine, and pantothenic acid.

[00279] In some embodiments, the recombinant microorganisms may be microorganisms that are non-fermenting yeast microorganisms, including, but not limited to those, classified into a genera selected from the group consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida, In a specific embodiment, the non-fermenting yeast is C, xestobii.

Isobutanoi-Producing Yeast Microorganisms

[00280] Yeast cells convert sugars to produce pyruvate, which is then utilized in a number of pathways of cellular metabolism. In recent years, yeast cells have been engineered to produce a number of desirable products via pyruvate-driven biosynthetic pathways, including isobutanoi, an important commodity chemical and biofuel candidate (See, e.g., commonly owned and co-pending patent publications, US 2009/0226991 , US 2010/0143997, US 201 1/0020889, US 201 1/0076733, and WO 2010/075504),

[00281] As described herein, the present application relates to recombinant microorganisms for producing isobutanoi, wherein said recombinant microorganisms comprise an isobutanoi producing metabolic pathway. In one embodiment, the isobutanoi producing metabolic pathway to convert pyruvate to isobutanoi can be comprised of the following reactions:

2 pyruvate→ acetolactate + CO2

acetolactate + NAD(P)H→ 2,3-dihydroxyisovaierate + NAD(P) ⁺ 2,3-dihydroxyisovaierate→ alpha-ketoisova!erate

aipha-ketoisovalerate→ isobutyraldehyde + CO2

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364561 vl/CO isobutyra!dehyde +NAD(P)H→ isobutanol + NADP,

[00282] In one embodiment, these reactions are carried out by the enzymes 1 ) Acetolactate synthase (ALS), 2) Ketol-acid reductoisomerase (KARI), 3) Dihydroxy- acid dehydratase (DHAD), 4) 2-keto-acid decarboxylase, e.g., Keto-isovalerate decarboxylase (KIVD), and 5) an Alcohol dehydrogenase (ADH) (Figure 1 ). in some embodiments, the recombinant microorganism may be engineered to overexpress one or more of these enzymes. In an exemplary embodiment, the recombinant microorganism is engineered to overexpress ail of these enzymes.

[00283] Alternative pathways for the production of isobutanoi in yeast have been described in WO/2007/050871 and in Dickinson et a/,, 1998, J Biol Chem 273:25751 -6. These and other isobutanol producing metabolic pathways are within the scope of the present application. In one embodiment, the isobutanol producing metabolic pathway comprises five substrate to product reactions. In another embodiment, the isobutanoi producing metabolic pathway comprises six substrate to product reactions. In yet another embodiment, the isobutanol producing metabolic pathway comprises seven substrate to product reactions.

[00284] In various embodiments described herein, the recombinant microorganism comprises an isobutanol producing metabolic pathway. In one embodiment, the isobutanol producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanoi. In another embodiment, the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanoi producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, all of the isobutanoi producing metabolic pathway steps in the conversion of pyruvate to isobutanoi are converted by exogenously encoded enzymes.

[00285] In one embodiment, one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol. In one embodiment, the

84

364561 yl/CO recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least one isobutanoi pathway enzyme localized in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least two isobutanoi pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least three isobutanoi pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least four isobutanoi pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with five isobutanoi pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with all isobutanoi pathway enzymes localized in the cytosol. Isobutanoi producing metabolic pathways in which one or more genes are localized to the cytosol are described in commonly owned and copending publication, US 201 1/0078733, which is herein incorporated by reference in its entirety for ail purposes.

[00286] As is understood in the art, a variety of organisms can serve as sources for the isobutanoi pathway enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. iactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including V. spp. stipitis, Toruiaspora pretonensis, Issatchenkia orientalis, Schizosaccharomyces spp., including S. pombe, Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustiiago spp. Sources of genes from anaerobic fungi include, but not limited to, Piromyces spp., Orpinomyces spp., or Neocaliimastix spp. Sources of prokaryotic enzymes that are useful include, but not limited to, Escherichia spp., Zymomonas spp., Staphylococcus spp., Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Siackia spp., Lactococcus spp., Enterobacter spp., Streptococcus spp., Salmonella spp., Bacteroides spp., Methanococcus spp., Eryihrobacter spp., Sphingomonas spp., Sphingobium spp., and Novosphingobium spp.

[00287] In some embodiments, one or more of these enzymes can be encoded by native genes. Alternatively, one or more of these enzymes can be encoded by heterologous genes. For example, ALS can be encoded by the alsS gene of B,

85

364561 yl/CO subti!is, aisS of L. lactis, or the HvK gene of K. pneumonia. For example, KIVD can be encoded by the ksvD or kdcA gene of L. lactis. For example, ADH can be encoded by ADH2, ADH6, or ADH7 of S. cerevisiae or the adhA gene of L. lactis.

[00288] In an exemplary embodiment, pathway steps 2 and 5 of the isobutanoi pathway may be carried out by KARI and ADH enzymes that utilize NADH (rather than NADPH) as a cofactor. The present inventors have found that utilization of NADH-dependent KARI (NKR) and ADH enzymes to catalyze pathway steps 2 and 5, respectively, surprisingly enables production of isobutanoi at theoretical yield and/or under anaerobic conditions. An example of an NADH-dependent isobutanoi pathway is illustrated in Figure 2. Thus, in one embodiment, the recombinant microorganisms of the present application may use an NKR to catalyze the conversion of acetoiactate to produce 2,3-dihydroxyisovalerate. In another embodiment, the recombinant microorganisms of the present application may use an NADH-dependent ADH to catalyze the conversion of isobutyraidehyde to produce isobutanoi. In yet another embodiment, the recombinant microorganisms of the present application may use both an NKR to catalyze the conversion of acetoiactate to produce 2,3-dihydroxyisovalerate, and an NADH-dependent ADH to catalyze the conversion of isobutyraidehyde to produce isobutanoi.

[00289] In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutanoi. In one embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutyraidehyde. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to keto-isovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to acetoiactate.

[00290] Furthermore, any of the genes encoding the foregoing enzymes (or any others mentioned herein (or any of the regulatory elements that control or modulate expression thereof)) may be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast and other microorganisms, e.g., bacterial microorganisms.

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364561 vl/CO Methods in General!

Identification of an Nfs1 or Isd1 1 Protein in a Microorganism

[00291] Any method can be used to identify genes that encode for proteins with Nfs1 and/or Isd1 1 activity. Generally, genes that are homologous or similar to a known NFS1 or ISD11 gene, e.g., S. cerevisiae NFS1 (encoding for Nfs1 ) or S. cerevisiae ISD11 (encoding for Isd1 1 ) can be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar NFS1 genes and/or homologous or similar Nfs1 proteins will have functional, structural, or genetic similarities. Likewise, homologous or similar ISD11 genes and/or homologous or similar isd1 1 proteins will have functional, structural, or genetic similarities. A representative listing of Nfs1 proteins may be found in SEQ ID NOs: 227, 229, 231 , 233, 235, 237, 239, 241 , 243, 245, 247, and 249, encoded by SEQ ID NOs: 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, and 248, respectively. Likewise, a representative listing of Isd1 1 proteins may found in SEQ ID NOs: 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271 , and 273, encoded by SEQ ID NOs: 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272, respectively.

[00292] Techniques known to those skilled in the art may be suitable to identify homologous genes and homologous enzymes. Generally, analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes. For example, to identify homologous or analogous genes, or proteins, techniques may include, but are not limited to, cloning a NFS1 and/or ISD11 gene by PGR using primers based on a published sequence of a gene/protein or by degenerate PGR using degenerate primers designed to amplify a conserved region among NFS1 and/or ISD11 genes. Further, one skilled in the art can use techniques to identify homologous or analogous genes, or proteins, with functional homology or similarity. For instance, the computer program BLAST may be used for such a purpose. To identify homologous or similar genes and/or homologous or similar proteins, analogous genes and/or analogous proteins, techniques also include comparison of data concerning a candidate gene or protein with databases such as BRENDA, KEGG, the Saccharomyces Genome Database, or M eta CYC. The candidate gene or protein may be identified within the above mentioned databases in accordance with the teachings herein.

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364561 vl/CO Identification of an Aft Protein in a Microorganism

[00293] Any method can be used to identify genes that encode for proteins with Aft activity, Aft1 and Aft2 enhance cellular iron availability. Generally, genes that are homologous or similar to a known AFT gene, e.g. S. cerevisiae AFT1 (encoding for SEQ ID NO: 2) or S. cerevisiae AFT2 (encoding for SEQ ID NO: 4) can be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar AFT genes and/or homologous or similar Aft proteins will have functional, structural, or genetic similarities. Techniques known to those skilled in the art may be suitable to identify homologous genes and homologous enzymes. Generally, analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes. For example, to identify homologous or analogous genes, proteins, or enzymes, techniques may include, but not limited to, cloning a AFT gene by PGR using primers based on a published sequence of a gene/enzyme or by degenerate PGR using degenerate primers designed to amplify a conserved region among AFT genes. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity. For instance, the computer program BLAST may be used for such a purpose. To identify homologous or similar genes and/or homologous or similar proteins, analogous genes and/or analogous proteins, techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC. The candidate gene or enzyme may be identified within the above mentioned databases in accordance with the teachings herein.

Genetic Insertions and Deletions

[00294] Any method can be used to introduce a nucleic acid molecule into yeast and many such methods are well known. For example, transformation and electroporation are common methods for introducing nucleic acid into yeast cells. See, e.g., Gietz et a/., 1992, Nuc Acids Res. 27: 69-74; Ito et a/., 1983, J. Bacterioi. 153: 183-8; and Becker et ai., 1991 , Methods in Enzymo!ogy 194: 182-7.

[00295] In an embodiment, the integration of a gene of interest into a DNA fragment or target gene of a yeast microorganism occurs according to the principle of homologous recombination. According to this embodiment, an integration cassette containing a module comprising at least one yeast marker gene and/or the

88

364561 vl/CO gene to be integrated (interna! module) is flanked on either side by DNA fragments homologous to those of the ends of the targeted integration site (recombinogenic sequences). After transforming the yeast with the cassette by appropriate methods, a homologous recombination between the recombinogenic sequences may result in the internal module replacing the chromosomal region in between the two sites of the genome corresponding to the recombinogenic sequences of the integration cassette. (Orr-Weaver et ai, 1981 , PNAS USA 78: 6354-58).

[00296] In an embodiment, the integration cassette for integration of a gene of interest into a yeast microorganism includes the heterologous gene under the control of an appropriate promoter and terminator together with the selectable marker flanked by recombinogenic sequences for integration of a heterologous gene into the yeast chromosome. In an embodiment, the heterologous gene includes an appropriate native gene desired to increase the copy number of a native gene(s). The selectable marker gene can be any marker gene used in yeast, including but not limited to, HIS3, TRP1, LEU2, URA3, bar, ble, hph, and kan. The recombinogenic sequences can be chosen at will, depending on the desired integration site suitable for the desired application.

[00297] In another embodiment, integration of a gene into the chromosome of the yeast microorganism may occur via random integration (Kooistra et a/., 2004, Yeast 21 : 781 -792).

[00298] Additionally, in an embodiment, certain introduced marker genes are removed from the genome using techniques well known to those skilled in the art. For example, URA3 marker loss can be obtained by plating URA3 containing cells in FOA (5-fluo!O-Q! tic acid) containing medium and selecting for FOA resistant colonies (Boeke et a!., 1984, Moi. Gen. Genet 197: 345-47).

[00299] The exogenous nucleic acid molecule contained within a yeast ceil of the disclosure can be maintained within that ceil in any form. For example, exogenous nucleic acid molecules can be integrated info the genome of the ceil or maintained in an episomai state that can stably be passed on ("inherited") to daughter ceils. Such extra-chromosomal genetic elements (such as plasmids, mitochondria! genome, etc) can additionally contain selection markers that ensure the presence of such genetic elements in daughter cells. Moreover, the yeast cells can be stab!y or transiently transformed. In addition, the yeast cells described herein can contain a single copy, or multiple copies of a particular exogenous nucleic acid molecule as described above.

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364561 yl/CO Reduction of Enzymatic Activity

[00300] Yeast microorganisms within the scope of the invention may have reduced enzymatic activity such as reduced 3-KAR, ALDH, PDC, or GPD activity. The term "reduced" as used herein with respect to a particular enzymatic activity refers to a lower ievei of enzymatic activity than that measured in a comparabie yeast ceil of the same species. The term reduced also refers to the elimination of enzymatic activity as compared to a comparabie yeast cell of the same species. Thus, yeast cells lacking 3-KAR, ALDH, PDC, or GPD activity are considered to have reduced 3-KAR, ALDH, PDC, or GPD activity since most, if not all, comparable yeast strains have at least some 3-KAR, ALDH, PDC, or GPD activity. Such reduced enzymatic activities can be the result of lower enzyme concentration, lower specific activity of an enzyme, or a combination thereof. Many different methods can be used to make yeast having reduced enzymatic activity. For example, a yeast ceil can be engineered to have a disrupted enzyme-encoding locus using common mutagenesis or knock-out technology. See, e.g., Methods in Yeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Stems, Cold Spring Harbor Press (1998). in addition, certain point-mutation(s) can be introduced which results in an enzyme with reduced activity. Also included within the scope of this invention are yeast strains which when found in nature, are substantially free of one or more activities selected from 3-KAR, ALDH, PDC, or GPD activity.

[00301] Alternatively, antisense technology can be used to reduce enzymatic activity. For example, yeast can be engineered to contain a cDNA that encodes an antisense molecule that prevents an enzyme from being made. The term "antisense molecule" as used herein encompasses any nucleic acid molecule that contains sequences that correspond to the coding strand of an endogenous polypeptide. An antisense molecule also can have flanking sequences (e.g., regulatory sequences). Thus antisense molecules can be ribozymes or antisense oligonucleotides. A ribozyme can have any general structure including, without limitation, hairpin, hammerhead, or axhead structures, provided the molecule cleaves RNA.

[00302] Yeasts having a reduced enzymatic activity can be identified using many methods. For example, yeasts having reduced 3-KAR, ALDH, PDC, or GPD activity can be easily identified using common methods, which may include, for example, measuring for the formation of the by-products produced by such enzymes via liquid chromatography.

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364561 vl/CO Qverexpress ion of Heterologous Genes

[00303] Methods for overexpressing a polypeptide from a native or heterologous nucleic acid molecule are well known. Such methods include, without limitation, constructing a nucleic acid sequence such that a regulatory element promotes the expression of a nucleic acid sequence that encodes the desired polypeptide. Typically, regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription. Thus, regulatory elements include, without limitation, promoters, enhancers, and the like. For example, the exogenous genes can be under the control of an inducible promoter or a constitutive promoter. Moreover, methods for expressing a polypeptide from an exogenous nucleic acid molecule in yeast are well known. For example, nucleic acid constructs that are used for the expression of exogenous polypeptides within Kluyveromyces and Saccharomyces are well known (see, e.g., U.S. Pat. Nos. 4,859,596 and 4,943,529, for Kluyveromyces and, e.g., Gellissen et ai, Gene 190(1 ):87-97 (1997) for Saccharomyces). Yeast piasmids have a selectable marker and an origin of replication. In addition certain piasmids may also contain a centromeric sequence. These centromeric piasmids are generally a single or low copy plasmid. Piasmids without a centromeric sequence and utilizing either a 2 micron (S. cerevisiae) or 1 .6 micron (K. lactis) replication origin are high copy piasmids. The selectable marker can be either prototrophic, such as HIS3, TRP1, LEU2, URA3 or ADE2, or antibiotic resistance, such as, bar, ble, hph, or kan.

[00304] In another embodiment, heterologous control elements can be used to activate or repress expression of endogenous genes. Additionally, when expression is to be repressed or eliminated, the gene for the relevant enzyme, protein or RNA can be eliminated by known deletion techniques.

[00305] As described herein, any yeast within the scope of the disclosure can be identified by selection techniques specific to the particular enzyme being expressed, over-expressed or repressed. Methods of identifying the strains with the desired phenotype are well known to those skilled in the art. Such methods include, without limitation, PGR, RT-PCR, and nucleic acid hybridization techniques such as Northern and Southern analysis, altered growth capabilities on a particular substrate or in the presence of a particular substrate, a chemical compound, a selection agent and the like. In some cases, immunohistochemistry and biochemical techniques can be used to determine if a cell contains a particular nucleic acid by detecting the

91

364561 yl/CO expression of the encoded polypeptide. For example, an antibody having specificity for an encoded enzyme can be used to determine whether or not a particular yeast cell contains that encoded enzyme. Further, biochemical techniques can be used to determine if a ceil contains a particular nucleic acid molecule encoding an enzymatic polypeptide by detecting a product produced as a result of the expression of the enzymatic polypeptide. For example, transforming a cell with a vector encoding acetoiactate synthase and detecting increased acetolactate concentrations compared to a cell without the vector indicates that the vector is both present and that the gene product is active. Methods for detecting specific enzymatic activities or the presence of particular products are well known to those skilled in the art. For example, the presence of acetolactate can be determined as described by Hugenholtz and Starrenburg, 1992, AppL Micro. Blot, 38:17-22.

Methods for the Overexpression of NFS1 and/or ISD11 Genes

[00306] Overexpression of the NFS1 and/or ISD11 genes may be accomplished by any number of methods. In one embodiment, overexpression of the NFS1 and/or

ISD11 genes may be accomplished with the use of plasmid vectors that function in yeast. !n exemplary embodiments, the expression of NFS1, ISD11, and/or homologous genes may be increased by overexpressing the genes on a CEN plasmid or alternative piasmids with a similar copy number. In one embodiment,

NFS1 or a homoiog thereof is overexpressed on a CEN plasmid or alternative piasmids with a similar copy number. In another embodiment, ISD11 or a homoiog thereof is overexpressed on a CEN plasmid or alternative piasmids with a similar copy number. In yet another embodiment, NFS1 and ISD11 or homologs thereof are overexpressed on a CEN plasmid or alternative piasmids with a similar copy number.

[00307] In further embodiments, expression of genes from single or multiple copy integrations into the chromosome of the ceil may be useful. Use of a number of promoters, such as TDH3, TEF1, CCW12, PGK1, and EN02, may be utilized. As would be understood in the art, the expression level may be fine-tuned by using a promoter that achieves the optimal expression {e.g., optimal overexpression) level in a given yeast. Different levels of expression of the genes may be achieved by using promoters with different levels of activity, either in single or multiple copy integrations or on piasmids. An example of such a group of promoters is a series of truncated

PDC1 promoters designed to provide different strength promoters. Alternatively, promoters that are active under desired conditions, such as growth on glucose, may

92

364561 vl/CO be used. For example, a promoter from one of the glycolytic genes, the PDC1 promoter, and a promoter from one of the ADH genes in S. cerevisiae may all be useful. Also, embodiments are exemplified using the yeast S. cerevisiae. However, other yeasts, such as those from the genera listed herein may also be used.

Methods for the Qverexpression of AFT Genes

[00308] Qverexpression of the AFT1 and AFT2 genes may be accomplished by any number of methods. In one embodiment, overexpression of the AFT1 and AFT2 genes may be accomplished with the use of piasmid vectors that function in yeast. In exemplary embodiments, the expression of AFT1, AFT2, and/or homologous genes may be increased by overexpressing the genes on a CEN piasmid or alternative piasmids with a similar copy number. In one embodiment, AFT1 or a homolog thereof is overexpressed on a CEN piasmid or alternative piasmids with a similar copy number. In another embodiment, AFT2 or a homolog thereof is overexpressed on a CEN piasmid or alternative piasmids with a similar copy number. In yet another embodiment, AFT1 and AFT2 or homologs thereof are overexpressed on a CEN piasmid or alternative piasmids with a similar copy number.

[00309] In further embodiments, expression of genes from single or multiple copy integrations into the chromosome of the cell may be useful. Use of a number of promoters, such as TDH3, TEF1, CCW12, PGK1, and EN02, may be utilized. As would be understood in the art, the expression level may be fine-tuned by using a promoter that achieves the optimal expression (e.g. optimal overexpression) level in a given yeast. Different levels of expression of the genes may be achieved by using promoters with different levels of activity, either in single or multiple copy integrations or on piasmids. An example of such a group of promoters is a series of truncated PDC1 promoters designed to provide different strength promoters. Alternatively promoters that are active under desired conditions, such as growth on glucose, may be used. For example a promoter from one of the glycolytic genes, the PDC1 promoter, and a promoter from one of the ADH genes in S. cerevisiae may ail be useful. Also, embodiments are exemplified using the yeast S. cerevisiae. However, other yeasts, such as those from the genera listed herein may also be used.

[00310] As described herein, overexpression of the Aft1 protein or a homolog thereof may be obtained by expressing a constitutively active Aft1 or a homolog thereof, !n one embodiment, the constitutively active Aft1 or a homolog thereof comprises a mutation at a position corresponding to the cysteine 291 residue of the

93

364561 yl/CO native S. cerevisiae Aft1 (SEQ ID NO: 2). In a specific embodiment, the cysteine 291 residue is replaced with a phenylalanine residue.

[00311] As described herein, overexpression of the Aft2 protein or a homolog thereof may be obtained by expressing a constitutively active Aft2 or a homolog thereof. In one embodiment, the constitutively active Aft2 or a homolog thereof comprises a mutation at a position corresponding to the cysteine 187 residue of the native S. cerevisiae Aft2 (SEO ID NO: 2). In a specific embodiment, the cysteine 187 residue is replaced with a phenylalanine residue.

Increase of Enzymatic Activity

[00312] Yeast microorganisms of the invention may be further engineered to have increased activity of enzymes. The term "increased" as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable yeast cell of the same species. For example, overexpression of a specific enzyme can lead to an increased level of activity in the ceils for that enzyme. Increased activities for enzymes involved in glycolysis or the isobutanoi pathway would result in increased productivity and yield of isobutanol.

[00313] Methods to increase enzymatic activity are known to those skilled in the art. Such techniques may include increasing the expression of the enzyme by increased copy number and/or use of a strong promoter, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the Km for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231 ), ed. Arnold and Georgiou, Humana Press (2003).

Methods of Using Recombinant Microorganisms for High-Yield Fermentations

[00314] For a biocatalyst to produce a beneficial metabolite most economically, it is desirable to produce said metabolite at a high yield. Preferably, the only product produced is the desired metabolite, as extra products (i.e. by-products) lead to a reduction in the yield of the desired metabolite and an increase in capital and operating costs, particularly if the extra products have little or no value. These extra products also require additional capital and operating costs to separate these products from the desired metabolite.

[00315] In one aspect, the present invention provides a method of producing a beneficial metabolite derived from a DHAD-requiring biosynthetic pathway. In one embodiment, the method includes cultivating a recombinant microorganism

94

364561 yl/CO comprising a DHAD-requiring biosynthetic pathway in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the beneficial metabolite is produced and optionally, recovering the metabolite. In an exemplary embodiment, said recombinant microorganism has been engineered to overexpress one or more polynucleotides encoding one or more Nfs1 proteins or homologs thereof and/or one or more polynucleotides encoding one or more Isd1 1 proteins or homologs thereof. The beneficial metabolite may be derived from any DHAD- requiring biosynthetic pathway, including, but not limited to, biosynthetic pathways for the production of isobutanoi, 3-methyi-1 -butanoi, 2~methyl-1 -butanoi, valine, isoleucine, leucine, and pantothenic acid. In a specific embodiment, the beneficial metabolite is isobutanoi.

[00316] In another aspect, the present invention provides a method of producing a beneficial metabolite derived from a DHAD-requiring biosynthetic pathway. In one embodiment, the method includes cultivating a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the beneficial metabolite is produced and optionally, recovering the metabolite. In an exemplary embodiment, said recombinant microorganism has been engineered to overexpress a polynucleotide encoding Aft1 (SEQ ID NO: 2) and/or Aft2 (SEQ ID NO: 4) or a homoiog thereof. The beneficial metabolite may be derived from any DHAD- requiring biosynthetic pathway, including, but not limited to, biosynthetic pathways for the production of isobutanoi, 3-methyi-1 -butanoi, 2-methyl-1 -butanoi, valine, isoleucine, leucine, and pantothenic acid. In a specific embodiment, the beneficial metabolite is isobutanoi.

[00317] In a method to produce a beneficial metabolite from a carbon source, the yeast microorganism is cultured in an appropriate culture medium containing a carbon source. In certain embodiments, the method further includes isolating the beneficial metabolite from the culture medium. For example, isobutanoi may be isolated from the culture medium by any method known to those skilled in the art, such as distillation, pervaporation, or liquid-liquid extraction

[00318] In one embodiment, the recombinant microorganism may produce the beneficial metabolite from a carbon source at a yield of at least 5 percent theoretical. In another embodiment, the microorganism may produce the beneficial metabolite from a carbon source at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30

95

364561 yl/CO percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 80 percent, at least about 85 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97,5% theoretical. In a specific embodiment, the beneficial metabolite is isobutanoi.

[00319] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference for ail purposes.

General Materials and Methods for Examples

[00320] Media: Media used were standard yeast medium (for example Sambrook, J., Russei, D.W. Molecular Cloning, A Laboratory Manual. 3rd ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press and Guthrie, C. and Fink, G.R. eds. Methods in Enzymoiogy Part B: Guide to Yeast Genetics and Molecular and Cell Biology 350:3-623 (2002)). YP medium contains 1 % (w/v) yeast extract, 2% (w/v) peptone. YPD is YP containing 2% (w/v) glucose.

[00321] S. cerevisiae Transformations: The yeast strain of interest was grown on YPD medium. The strain was re-suspended in 100 mM lithium acetate. Once the ceils were re-suspended, a mixture of DNA (final volume of 15 pL with sterile water), 72 pL 50% w/v PEG, 10 pL 1 M lithium acetate, and 3 pL of denatured salmon sperm DNA (10 mg/rriL) was prepared for each transformation. In a 1 .5 mL tube, 15 pL of the cell suspension was added to the DNA mixture (100 pL), and the transformation suspension was vortexed for 5 short pulses. The transformation was incubated for 30 min at 30 ^C C, followed by incubation for 22 min at 42°C. The ceils were collected by centrifugation (18,000 rcf, 10 sec, 25°C). The cells were resuspended in 1 mL YPD and after an overnight recovery shaking at 30°C and 250 rpm, the ceils were spread over YPD + 0.2 g/L G418 + 0.1 g/L hygromycin selective plates. Transformants were then single colony purified onto selective plates containing appropriate antibiotics.

[00322] Preparation of Yeast Lysate: Cells were thawed on ice and resuspended in lysis buffer (50 mM Tris pH 8.0, 5 mM MgS0 ₄ ) such that the result was a 20% cell suspension by mass. 1000 pL of glass beads (0.5 mm diameter) were added to a

96

364561 yl/CO 1 .5 mL microcentrifuge tube and 875 pL of ceil suspension was added. Yeast ceils were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, PA), mixing 8 X 1 min each at full speed with 1 min incubations on ice between each bead-beating step. The tubes were centrifuged for 10 min at 23,500 rcf at 4 ^" C and the supernatant was removed for use. The iysates were heid on ice until assayed.

[00323] DHAD Assay: Each sample was diluted in DHAD assay buffer (50 mM Tris pH 8, 5 mM MgS0 ₄ ) to a 1 :10 and a 1 :40 to 1 :100 dilution. Three samples of each lysate were assayed, along with no lysate controls. 10 pL of each sample (or DHAD assay buffer) was added to 0.2 mL PGR tubes. Using a multi-channel pipette, 90 pL of the substrate was added to each tube (substrate mix was prepared by adding 4 mL DHAD assay buffer to 0.5 mL 100 mM DHIV). Samples were put in a thermocycler (Eppendorf Mastercycler) at 35°C for 30 min followed by a 5 min incubation at 95°C. Samples were cooled to 4°C on the thermocycler, then centrifuged at 3000 rcf for 5 min. Finally, 75 pL of supernatant was transferred to new PCR tubes and submitted to analytics for analysis by Liquid Chromatography, method 2. DHAD activity units were calculated as pmoi KIV produced/min/mg total ceil lysate protein in the assay.

[00324] Protein Concentration Determination. Yeast lysate protein concentration was determined using the BioRad Bradford Protein Assay Reagent Kit (Cat# 500- 0006, BioRad Laboratories, Hercules, CA) and using BSA for the standard curve. Briefly, 10 pL standard or lysate were added into a microcentrifuge tube. The samples were diluted to fit in the linear range of the standard curve (1 :40). 500 pL of 1 :4 diluted and filtered Bio-Rad protein assay dye was added to the blank and samples and then vortexed. Samples were incubated at room temperature for 6 min, transferred into cuvettes and the OD ₅₉₅ was determined in a spectrophotometer. The linear regression of the standards was then used to calculate the protein concentration in each sample.

[00325] Gas Chromatography: Analysis of volatile organic compounds including isobutanol, was performed on a HP 5890/8890/7890 gas chromatograph fitted with an HP 7873 Autosamp!er, a ZB-FFAP column (Phenomenex; 30 m length, 0.32 mm ID, 0.25 pM film thickness) or equivalent connected to a flame ionization detector (FID). The temperature program was as follows: 200°C for the injector, 300°C for the detector, 100°C oven for 1 min, 70°C/min gradient to 230°C, and then hold for 2.5 min. Analysis was performed using authentic standards (>99%, obtained from Sigma-Aldrich) and a 5-point calibration curve with 1 -pentanol as the internal

97

364561 yl/CO standard.

[00326] Liquid Chromatography, Method 1 : Analysis of organic acid metabolites, specifically pyruvate, acetate, 2,3-dihydroxy-isovalerate, and 2,3-butanediol, was performed on an HP-1200 High Performance Liquid Chromatography system equipped with two Rezex RFQ 150 x 4.6 mm columns in series. Organic acid metabolites were detected using an HP-1 100 UV detector (210 nm) and refractive index. The column temperature was 60°C. This method was isocratic with 0.0180 N H2SO4 in Milii-Q water as mobile phase. Flow was set to 1 .1 mL/min. Injection volume was 20 pL and run time was 16 min. Analysis was performed using authentic standards (>99%, obtained from Sigma-Aldrich, with the exception of DH!V (2,3- dihidroxy-3-methyi-butanoate, CAS 1756-18-9), which was custom synthesized at Caltech (Cioffi, E. ef a/. Anal Biochem 104 pp.485 (1980)), and a 5-point calibration curve.

[00327] Liquid Chromatography, Method 2: Analysis of 2-keto-isovalerate (KIV), the product indicating DHAD activity, was measured using liquid chromatography. DNPH reagent (12 mM 2,4 ~ Dinitrophenyl Hydrazine, 20 mM Citric Acid pH 3.0, 80% Acetonitrile, 20% MiliiQ H ₂ Q) was added to each sample in a 1 :1 ratio. Samples were incubated for 30 min at 70°C in a thermo-cycier (Eppendorf, Mastercycler). Analysis of KIV was performed on an HP-1200 High Performance Liquid Chromatography system equipped with an Eclipse XDB C-18 reverse phase column (Agilent) and a C- 18 reverse phase column guard (Phenomenex). K!V was defected using an HP-1 100 UV detector (360 nm). The column temperature was 50°C. This method was isocratic with 70% acetonitrile 2.5% phosphoric acid (4%), 27.5% water as mobile phase. Flow was set to 3 mL/min. Injection size was 10 L and run time was 2 min.

Example 1 : Qverexpression of AFT1 Increases DHAD Activity and Isobutanoi Productivity , Titer, a no Yield in Fermentation Vessels

[00328] The purpose of this example is to demonstrate that overexpression of AFT1 increases DHAD activity, isobutanoi titer, productivity, and yield.

[00329] Media: Medium used for the fermentation was YP + 80 g/L glucose + 0.2 g/L G418 + 0.1 g/L hygromycin + 100μΜ CuS0 ₄ 5H ₂ 0 + 1 % v/v ethanol. The medium was filter sterilized using a 1 L bottle top Corning PES 0.22pm filter (431 174). Medium was pH adjusted to 6.0 in the fermenter vessels using 6N KOH.

[00330] Vessel Preparation and Operating Conditions: Batch fermentations were conducted using six 2 L top drive motor DasGip vessels with a working volume of 0.9

98

364561 vl/CO L per vessel. Vessels were sterilized, along with the appropriate dissolved oxygen probes and pH probes, for 80 min at 121 °C. pH probes were calibrated prior to sterilization, however, dissolved oxygen probes were calibrated post sterilization in order to allow for polarization.

[00331] Process Control Parameters: Initial volume, 900 mL. Temperature, 30°C. pH 8.0, pH was controlled using 6N KOH and 2N H ₂ 80 ₄ (Table 4).

Table 4. Process control parameters.

^' Oxygen transfer rate increased from 0.5 mM/h to 1.8 rrsM/h by increase in agitation from 300 rpm to 400 rpm 56 h post inoculation.

[00332] Fermentation: The fermentation was run for 1 19 h . Vessels were sampled 3 times daily. Sterile 5 mL syringes were used to collect 3 mL of fermenter culture via a sterile sample port. The sample was placed in a 2 mL microfuge tube and a portion was used to measure cell density (OD ₆ oo) on a Genesys 10 spectrophotometer (Thermo Scientific). The remaining sample was filtered through a 0.22 prn pore-size Corning filter. The supernatant from each vessel was refrigerated in a 96-weil, deep well plate, and stored at 4°C prior to gas and liquid chromatography analysis (see General Methods).

[00333] Qff-gas Measurements: On-line continuous measurement of each fermenter vessel off-gas by mass spectrometry analysis was performed for oxygen, isobutanoi, ethanol, carbon dioxide, and nitrogen throughout the experiment. Fermentor off-gas was analyzed by Prima dB mass spectrometer (Thermo, Waltham, MA) for nitrogen, oxygen, argon, carbon dioxide, isobutanoi, ethanol, and isobutyraldehyde. A reference stream of similar composition to the inlet fermentor air was also analyzed. The mass spectrometer cycles through the reference air and fermentor off-gas streams (one by one) and measures percent concentration of these gases after an 8.3 min settling time to ensure representative samples. Equation 1 is a derived value expression input into the mass spectrometer software to determine OTR using percent oxygen and percent nitrogen from the reference air

99

364561 vl/CO (% Ozr, and % Η ₂ ι _η } and fermentor off-gas (% 02out and % N ₂₀ ut)- Nitrogen is not involved in cellular respiration, and therefore, can be used to compensate for outlet oxygen dilution caused by the formation of C0 ₂ . The inlet flow is calculated from Equation 2 based on the ideal gas law and is standardized to 1 .0 sLph flow rate and 1 ,0 L fermentor working volume to yield a derived value OTR in mmol/L/h from the mass spectrometer. This derived value OTR is then multiplied by actual inlet flow rate (sLph) and divided by actual working volume (L) in fermentation spreadsheets to obtain an OTR for specific operating conditions.

OSS s&

[00334] See the General Methods for a description of how the yeast transformations were performed, as well as a description of how the yeast Iysate was prepared. The DHAD assay and protein concentration assay are also described in the general methods section. Strains, piasmids, and the gene/protein sequences used in Example 1 are described in Tables 5, 8, and 7, respectively.

Table 5. Genotype of strain disclosed in Example 1 .

GEVO Number Genotype

S.cerevisiae CEN.PK2, MATa ura3 Ieu2 his3 trpl

pdc1A::[Pcupi: s aisSI COSC:T _C YCI: PPGKI- Li kivD2: PFNO?: Sp HiS5] pdc5A::[LEU2-bla-P _T EFi: ILV3AN: P _TDH3 : EcJvC__coSc ^Q110i7 ]

GEV02843

pdc6A::[URA3: Ma; P _TE FI: LI_kivD2: Pj _DH3 : Dm_ADH ]

{evolved for C2 supplement-independence, glucose tolerance and faster growth}

Table 6. Piasmids disclosed in Example 1 .

100

364561 vl/CO PPGK empty

groR

PJEFI- empty

pGV2472 CEN piasmid expressing AFT1

PpGKi.empty

CEN on, bia, HygroR

Table 7. Nucleotide and amino acid sequences of genes and proteins disclosed in Examples.

[00335] GEV02843 was co-transformed with two plasmids (Table 8). GEV03342 contains plasmids pGV2227 and pGV2196; GEV03343 contains plasmids pGV2227 and pGV2472.

Table 8, Indicates the strains containing plasmids transformed together into strain GEVG2843.

[00336] DHAD Assay Results: The in vitro DHAD enzymatic activity of iysates from the microaerobic fermentation of GEV03342 and GEV03343 were carried out as described above. Overexpression of AFT1 from a CEN piasmid resulted in a three-fold increase in specific DHAD activity (U/mg total ceil lysate protein). Data is presented as specific DHAD activity (U/mg total cell lysate protein) averages from technical triplicates with standard deviations. DHAD activity for GEV03342 (control) was 0.066 ± 0.005 U/mg and DHAD activity for GEV03343 (AFT1 over-expressed)

101

364561 vl/CO was 0.215 ± 0.008 U/mg at the end of the fermentation (1 19 h).

[00337] isobutanol Results: Isobutanol titers, rates and yields were calculated based on the experiment run in batch fermentors. Table 9 shows the increase in isobutanol titer, rate and yield in the strain overexpressing the AFT† gene. The overexpression of AFT1 from a CEN plasmid (GEV03343) resulted in an increase in isobutanol titer, an increase in isobutanol yield, and an increase in isobutanol rate.

Table 9. Isobutanol titer, rate and yield for replicate fermentation experiments.

[00338] Change in metabolic by-products: The strain transformed with the AFT1 gene expressed on the CEN plasmid (GEV03343) produced less pyruvate, acetate, DHIV (dihydroxyisovalerate)/DH2MB (2,3-dihydroxy-2-methyibutanoic acid), and 2,3- butanediol than the strain with the control plasmid (GEV03342) during the fermentation. There was a six fold decrease in pyruvate, one fold decrease in acetate, one and a half fold decrease in DHIV/DH2 B, and six fold decrease in 2,3- butanedio!.

Example 2 : Overexpression of AFT2 Increases D HAD Activity

[00339] The purpose of this example is to demonstrate that overexpression of

AFT2 increases DHAD activity. Methods of strain construction and cloning techniques are described in Example 1 . Strain GEV02843 is described in Table 5.

Table 10. Plasmids disclosed in Example 2.

Methods

102

364561 yl/CO [00340] Methods for yeast transformations and the preparation of yeast iysates are described in the general methods. The DHAD assay, the liquid chromatography, method 2, assay, and assays for measuring protein concentration are described in the general methods.

[00341] Results for DHAD Activity: Data is presented as specific DHAD activity (U/mg total cell iysate protein) averages from biological and technical triplicates with standard deviations. DHAD activity in GEV02843 (Table 5) transformed with pGN 2247 + pGV2196 (no AFT2) was 0.358 ± 0.009 U/mg, DHAD activity for pGV2247 + pGV2827 (contains AFT2) was 0.677 ± 0.072 U/mg. The overexpression of AFT2 increased the amount of DHAD activity in the strain.

Example 3: Overexpression of AFT1 Increases DHAD Activity for DHAD Enzymes from Multiple Organisms

[00342] The purpose of this example is to demonstrate that overexpression of AFT1 increases DHAD activity for DHAD enzymes from multiple organisms.

[00343] Strains and p!asmids used in Example 4 are described in Tables 1 1 and 12, respectively.

Table 11. Genotype of strains disclosed in Example 3.

103

364561 vl/CO

104

364561 vl/CO

glucose tolerance and faster arowth}

Table 12. Plasmids disclosed in Example 3.

Contains 6-his tags as compared to Ec_ilvC_coSc'

[00344] Shake Flask Fermentations: Fermentations were performed to compare the DHAD enzyme activity of strains GEV03879, GEVO3880, GEV03881 , GEV03928, GEVG3929, GEV0393G, GEV03931 and GEV03932, which overexpress AFT1 from S. cerevisiae from plasmid pGV2472, with strains GEV03873, GEV03874, GEV03875, GEV03878, GEV03877, and GEV03878, which do not overexpress AFT1. Strains GEV03873, GEV03874, GEV03879, GEVO3880 and GEV03881 express the Lactococcus lactis IlvD protein (LIJ!vD) from the LIJlvD gene on pGV2603. Strains GEV03875, GEV03928 and GEV03929 express the Neurospora crassa MvD2 protein (Nc !ivD2) from the Nc ilvD2 gene on pGV2607. Strains GEV03876, GEV03877, GEV03878, GEVO3930, GEV03931 and GEV03932 express the Streptococcus mutans livD protein (Sm IlvD) from the Sm ivD gene on pGV2608. These plasmids were ail present in the same host background strain, GEV03626.

[00345] Strains containing plasmid pGV2472 were maintained and grown in media

105

364561 vl/CO containing both 0.2 g/L G418 and 0.1 g/L hygromycin while strains lacking pGV2472 were maintained and grown in media containing 0.2 g/L G418. Yeast strains were inoculated from ceil patches or from purified single colonies from YPD supplemented with 0.2 g/L G418 medium agar plates or from YPD supplemented with 0.2 g/L G418 and 0.1 g/L hygromycin medium agar plates into 3 rrsL of growth medium in 14 mL round-bottom snap-cap tubes to provide three replicates of strains carrying each plasmid or plasmid combination. The growth media used were YPD + 0.2 g/L G418 + 1 % v/v ethanol medium for strains lacking pGV2472 and YPD + 0.2 g/L G418 + 0.1 g/L hygromycin + 1 % v/v ethanol medium for strains containing pGV2472. The cultures were incubated for up to 24 h shaking at an angle at 250 rpm at 30°C. Separately for each tube culture, these overnight cultures were used to inoculate 50 mL of medium in a 250 mL baffled flask with a sleeve closure to an OD ₆ oo of 0.1 . The media used were YP + 50 g/L glucose + 0.2 g/L G418 + 1 % v/v ethanol medium for strains lacking pGV2472 and YP + 50 g/L glucose + 0.2 g/L G418 + 0.1 g/L hygromycin + 1 % v/v ethanol medium for strains containing pGV2472. These flask cultures were incubated for up to 24 h shaking at 250 rpm at 30°C. The ceils from these flask cultures were harvested separately for each flask culture by ceritrifugation at 3000 rcf for 5 min and each ceil pellet was resuspended separately in 5 mL of YP medium supplemented with 80 g/L glucose, 1 % v/v stock solution of 3 g/L ergosteroi and 132 g/L Tween 80 dissolved in ethanol, 200 mM MES buffer, pH 6.5, and 0.2 g/L G418. Each ceil suspension was used to inoculate 50 mL of YP medium supplemented with 80 g/L glucose, 1 % v/v stock solution of 3 g/L ergosteroi and 132 g/L Tween 80 dissolved in ethanol, 200 mM MES buffer, pH 6.5, and 0.2 g/L G418 in a 250 mL non-baffled flask with a vented screw-cap to an ODeoo of approximately 5. These fermentations were incubated shaking at 250 rpm at 30°C. After 73 h of incubation, the ceils from half of each fermentation culture were harvested by ceritrifugation at 3000 rcf for 5 min at 4°C. Each ceil pellet was resuspended in 25 mL of cold MilliQ water and then harvested by centrifugation at 3000 rcf for 5 min at 4°C. The supernatant was removed from each pellet and the tubes containing the pellets were frozen at ~80°C.

[00346] Ceil iysate production, total protein quantification, DHAD assays and liquid chromatography, method 2, were performed as described in the general methods.

[00347] Qverexpression of S. cerevisiae AFT1 Increased the DHAD Activity of Strains Expressing Different DHAD Enzymes: Qverexpression of S. cerevisiae AFT1 increased the DHAD enzyme activity of strains expressing the L lactis livD, N,

106

364561 yl/CO crassa i!vD2 and S, mutans IlvD DHADs by at least 2.5-io!d (Table 13). DHAD enzyme activities of the strains expressing the different DHADs were similar in the absence of AFT1 overexpression but were at different increased enzyme activity levels in the strains expressing the different DHADs together with AFT1 overexpression. This demonstrates that AFT1 overexpression increases the activity of multiple DHAD enzymes from several different organisms.

Table 13, DHAD enzyme activity results from shake flask fermentations demonstrating increased DHAD activity from S. cerevisiae expressing DHAD enzymes from L. lactis, N. crassa and S. rnutans and overexpressing AFT1.

Activity

[00348] The purpose of this example is to demonstrate that overexpression of S. cerevisiae AFT1 (Sc_AFT1) and S. cerevisiae AFT2 (Sc__AFT2) increases DHAD activity.

[00349] Standard molecular biology methods for cloning and plasmid construction were generally used _. , unless otherwise noted (Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual, 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press). Cloning techniques included gel purification of DNA fragments (using the Zymoclean Gel DNA Recovery Kit, Cat# D4002, Zymo Research Corp, Orange, CA).

[00350] S. cerevisiae Transformations: Co-transformations with the CEN and 2μ plasmids into S. cerevisiae strains are described below. Briefly, the S. cerevisiae strain GEVO2843 (Table 5) was grown on YPD medium. From the plate, the strain was re-suspended in 100 mM lithium acetate. Once the cells were re-suspended, a mixture of DNA (final volume of 15 pL with sterile water), 72 L 50% w/v PEG, 10 μί 1 M lithium acetate, and 3 pL of denatured salmon sperm DNA (10 mg/mL) was prepared for each transformation. In a 1 .5 mL tube, 15 μί, of the cell suspension was added to the DNA mixture (100 μί), and the transformation suspension was vortexed for 5 short pulses. The transformation was incubated for 30 min at 30°C, followed by incubation for 22 min at 42°C. The ceils were collected by centrifugation (18,000 rcf, 10 sec, 25°C). The cells were resuspended in 1 mL YPD and after an overnight

107

364561 yl/CO recovery shaking at 30°C and 250 rpm, the cells were spread over YPD supplemented with 0.2 g/L G418 and 0.1 g/L hygromycin selective plates. Transformants were then single colony purified onto G418 and hygromycin selective plates.

[00351] Shake Flask Fermentation: Fermentations for the AFT1iAFT2 transformant strains were performed. Starter cultures with each transformed strain were inoculated into 3 mL YPD with 0.1 g/L hygromycin, 0.2 g/L G418, 1 % v/v EtOH and incubated shaking at 250 rpm at 30°C. Pre-cuitures for the fermentations were inoculated to 0.05 OD ₆ oo nto 50 mL YPD (8% w/v glucose) with 200 mM IVIES, 0.1 g/L hygromycin, 0.2 g/L G418, 1 % v/v stock solution of 3 g/L ergosterol and 132 g/L Tween 80 dissolved in ethanoi, and 20μΜ CuS0 ₄ at pH 6.5 in 250 mL baffled flasks, shaking at 250 rpm at 30°C. Fermentation cultures were inoculated to 4.0 - 5.0 ODeoo into 50 mL YPD (8% w/v glucose) with 200 mM IVIES, 0.1 g/L hygromycin, 0.2 g/L G418, 1 % v/v stock solution of 3 g/L ergosterol and 132 g/L Tween 80 dissolved in ethanoi, and 20μΜ CuS0 ₄ at pH 6.5 in 250 mL unbaffled flasks, shaking at 75 rpm at 30°C. Ail cultures were done in biological triplicate.

[00352] Preparation of Yeast Lysate: 50 mL of ceils were spun down at 4°C, 3000 rcf for 5 min from the 72hr timepoint of the fermentation. The medium was decanted and the cells were resuspended in 10 mL of cold Mi!liQ water. The cells were centrifuged a second time at 4°C, 3000 rcf for 5 min. The medium was again decanted and the cells were centrifuged at 4°C, 3000 rcf for 5 min. Remaining media was removed and the cell pellet was frozen at -80°C. Ceils were thawed on ice and resuspended in lysis buffer (50 mM Tris pH 8.0, 5 mM MgS0 ₄ ) such that the result was a 20% cell suspension by mass. 1000 pL of glass beads (0.5 mm diameter) were added to a 1 .5 mL microcentrifuge tube and 875 L of ceil suspension was added. Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, PA), mixing 8 X 1 min each at full speed with 1 min incubations on ice between each bead-beating step. The tubes were centrifuged for 10 min at 23,500 rcf at 4 ^° C and the supernatant was removed for use. The lysates were held on ice until assayed.

[00353] DHAD Assay: each sample was diluted in DHAD assay buffer (50 mM Tris pH 8, 5 mM MgS0 ₄ ) to a 1 :10 and 1 :100 dilution. Three samples of each lysate were assayed, along with no lysate controls. 10 pL of each sample (or DHAD assay buffer) was added to 0.2 mL PGR tubes. Using a multi-channel pipette, 90 pL of the substrate was added to each tube (substrate mix was prepared by adding 4 mL

108

364561 yl/CO DHAD assay buffer to 0.5 mL 100 mM DHIV). Samples were put in a thermocycier (Eppendorf Mastercycler) at 35°C for 30 min followed by a 5 min incubation at 95°C. Samples were cooled to 4°C on the therniocycler, then centrifuged at 3000 rcf for 5 min. Finally, 75 pL of supernatant was transferred to new PGR tubes and submitted to analytics for analysis by Liquid Chromatography, method 2. Yeast lysate protein concentration was determined as described under General Methods.

[00354] Liquid Chromatography, method 2: DNPH reagent (4:1 of 15 mM 2,4 - Dinitrophenyl Hydrazines 00 mM Citric Acid pH 3.0) was added to each sample in a 1 :1 ratio. Samples were incubated for 30 min at 70°C in a thermo-cycier (Eppendorf, Mastercycler). Analysis of keto-isovalerafe and isobutyra!dehyde was performed on an Agilent 1200 High Performance Liquid Chromatography system equipped with an Eclipse XDB C-18 reverse phase column (Agilent) and a C-18 reverse phase column guard (Phenomenex). Ketoisovaierate and isobufyraidehyde were detected using an Agilent 1 100 UV detector (380 nm). The column temperature was 50°C. This method was isocratic with 70% acetonitriie 2.5% phosphoric acid (0.4%), 27.5% water as mobile phase. Flow was set to 3 mL/min. Injection size was 10 pL and run time was 2 min.

[00355] Results for DHAD Activity: Data is presented as specific DHAD activity (U/mg total cell lysate protein) averages from biological and technical triplicates with standard deviations. DHAD activity in GEV02843 transformed with pGV2247 (Table 10) + pGV2198 (empty vector, Table 8) was 0.358 ± 0.009 U/mg. DHAD activity for GEV02843 transformed with pGV2247 + pGV2626 (CEN plasmid that contains Sc_AFT1 and Sc_AFT2; Genotype: P _TDH 3:Sc_AFT1 , PTEF empty, P _PGK1 :Sc__AFT2, CEN ori, bla, HygroR) was 0.902 ± 0.032 U/mg. The simultaneous overexpression of Sc AFT1 and Sc AFT2 increased the amount of DHAD activity in the strain.

Example 5. AFT1 Expression Increases DHAD Activity Independentl of DHAD Protein Levels

[00356] The following example illustrates that overexpression of the AFT1 gene in Saccharomyces cerevisiae leads to increased DHAD activity independently of DHAD protein levels.

14. Genotype of strains disclosed in Example 5.

109

364561 vl/CO 3EVO3901 MATa ura3' Ieu2 his3 trpl gpd1::T _K , _URA3 gpd2::T _K , _URA3 tma29::T _{K! URA} v

pdc1::Pp _DC1 :U_kivD2_coSc5:P _FBA1 :lEU2)T _LEU2 :P^H^

2_C ^Q EC:PENO2- SP_ IS5 pdc5::T _K i_uRA3 _

pdc6::P _TDH3 :8c _^ AFT1:P _£N 02:LLadhA ^RE1 :T _Kl ^^^

C2 supplement-independence, glucose tolerance and faster growth} [pGV2603j

[00357] Media: Medium used was standard yeast medium (for example Sambrook, J,, Russei, D.W. Molecular Cloning, A Laboratory Manual. 3rd ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press and Guthrie, C. and Fink, G.R. eds. Methods in Enzymology Part B: Guide to Yeast Genetics and Molecular and Cell Biology 350:3-623 (2002)). YP medium contains 1 % (w/v) yeast extract, 2% (w/v) peptone. YPD is YP containing 2% (w/v) glucose.

[00358] Fermentations in benchtop fermentors: Fermentations in benchtop fermentors were performed to compare the DHAD enzyme activity and DHAD protein level of GEV03882 (no AFT1 overexpression) to GEVO3901 (AFT1 overexpression). For these fermentations, 1 mL from thawed frozen stocks of the strains were transferred to 500 mL baffled flasks containing 80 mL of YP medium supplemented with 80 g/L glucose, 5 g/L ethanol, 0.5 g/L MgS0 ₄ and 0.2 g/L G418 and incubated for 24 h at 30°C in an orbital shaker at 250 rpm. The flask culture for each strain was transferred to duplicate 2-L top drive motor fermentor vessels with a working volume of 0.9 L of YP medium supplemented with 80 g/L glucose, 5 g/L ethanol, 0.5 g/L MgS0 ₄ and 0.2 g/L G418 per vessel for a starting OD ₆ QO of 0.5. Fermentors were operated at 30°C and pH 6.0 controlled with 6N KOH and 2N H ₂ S0 ₄ in a 2-phase aerobic condition based on oxygen transfer rate (OTR). Initially, fermentors were operated at a growth phase OTR of 10 mM/h by fixed agitation of 700 rpm and an air overlay of 5 sL/h. Cultures were grown for 20 h to approximately 10-13 ODeoo then immediately switched to a production aeration OTR = 0.5 mM/h by reducing agitation from 700 rpm to 300 rpm for the period of 20 h to 70.5 h.

[00359] Sample Collection: Samples from each fermentor were collected at 15.5 h, 20 h, 27 h, 48.5 h and 70.5 h to measure optical density at 600 nm (OD ₆ oo). A volume of culture equal to 150 OD600 was then collected from each fermentor at each time point using 60 mL sterile syringes via a sterile sample port on each vessel and placed on ice in 500 mL centrifuge bottles. The samples were centrifuged at 4000 rcf for 10 min at 4°C to pellet the ceils. The ceil pellets were then separately resuspended in 60 mL cold deionized water for DHAD enzyme assays or cold deionized water containing Yeast/Fungal Protease Arrest (GBiosciences) for DHAD protein quantification and separated into 10 mL aliquots which were centrifuged at

110

364561 yl/CO 4000 rcf for 10 min at 4°C to pellet the cells. The supernatant was removed from each pellet and the resulting cell pellets were stored frozen at -80°C until used to prepare cell lysates.

[00360] Cell Lysate Production: Ceil lysates were prepared for each frozen sample pellet in lysis buffer U1 , which contains 0.1 sodium phosphate, pH 7.0, 5 rnlVI gCi2 and 1 mM DTT, for DHAD enzyme assays or lysis buffer U1 containing Yeast/Fungal Protease Arrest (GBiosciences) for DHAD protein quantification. Each ceil pellet was individually suspended to 20% (w/v) in the appropriate lysis buffer and 1 mL of that cell suspension was added together with 1000 pL of 0.5 mm diameter glass beads to a 1 .5 mL microcentrifuge tube. The yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc., Newtown, PA) by mixing for six 1 -min cycles at full speed with 1 -min incubations on ice between each cycle. The tubes were then centrifuged for 10 min at 23,500 rcf at 4°C and the supernatant was removed. Samples for DHAD enzyme assays were held on ice until assayed on the same day and samples for DHAD protein quantification were frozen at -20°C. Yeast lysate protein concentration was determined as described under General Methods.

[00361] DHAD Assay: Each cell lysate sample was diluted 1 :10 in DHAD assay buffer (50 mM Tris, pH 8, 5 mM MgS0 ₄ ). Three samples of diluted lysate were assayed, along with three controls of DHAD assay buffer containing no lysate. 10 pL of each sample or control was added to 0.2 mL PGR tubes. Using a multi-channel pipette, 90 pL of substrate mix, prepared by adding 4 mL DHAD assay buffer to 0.5 mL 100 mM DHIV, was added to each tube. These tubes were placed in an Eppendorf Mastercycler thermocycler and incubated at 35°C for 30 min followed by incubation at 95 ^C C for 5 min then cooled to 4°C in the thermocycler and centrifuged at 3000 rcf for 5 min. 75 pL of supernatant from each tube was transferred to separate new PGR tubes and submitted for liquid chromatography analysis for keto- isovalerate quantification. The DHAD activity was calculated as μπηοί K!V produced/min/mg total cell lysate protein in the assay.

[00362] Liquid Ch roma tog ra phy for Keto- 1 so va I era te Quantification: 100 pL of DNPH reagent, containing 12 mM 2,4-dinitrophenyi hydrazine, 10 mM citric acid, pH 3.0, 80% Acetonifrile and 20% MiiliQ H ₂ 0, was added to 100 pL of each sample. The mixtures were then incubated for 30 min at 70°C in an Eppendorf Mastercycler thermocycler. Analysis of keto-isovalerate (KiV) was performed on an HP-12G0 High Performance Liquid Chromatography system equipped with an Eclipse XDB C-18 reverse phase column (Agilent) and a C-18 reverse phase column guard

111

364561 yl/CO (Phenomenex). Keto-isova!erate (KIV) was detected using an HP-1 100 UV detector at 210 nm. The column temperature was 50°C. This method was isocratic with 70% acetonitrile to water as mobile phase with 2.5% dilute phosphoric acid (4%). Flow was set to 3 mL/min. Injection size was 10 pL and the run time was 2 min.

[00363] DHAD Protein Quantification: Ceil lysate samples were prepared for gel electrophoresis by mixing with appropriate volumes of 4X LDS loading buffer (Invitrogen) and 10X reducing agent solution (Invitrogen) and MiliiQ water, followed by incubation at 70°C for 10 min. Prepared samples were run on 4-12% acryiamide Bis-Tris gels (Invitrogen) at 200V for 55 min on the Novex Gel Midi System (Invitrogen) and protein was subsequently transferred from the gel to PVDF membrane with the Novex Semi-Dry Blotter (Invitrogen). Gel electrophoresis and protein transfer were performed according to the manufacturer's recommendations. PVDF membranes with transferred proteins were blocked in 2% ECL Advance Blocking Agent (GE Healthcare) diluted in filtered TBST (150 mM NaCI, 10 mM Tris- HCI, pH 7.5, 0.5% v/v Tween 20) for 1 h at room temperature under mild agitation. Membranes were then probed with a 1 :500 dilution of rabbit anti-LM!vD or a 1 :500 dilution of rabbit anti-Sc__llv3 serum for 1 h at room temperature under mild agitation. Membranes were washed with filtered TBST for 15 min, followed by three 5 min washes with additional filtered TBST. Membranes were then incubated with a 1 :5000 dilution of goat anti-rabbit AlexaFiuor 633-tagged secondary antibody (Invitrogen) for 1 h at room temperature under mild agitation while protected from light. Membranes were washed with TBST as described above while protected from light and then were dried and scanned on a Storm 880 fluorescence imaging system (Molecular Dynamics) using the 635 nm laser at 300V and 100pm resolution. ImageQuant software (GE Healthcare) was used to perform standardized densitometry to quantify relative levels of protein expression, reported as integrated band intensity from the blots.

[00364] Overexpression of AFT1 Increases DHAD Activity Without Increasing DHAD Protein Levels: DHAD enzyme activity and DHAD protein levels from benchtop fermentor fermentations are summarized in Tables 15 and 16. AFT1- overexpressing strain GEVO3901 contains at least 1 .5-foid higher DHAD enzyme activity at all fermentation sample time points compared with strain GEV03882 with no AFT1 overexpression (Table 15). The ratio of DHAD enzyme activity in GEVO3901 overexpressing AFT1 compared to DHAD enzyme activity in strain GEV03882 with no AFT1 overexpression was higher during the growth phase of the

112

364561 yl/CO fermentation (3.7 at 15.5 h, 3.8 at 20 h) than during the production phase of the fermentation (2.8 at 27 h, 1 .5 at 48.5 h and 1 .8 at 70.5 h).

[00365] DHAD protein ievels from yAFTf-overexpressing strain GEVO3901 were not substantiaiiy different from strain GEV03882 with no AFT1 overexpression at any of the fermentation sample time points (Table 18). Neither the LIJlvD nor the Scjlv3 DHAD protein levels were substantially different from GEVO3901 overexpressing AFT1 compared with GEV03882 without AFT1 overexpression at any fermentation sample time point.

Table 15. DHAD enzyme activity results from fermentation samples demonstrating increased DHAD activity with AFT1 overexpression,

Table 16. DHAD protein level determinations from fermentation samples demonstrating no increase in DHAD protein Ievels with AFT1 overexpression.

Example 6: Mutating Sc AFT1 or Sc AFT2 to Sc AFT1 or Sc AFT2 Alleles

[00366] A point mutation in Sc Aft1 and Sc Aft2 causes derepression of transcriptional activation in the presence of iron. Sc_Aft1 -1 ^up mutation changes

Cys291 Phe (Yamaguchi-lwia et al. 1995 EMBO Journal 14: 1231 -9). The Sc_Aft2-

1 ^UP mutation changes Cys187Phe (Rutherford et al. 2001 PNAS 98: 14322-7). The purpose of this example is to demonstrate that mutating the endogenous copy of Sc__AFT1 or Sc__AFT2 into the Sc_AFT1-1 ^up or Sc__AFT2~1 ^up mutant alleles generally mimics the overexpression of Sc__AFT1 or Sc__AFT2 by increasing DHAD activity and isobutanol titers in yeast strains carrying an isobutanol producing metabolic pathway.

[00367] In this example, Sc__AFT1 and Sc__AFT2 are replaced in the genome by

113

364561 vl/CO Sc__AFT1-1 ^up and 8ο_ΑΡΤ2~1 ^υρ alleles, either individually or together. Figure 4 and Figure 5 show the constructs for the allelic replacement for Sc___AFT1~1 ^up (SEG ID NO: 82) and Sc_AFT2-1 ^up (SEQ ID NO: 83). These constructs are synthesized by DNA2.Q. The constructs are transformed into GEV02843 (Table 5) either with pGV2227 (Table 8) or pGV2196 (empty vector control, Table 8) to yield GEVO6209 and GEVO6210 (Table 17).

[00368] Yeast Transformations: Transformations of either the linear Sc_AFT1~1 ^up or the Sc_AFT2-1 ^up constructs or pGV2227(or pGV2196) into GEV02483 are described below. Briefly, the S. cerevisiae strain GEV02843 is grown on YPD medium. The strain is re-suspended in 100 m lithium acetate. Once the cells are re-suspended, a mixture of DNA (final volume of 15 pL with sterile water), 72 pL 50% w/v PEG, 10 pL 1 lithium acetate, and 3 pL of denatured salmon sperm DNA (10 mg/mL) is prepared for each transformation. In a 1 .5 mL tube, 15 pL of the cell suspension is added to the DNA mixture (100 pL), and the transformation suspension is vortexed for 5 short pulses. The transformation is incubated for 30 min at 30°C, followed by incubation for 22 min at 42°C. The cells are collected by centrifugation (18,000 rcf, 10 sec, 25°C). The ceils are resuspended in 1 mL YPD and after an overnight recovery shaking at 30°C and 250 rpm, the transformants are spread over YPD supplemented with 0.2 g/L G418 selective plates. Transformants are then single colony purified onto G418 selective plates. GEV02483 containing pGV2227 or pGV2198 and transformed with the linear AFT ^UP constructs are plated onto YPD with 0.2 g/L G418 and 0.1 g/L hygromycin.

17. Genotype of strains disclosed in Example 6.

Strains that grow on 0.2 g/L G418 and 0.1 g/L hygromycin are further

114

364561 vl/CO screened by PGR to determine if the integration has replaced Sc_AFT1 or Sc__AFT2.

[00370] For AFT1 : The primer AFT1 UP forward (SEQ ID NO: 64) is used with the primer pENO2R (SEQ ID NO: 65) to yield a 599 base pair product that will not be present in the parental strain. The primer AFT1 UP forward is used with primer AFTI termR (SEQ ID NO: 66) to ensure that the parental Sc__AFT1 does not remain in the strain. If integrated correctly, these primers give an approximately 2210 base pair product; if the parental Sc AFT1 remains in the strain the product size is 584 base pairs. Finally, the 8ο__ΑΡΤ1-1 ^υΗ gene is amplified using the AFT1 UPfuliF (SEQ !D NO: 67) and pENO2R primers. This product is submitted for sequencing using the AFT1 UPsequence1 (SEQ ID NO: 68) and AFT1 UPsequence2 (SEQ ID NO: 69) primers to ensure that the proper mutation is in the genome.

[00371] For AFT2: Primer AFT2Upforward (SEQ ID NO: 70) is used with primer pENO2R to yield an approximately 350 base pair product that will not be present in the parental strain. Primer AFT2UP forward is used with primer AFT2termR (SEQ ID NO: 71 ) to ensure that the parental Sc__AFT2 does not remain in the strain. If integrated correctly these primers give an approximately 1819 base pair product, !f the parental Sc_AFT2 remains in the strain the product size is 195 base pairs. Finally, the Sc__ AFT2~1 ^UF gene is amplified using the AFT2UPfullF (SEQ ID NO: 72) and pENO2R primers. This product is submitted for sequencing using the AFT2UPsequence1 (SEQ ID NO: 73) and AFT2UPsequence2 (SEQ ID NO: 74) primers to ensure that the proper mutation is in the genome.

[00372] Preparation of Yeast Ceils: Yeast strains are grown in 50 mL YPD with 0.2 g/L G418 (if carrying the AFT ^UP allele) to mid-log phase (1 -3 OD ₆ oo)- A volume of ceils so that 20 OD ₆ oo of ceils are acquired are spun down at 4°C, 3000 rcf for 5 min. The medium is decanted and the cells are resuspended in 10 mL of cold MilliQ water. The cells are centrifuged a second time at 4°C, 3000 rcf for 5 min. The medium is again decanted and the cells are centrifuged at 4°C, 3000 rcf for 5 min. The remaining medium is removed and the cell pellet is frozen at -80°C.

[00373] DHAD Assays are performed as described in the general methods section. Yeast lysate protein concentration was determined as described in the general methods section.

[00374] Gas Chromatography, Liquid chromatography method 1 and liquid chromatography method 2 are performed as described in the general methods section.

[00375] Shake-Flask Fermentation: Fermentations for the AFT1-1 ^UP and AFT2-1 ^up

115

364561 yl/CO transformant strains are performed. Starter cultures with each transformed strain are inoculated into 3 mL YPD with 0.2 g/L G418 and 1 % v/v EtOH and incubated shaking at 250 rpm at 30°C. Pre-cuitures for the fermentations are inoculated to 0.05 ODeoo into 50 mL YPD (8% w/v glucose) with 200 mM MES, 0.2 g/L G418, 1 % v/v stock solution of 3 g/L ergostero! and 132 g/L Tween 80 dissolved in ethanoi, and 20μΜ CuS0 ₄ at pH 6.5 in 250 mL baffled flasks, shaking at 250 rpm at 30°C. Fermentation cultures are inoculated to 5.0 ODeoo into 50 mL YPD (8% w/v glucose) with 200 mM MES, 0.2 g/L G418, 1 % v/v stock solution of 3 g/L ergosterol and 132 g/L Tween 80 dissolved in ethanoi, and 20μΜ CuS0 ₄ at pH 6.5 in 250 mL unbaffied flasks, shaking at 75 rpm at 30°C. All cultures are done in biological triplicate. Samples are collected at 24, 48 and 72 h and analyzed using the liquid chromatography, method 1 , and gas chromatography protocols.

[00376] Results for DHAD activity: Data is presented as specific DHAD activity (U/mg total cell lysate protein) averages from biological and technical triplicates with standard deviations. DHAD activity in GEV02843 transformed with pGV2227 is generally expected to be lower than that of GEVO2843 + pGV2227 transformed with either the Sc__AFT1-1 ^up or Sc__AFT2-1 ^up allele.

[00377] Results for Isobutanol Fermentation : Data is presented as specific isobutanol titer (g/L/O _D 6oo); averages from biological and technical triplicates with standard deviations. Isobutanol titers in GEVO2843 transformed with pGV2227 is generally expected to be lower than that of GEVO2843 + pGV2227 transformed with either the Sc__AFT1-1 ^UP or Sc_AFT2-1 ^up allele.

Example 7: Overexpression of AFT1 in S. cerevisiae Carrying an Isobutanol Producing Metabolic Pathway Increases AFT Regulon Genes as Measured by mRNA

[00378] The purpose of this example is to demonstrate that overexpression of AFT1 in strains expressing an isobutanol producing metabolic pathway increases the expression of genes in the AFT regulon in fermentation vessels. This in turn increases DHAD activity and isobutanol titer, productivity, and yield.

[00379] Media: Medium used was standard yeast medium (for example Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3rd ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press and Guthrie, C. and Fink, G.R. eds. Methods in Enzymoiogy Part B: Guide to Yeast Genetics and Molecular and Cell Biology 350:3-623 (2002)). YP medium contains 1 % (w/v) yeast extract, 2% (w/v) peptone. YPD is YP containing 2% (w/v) glucose. Medium used for the

116

364561 yl/CO fermentation was YP with 80 g/L glucose, 0.2 g/L G418, 0.1 g/L hygrornycin, 100μΜ CuS0 ₄ .5H ₂ 0 and 1 % v/v ethanol. The medium was filter sterilized using a 1 L bottle top Corning PES 0.22μηι filter (431 174). Medium was pH adjusted to 6.0 in the fermenter vessels using 6N KOH.

[00380] Fermentation vessel preparation and operating conditions: Batch fermentations were conducted using six 2 L top drive motor DasGip vessels with a working volume of 0.9 L per vessel. Vessels were sterilized, along with the appropriate dissolved oxygen probes and pH probes, for 80 min at 121 °C. pH probes were calibrated prior to sterilization, however, dissolved oxygen probes were calibrated post sterilization in order to allow for polarization.

[00381] Process co trol para meters : Initial volume, 900 mL. Temperature, 30°C. pH 8.0, pH was controlled using 6N KOH and 2N H ₂ SO ₄ (Table 20).

Table 18. Process Control Parameters.

Oxygen transfer rate increased from 0.5 mM/h to 1 .8 mM/h by increase in agitation from 300 rpm to 400 rpm 58 h post inoculation.

[00382] Fermentation: The fermentation was run for 1 19 h. Vessels were sampled 3 times daily. Sterile 5 mL syringes were used to collect 3 mL of fermenter culture via a sterile sample port. The sample was placed in a 2 mL microfuge tube and a portion was used to measure cell density (OD ₆ oo) on a Genesys 10 spectrophotometer (Thermo Scientific). An additional 2 mL portion was taken in the same manner as described above, for use in qRT-PCR analysis. This sample was spun in a microcentrifuge for 1 min at 14,000 rpm.

[00383] Yeast Transformations: Co-transformations with the CEN and 2μ piasmids are described below. Briefly, the S. cerevisiae strain GEV02843 (Table 5) was

117

364561 vl/CO grown on YPD medium. The strain was re-suspended in 100 mM lithium acetate. Once the cells were re-suspended, a mixture of DNA (final volume of 15 pL with sterile water), 72 pL 50% w/v PEG, 10 pL 1 lithium acetate, and 3 pL of denatured salmon sperm DNA (10 mg/mL) was prepared for each transformation. In a 1 .5 mL tube, 15 pL of the cell suspension was added to the DNA mixture (100 pL), and the transformation suspension was vortexed for 5 short pulses. The transformation was incubated for 30 min at 30°C, followed by incubation for 22 min at 42°C. The ceils were collected by centrifugation (18,000 rcf, 10 sec, 25°C). The cells were resuspended in 1 mL YPD and after an overnight recovery shaking at 30°C and 250 rpm, the cells were spread over YPD supplemented with 0.2 g/L G418 and 0.1 g/L hygromycin selective plates. Transformants were then single colony purified onto G418 and hygromycin selective plates.

[00384] RNA preparation: RNA was isolated using the YeaS ar RNAKit™ (Zymo Research Corp. Orange, CA). Cells were resuspended in 80 p! of YR Digestion Buffer, 1 pi RNAsin (Promega, Madison, Wl) and 5 pi of Zymoiyase™ (provided with YeaStar RNAKit). The pellet was completely resuspended by repeated pipetting. The suspension was incubated at 37°C for 80 min. Following the incubation, 160 pi of YR Lysis Buffer was added to the suspension, which was then mixed thoroughly by vortexing. The mixture was centrifuged at 7,000 g for 2 min in a microcentrifuge, and the supernatant was transferred to a Zymo-Spin Column in a collection tube. The column was centrifuged at 10,000 g for 1 min in a microcentrifuge. To the column, 200 pi RNA Wash Buffer was added, and the column was centrifuged for 1 min at full speed in a microcentrifuge. The flow-through was discarded and 200 pi RNA Wash Buffer was added to the column. The column was centrifuged for 1 min at 14,000g in a microcentrifuge. The Zymo-Spin Column was transferred to a new RNase-free 1 .5 mL centrifuge tube, and 80 pi of DNase/RNase-free water was added directly to the column membrane and let stand for 1 min at room temperature. The RNA was eluted by centrifugation for 1 min at full speed in the microcentrifuge. Concentrations were determined by measuring the OD ₂ 6o with the NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA 02454). RNA was stored at - 80°C until use.

[00385] qRT-PCR analysis: RNA prepared from the fermentation samples (at a dilution of 5 ng/pi) was used as a template for one-step quantitative RT-PCR using the qScript One-Step SYBR Green qRT-PCR kit (Quanta Biosciences™ Gaithersburg, MD). Each PCR reaction contained 10 ng of RNA, 0.5 pL of 10 pM

118

364561 yl/CO forward primer, 0,5 pL of 10 μΜ reverse primer, 8.1 pL of sterile water, and 10 μί of the One-Step SYBR Green Master Mix, 0.5 pL RNAsin, and 0.4 pL of qScript One- Step Reverse Transcriptase. qRT-PCR was done in tripiicate for each sampie. For the purpose of normalizing the experimental samples, qRT-PCR was also done for the TFC1 housekeeping gene. Primers used to target the AFT regulon genes and for the TFC1 gene are presented in Table 19. The reactions were incubated in an Eppendorf Mastercycler ep thermocycler (Eppendorf, Hamburg, Germany) using the following conditions: 50°C for 10 min, 95°C for 5 min, 40 cycles of 95°0 for 15 sec and 80°C for 45 sec (amplification), then 95°C for 15 sec, 60°C for 15 sec, and a 20 min slow ramping up of the temperature until it reaches 95°C (melting curve analysis). The fluorescence emitted by the SYBR dye was measured at the 80°C incubation step during each of the 40 cycles, as well as during the ramping up to 95°C for melting curve analysis of the PCR product.

Table 19. Primers used for qRT-PCR analysis to target the AFT regulon.

[00386] Standard molecular biology methods for cloning and plasmid construction were generally used, unless otherwise noted (Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual, 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

[00387] Cloning techniques included gel purification of DNA fragments (using the Zymoclean Gel DNA Recovery Kit, Cat# D4002, Zymo Research Corp, Orange, CA).

[00388] GEVO2843 (Table 5) was co-transformed with two plasmids. GEVO3342 (Table 8) has plasmids pGV2227 (Table 8) and pGV2198 (empty vector, Table 6); GEV03343 (Table 8) has plasmids pGV2227 (Table 8) and pGV2472 (Table 6 -

119

364561 vl/CO contains Sc__AFT1).

[00389] In Table 20, the fold change data was normalized to the strain without Sc_AFT1 overexpression at 24 h. Thus, all data points for the strain without Sc_AFT1 overexpression at 24 h have been set to one. The overexpression of Sc__AFT1 in S. cerevisiae strains increased predicted Sc__AFT1 target genes, ENB1 (SEG ID NO: 123) and FET3 (SEQ ID NO: 91 ). SIV1F3 (SEQ ID NO: 159) is predicted to be more dependent on Sc AFT2 for expression and SMF3 had a much weaker response to the overexpression of Sc__AFT1 _s as can be seen in Table 20.

Table 20. Fold change in mRNA expression between strains with and without Sc_AFT1 overexpressed.

[00390] Overexpression of Sc__AFT1 increased gene expression of targeted genes in the AFT regulon. As shown in Example 1 , the increased expression of Sc__AFT1 in these strains also caused increased isobutanoi titers, production rates and yields and DHAD activity in fermentations. Thus, it is likely that one or more genes in the AFT regulon impacts DHAD activity and isobutanoi production.

Example 8: Overexpression of Specific Genes in the AFT1 and AFT2 Regulons

[00391] The purpose of this example is to demonstrate that a specific gene or genes from the AFT1 or AFT2 regulon are important for an increase in DHAD activity and isobutanoi production.

[00392] Standard molecular biology methods for cloning and p!asmid construction are generally used, unless otherwise noted (Sambrook, J., Russei, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

[00393] Media: Medium used is described in the general methods section. Cloning techniques include gel purification of DNA fragments (using the Zymoclean Gel DNA Recovery Kit, Cat# D4002, Zymo Research Corp, Orange, CA).

120

364561 vl/CO [00394] AFT1 and AFT2 regulon genes presented in Table 21 are synthesized by DNA 2.0 (Menlo Park, CA, USA) removing any Hpa\ or Sad restriction sites within the genes. The synthesized AFT regulon genes are cloned behind the PGK1 promoter in pGV2196 (empty vector - Table 8) creating a series of 50 plasmids that are co-transformed with pGV2227 (Table 8) into S. cerevisiae strain GEV02843 (Table 5). Isobutanoi production from strain GEV02843 containing pGV2227 has been shown to be limited by DHAD activity. Thus, this provides a suitable background for detecting increases in DHAD activity and subsequent increases in the production of a metabolite from a DHAD-requiring biosynthetic pathway, such as an isobutanoi producing metabolic pathway.

Table 21. Genes in the AFT1 and AFT2 Regulon For Screening DHAD Activity

121

364561 vl/CO LAP4/APE 1 YSC1/API SEQ ID NO: 145 SEQ ID NO: 146

ECU SEQ ID NO: 147 SEQ ID NO: 148

OSW1 SEQ ID NO: 149 SEQ ID NO: 150

NFT1 SEQ ID NO: 151 SEQ ID NO: 152

YBR012C SEQ ID NO: 153 SEQ ID NO: 154

YQL083W SEQ ID NO: 155 SEQ ID NO: 156

ARA2 SEQ ID NO: 157 SEQ ID NO: 158

SMF3 SEQ ID NO: 159 SEQ ID NO: 160

MRS4 SEQ ID NO: 161 SEQ ID NO: 162

ISU1/NUA1 SEQ ID NO: 163 SEQ ID NO: 164

FET4 SEQ ID NO: 165 SEQ ID NO: 166

FET5 SEQ ID NO: 167 SEQ ID NO: 168

FTH1 SEQ ID NO: 169 SEQ ID NO: 170

CCC2 SEQ ID NO: 171 SEQ ID NO: 172

FRE4 SEQ ID NO: 173 SEQ ID NO: 174

ISU2 SEQ ID NO: 175 SEQ ID NO: 176

HMX1 SEQ ID NO: 177 SEQ ID NO: 178

PCL5 SEQ ID NO: 179 SEQ D NO: 180

ICY2 SEQ ID NO: 181 SEQ ID NO: 182

PRY1 SEQ ID NO: 183 SEQ ID NO: 184

YDL124W SEQ ID NO: 185 SEQ ID NO: 186

[00395] Yeast Transformations are performed as described in the general methods section.

[00396] Preparation of Yeast Cells for Enzyme Assays: Yeast strains are grown in 50 mL YPD with 0.2 g/L G418 and 0.1 g/L hygromycin to mid-log phase (1 -3 OD ₆ oo). A volume of cells so that 20 OD ₆ oo of cells are acquired are spun down at 4°C, 3000 rcf for 5 min. The medium is decanted and the cells are resuspend in 10 mL of cold MiiliQ water. The ceils are centrifuged a second time at 4°C, 3000 rcf for 5 min. The medium is again decanted and the cells are centrifuged at 4°C, 3000 rcf for 5 min. The remaining media is removed and the cell pellet is frozen at -8G°C.

[00397] Prepa ration of Yea st Lysate for E nzyme Assays : Cell pellets are thawed on ice. Y-PER Plus reagent (Thermo Scientific #78999) is added to each pellet at a ratio of 12.5 pL of reagent per one OD of ceils and the cells resuspended by vortexing. The suspension is gently agitated for 20 min at room temperature. After 20 min, a volume equal to the Y-PER Plus volume of universal lysis buffer (0.1 M Sodium Phosphate, pH 7.0, 5 mM MgCIa, 1 mM DTT) is added. The suspension is shaken for another 40 min. Samples are centrifuged at 5300 g for 10 min at room temperature. The clarified iysates are transferred to a fresh tube and kept on ice until assayed.

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364561 vl/CO [00398] DHAD Assays are performed as described in the genera! methods section, [00399] Yeast lysate protein concentration was determined as described in the genera! methods section,

[00400] Gas Chromatography, liquid chromatography method 1 and liquid chromatography method 2 are performed as described in the genera! methods section.

[00401] Shake-Flask Fermentation: Fermentations with the AFT regu!on gene transformant strains are performed. Starter cultures with each transformed strain are inoculated into 3 mL YPD supplemented with 0.2 g/L G418 and 1 % v/v EtOH and incubated shaking at 250 rpm at 30°C, Pre-cultures for the fermentations are inoculated to 0.05 OD ₆₀ o into 50 mL YPD (8% w/v glucose) with 200 mM MES, 0.2 g/L G418, 1 % v/v stock solution of 3 g/L ergosterol and 132 g/L Tween 80 dissolved in ethanoi, and 20μΜ CuS0 ₄ at pH 6.5 in 250 mL baffled flasks, shaking at 250 rpm at 30°C, Fermentation cultures are inoculated to 5.0 OD ₆ oo into 50 mL YPD (8% w/v glucose) with 200 mM MES, 0.2 g/L G418, 1 % v/v stock solution of 3 g/L ergosterol and 132 g/L Tween 80 dissolved in ethanoi, and 20μΜ CuS0 ₄ at pH 8,5 in 250 mL unbaffled flasks, shaking at 75 rpm at 30 ^C C. Ail cultures are done in biological triplicate. Samples are collected at 24, 48 and 72 h and analyzed using the liquid chromatography, method 1 , and gas chromatography protocols.

[00402] Results for DHAD activity: Data is presented as specific DHAD activity (U/mg total cell lysate protein) averages from biological and technical triplicates with standard deviations. DHAD activity in GEV02843 transformed with pGV2227 + pGV2198 (empty vector) is generally expected to be lower than that of GEV02843 transformed with either AFT1 or AFT2 genes. In addition, GEV02843 transformed with pGV2227 and clones containing AFT regulon genes that are important for increasing DHAD activity will generally have similar or higher DHAD activity to GEV02843 transformed with pGV2227 and the AFT1 or AFT2 genes,

[00403] Results for Isobutanoi Fermentation: Data is presented as specific isobutanoi titer (g/L/ODeoo); averages from biological and technical triplicates with standard deviations. Isobutanoi titers in GEV02843 transformed with pGV2227 + pGV2196 (empty vector) are generally expected to be lower than that of GEV02843 transformed with either AFT1 or AFT2 genes. In addition, GEV02843 transformed with pGV2227 and clones containing AFT regulon genes that are important for increasing DHAD activity will generally have similar or higher isobutanoi titers to GEV02843 transformed with pGV2227 and AFT1 or AFT2.

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364561 yl/CO Example 9: Overexpression of the Kiuyveromyces lactis AFT Increases DHAD Activity in K, !actis

[00404] The purpose of this example is to demonstrate that overexpression of AFT from K, lactis increases DHAD activity in K, lactis,

[00405] Standard molecular biology methods for cloning and plasmid construction were generally used, unless otherwise noted (Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

[00406] Cloning techniques included gel purification of DNA fragments (using the Zymoclean Gel DNA Recovery Kit, Cat# D4002, Zymo Research Corp, Orange, CA).

[00407] Strains and p!asmids used in Example 9 are described in Tables 22 and 23, respectively.

Table 22. Genotype of strains disclosed in Example 9.

[00408] K. lactis strains: K. lactis strain GEV01287 was transformed with pGV2273 to form GEV04378. KL AFT was PGR amplified from template DNA from strain GEV04378 using primers 0GV3432 (SEQ ID NO: 189) (contains Kpn\) and OGV3433 (SEQ ID NO: 190) (contains ΑνήΙ). Plasmid pGV2796 and the KL AFT PGR product were cut with Kpn\ and .Α τΙΙ and ligated together to form plasmid pGV2962. The linear fragment containing K/__ \F7:G418 was obtained by the

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364561 yl/CO restriction digest of pGV2962 with restriction enzymes, Sail, BglU and Pfo\. The linear KI__AFT:G418 (SEQ ID NO: 201 ) fragment was randomly integrated by transformation into GEV04378 to make GEV08169,

[00409] Yeast transformations - K. lactis K. lactis strain GEV01287 or GEV04378 was inoculated into a 3 mL YPD culture and incubated overnight at 250 rpm and 30 ^C C. A 50 mL YPD culture in a baffled 250 mL shake flask was inoculated and shaken at 30°C until the K. lactis strain GEV01287 reached an OD ₆ oo of 0,83 and K. lactis strain GEV04378 reached an GD ₆ oo of 0.79. Cells were made chemically competent by the following procedure. Ceils were collected by centrifugation at 2700 rcf for 2 min. To wash, cells were re-suspended with 50 mL of sterile miiliQ wafer and again centrifuged at 2700 rcf for 2 min. The wash was repeated by re- suspending cells with 25 mL sterile milliQ water, cells were collected by centrifugation at 2700 rcf for 2 min. Finally the cells were resuspend with 1 mL 100 mM lithium acetate (LiOAc) and transferred to sterile 1 .5 mL microcentrifuge tube. Cells were then collected by centrifugation in microfuge (set to max speed) for 10 sec. The supernatant was removed and the ceils were re-suspended with 4 times the pellet volume of 100 mM LiOAc. Once the cells were prepared, a mixture of DNA (approximately 1 ug for linear DNA fragment and about 500ng of plasmid DNA, wasbrought to 15 pL with sterile water), 72 pL 50% w/v PEG, 10 pL 1 M lithium acetate, and 3 pL of denatured salmon sperm DNA (10 mg/mL) was prepared for each transformation. In a 1 .5 mL tube, 15 pL of the cell suspension was added to the DNA mixture (100 pL), and the transformation suspension was vortexed for 5 short pulses. The transformation was incubated for 30 min at 30°C, followed by incubation for 22 min at 42°C. The cells were collected by centrifugation (18,000 rcf, 10 sec, 25°C). The ceils were resuspended in 1 mL YPD and, after an overnight recovery shaking at 30°C and 250 rpm, 200 pL of the GEVO1287 transformation wasspread over YPD supplemented with 0.1 g/L hygromycin. 200 pL of the GEVO4378 transformation was spread over YPD supplemented with 0.1 g/L hygromycin and 0.2 g/L G418. Transformants were selected at 30°C. Transformants were then single colony purified onto either hygromycin and G418 or hygromycin selective plates.

[00410] Preparation of Yeast Lvsafe: K. lactis strains GEVO4378 and GEVO6169 were inoculated into 3 mL of YPD with 0.1 g/L hygromycin and incubated at 30°C at 250 rpm overnight culture. After approximately 18 h a 50 mL YPD or YPD + 0.1 g/L hygromycin culture in a baffled 250 mL shake flask was inoculated and shaken at 250 rpm until the culture reached approximately 2-3 ODeoo- 20 OD ₆ oo of cells were

125

364561 yl/CO harvested in 15 mL Falcon tubes and centrifuged at 4°C, 3000 rcf for 5 min, The medium was decanted and the cells were re-suspended in 2 mL of ice-cold Mil!iQ water. The cells were cent ifuged a second time at 4°C, 3000 rcf for 5 min. The supernatant was again decanted, and the cells were centrifuged at 4°C, 3000 rcf for 5 min. The remaining medium was removed. The cell pellet was frozen at -80°C. The ceil pellets were thawed on ice and 750 pL of lysis buffer (0.1 M Sodium Phosphate, pH 7.0, 5 mM MgCb, 1 mM DTT) was used to re-suspend each pellet. 800 pL of re-suspended ceil pellet was added to a 1 .5 mL centrifuge tube with 1 mL of 0.5 mm glass beads. The tubes containing the glass beads and ceil suspension were put into the two bead beater blocks chilled to -20°C. The Retsch MM301 bead beater was set to 1 min and 300 1/sec frequency. To iyse the cells, the ceil suspensions were beat 6 times for 1 min each, with 2 min of cooling the tubes and the bead beater blocks on ice in between beatings. After bead beating, the tubes were centrifuged at 4°C at 21 ,500g for 10 min in a tabletop centrifuge. The supernatant was transferred into 1 .5 mL tubes and placed on ice for use in the DHAD assay. Yeast lysate protein concentration was determined as described under General Methods.

[00411] DHAD Assay: The assay was performed in triplicate for each sample, !n addition, a no lysate control with lysis buffer was included. To assay each sample, 10 pL of a 1 :10 dilution of lysate in lysis buffer (0.1 M Sodium Phosphate, pH 7.0, 5 mM MgC , 1 mM DTT) was mixed with 90 pL of assay buffer (5 pL of 0.1 M MgS0 ₄ , 10 pL of 0.1 M DHIV, and 75 pL 50 mM Tris pH 7.5), and incubated in a thermocycler for 30 min at 30°C, then at 95°C for 5 min. Insoluble material was removed from the samples by centrifugation at 3000 rcf for 5 min. The supernatants are transferred to fresh PGR tubes and submitted to analytics for analysis by liquid chromatography, method 2.

[00412] Liquid Chromatography, Method 2: DNPH reagent (4:1 of 15 mM 2,4 - Dinitrophenyl Hydrazine:! 00 mM Citric Acid pH 3.0) was added to each sample in a 1 :1 ratio. Samples were incubated for 30 min at 70°C in a thermo-cycler (Eppendorf, Mastercycler). Analysis of keto-isovaierate was performed on an Agilent 1200 High Performance Liquid Chromatography system equipped with an Eclipse XDB C-18 reverse phase column (Agilent) and a C-18 reverse phase column guard (Phenomenex). Ketoisovalerate were detected using an Agilent 1 100 UV detector (380 nm). The column temperature was 50°C. This method was isocratic with 70% acetonitrile 2.5% phosphoric acid (0.4%), 27.5% water as mobile phase. Flow was

126

364561 yl/CO set to 3 mL/rnin. Injection size was 10 pL and run time was 2 min.

[00413] DHAD Assay Results: The in vitro DHAD enzymatic activity of lysates from the microaerobic fermentation of K. lactis strains was determined as described above. All values are the specific DHAD activity (U/mg total cell iysate protein) as averages from technical triplicates. In K. lactis, overexpression of the KI_AFT gene resulted in an increase in DHAD activity (U/mg total cell Iysate protein). GEV04378 without Ki AFT overexpression had an activity of 0.053 ± 0.009 U/mg while GEV06169, overexpressing Ki_AFT had a specific activity of 0.131 ± 0.012 U/mg.

Example 10: Overexpression of the Kluyverornyces marxianus AFT

[00414] The purpose of this example is to demonstrate that overexpression of K. marxianus AFT {Km_AFT) is generally expected to increase DHAD activity in K, marxianus.

[00415] Standard molecular biology methods for cloning and plasmid construction are generally used, unless otherwise noted (Sambrook, J., Russei, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press). Cloning techniques include gel purification of DNA fragments (using the Zymociean Gel DNA Recovery Kit, Cat# D4002, Zymo Research Corp, Orange, CA).

[00416] Strains used in Example 10 are described in Table 24.

Table 24. Genotype of strains disclosed in Example 10.

[00417] In this example, the K. marxianus URA3 gene was deleted by transformation of GEVO1088 with a PGR fragment (SEQ ID NO: 191 ) of K. marxianus URA3 carrying a deletion of 348 base pairs that was amplified from pGV1799 (SEQ ID NO: 192) using primers 0GV394 (SEQ ID NO: 193) and 0GV395 (SEQ ID NO: 194). The K. marxianus ura3 deletion strain transformants were selected by plating on 5-FOA (5-fluoroorotic acid) plates (For 500 mL: 10 g agar, 400 rnL dH ₂ O, 0.5 g 5-FOA (in 5 mL DMSO), 50 mL 10Xa.a (14g yeast synthetic drop-out supplement (US Biological) dissolved in 1 L water), 3.35 g YNB, 10 g glucose, 10 mL

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364561 vl/CO 50X HIS (0,95g histidine/250 rnL H ₂ 0), 10 rnL 50X TRP (1 .9 g in 500 mL H ₂ G), 10 mL 10X LEU (4.75 g Leucine/250 mL H ₂ 0), 3.15 mL 25X URA(0.47S g uracil/250 mL H ₂ 0). The 5-FOA resistant colonies were confirmed for the correct phenotype (auxotrophic for uracil). PGR demonstrated a partial deletion of approximately 200 bp in the ura3 gene and this strain was named GEV01947.

[00418] A linear DNA fragment containing Km__AFT _s LI lvD, and a G418 resistance marker (SEQ !D NO: 195, Figure 6) is synthesized by DNA2.0. The fragment is randomly integrated by transformation into K. marxianus strain GEV01947 to obtain GEV08222. A linear fragment containing LI jlvD and a G418 marker is also synthesized by DNA2.0 (SEQ ID NO: 196, Figure 7) and is randomly integrated by transforming K. marxianus strain GEV01947 to obtain GEVO8223.

[00419] Transformations are carried out as follows: K, marxianus strain GEV01947 is incubated in 50 mL of YPD medium (1 % (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose) shaking at 250 RPM at 30°C until the culture is at an ODeoo of approximately 5. The ceils are collected in a sterile 50 mL conical tube by centrifugation (1800 rcf, 5 min at room temperature). The cells are then resuspended in 10 mL of eiectroporation buffer (10 mM Tris-HCI, 270 mM sucrose, 1 mM MgCI ₂ , pH 7.5), and collected at 1600 rcf for 5 min at room temperature. The ceils are then resuspended in 10 mL IB (YPD medium, 25 mM DTT, 20 mM HEPES, pH 8.0; prepared fresh by diluting 100 pL of 2.5M DTT and 200 pL of 1 M HEPES, pH 8.0 into 10 mL of YPD) and are incubated for 30 min, 250 RPM, 30°C (tube standing vertical). The cells are collected at 1600 rcf for 5 min at room temperature and resuspended in 10 mL of chilled eiectroporation buffer. The cells are then pelleted at 1600 rcf for 5 min at 4°C. The ceils are then resuspended in 1 mL of chilled eiectroporation buffer and transferred to a microfuge tube. The cells are collected by centrifugation at >10,000 rcf for 20 sec at 4 ^C C. The cells are then resuspended in an appropriate amount of chilled eiectroporation buffer for a final biomass concentration of 30 ODeoo/mL. 400 pL of cell suspension is added to a chilled eiectroporation cuvette (0.4cm gap) and 50 pL of DNA (SEQ ID NO: 195 or SEQ ID NO: 196 or water control) is added and mixed by pipetting up and down, and the cuvette is incubated on ice for 15-30 min. The samples are then electroporafed at 1 .8 kV, 1000 Ohm, 25 pF. The samples are transferred to a 50 mL tube with 1 mL YPD medium, and the samples are incubated for 4 h at 250 rpm at 30°C. 200 pL of each transformation culture are spread onto YPD plates containing 0.2 g/L G418 and the plates are incubated at 30°C until individual colonies develop.

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364561 yl/CO [00420] K. marxianus strain GEV06222 is verified by colony PGR for the integration of Km__AFT using primers PGK1 F (SEQ ID NO: 197) and KmAFTR (SEQ ID NO: 198) (yielding an approximately 325 base pair product) and integration of LIJlvD using primers OGV2107 (SEQ ID NO: 199) and OGV2108 (SEQ ID NO: 200) (yielding an approximately 104 base pair product), K. marxianus strain GEVO8223 is verified by colony PGR for the integration of LIJlvD using primers oGV2107 and OGV2108.

[00421] Next, K. marxianus strains GEVO1947, GEVO8222 and GEVO8223 are inoculated into 3 mL of YPD medium (1 % (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose) and incubated at 30°C at 250 rpm. After approximately 18 h, a 50 mL YPD culture in a baffled 250 mL shake flask is inoculated and shaken at 250 rpm until the culture reaches approximately 2-3 OD ₆ oc- Cell pellets are prepared by taking 20 OD units of culture [OD ₆ oonm ^x volume (mL) = 20] and centrifuging the appropriate volume at 3000 rpm and 4°C for 5 min, The medium is decanted and the cells are resuspended in 2 mL of ice-cold MilliQ water. The ceils are centrifuged a second time at 4°C, 3000 rcf for 5 min. The supernatant is again decanted, and the ceils are centrifuged at 4°C, 3000 rcf for 5 min. The remaining medium is removed. The ceil pellet is frozen at -80°C. To prepare lysate, the cell pellets are thawed on ice and 750 μί of lysis buffer (0.1 M Sodium Phosphate, pH 7.0, 5 mM MgCI ₂ , 1 mM DTT) is used to re-suspend each pellet. 800 pLof re-suspended cell pellet is added to a 1 .5 mL centrifuge tube with 1 mL of 0.5 mm glass beads. The tubes containing the glass beads and cell suspension are put into the two bead beater blocks chilled to -20°C. A Retsch M 301 bead beater is set to 1 min and 300 1/sec frequency. To !yse the ceils, the cell suspensions are beat 6 times for 1 min each, with 2 min of cooling the tubes and the bead beater blocks on ice in between beatings. After bead beating, the tubes are centrifuged at 4°C at 21 ,500g for 10 min in a tabietop centrifuge. The supernatant is transferred into 1 .5 mL tubes and placed on ice for use in the DHAD activity assay, Yeast lysate protein concentration is determined as described under General Methods.

[00422] DHAD assays are performed as described in the general methods sectionLiquid chromatography method 2 is performed as described in the general methods section,

[00423] Results for DHAD activity: Data is presented as specific DHAD activity (U/mg total cell lysate protein) averages from biological and technical triplicates with standard deviations. DHAD activity in GEVO8223, containing DHAD is generally

129

364561 yl/CO expected to be lower than that of GEV06222 containing both Km_AFT and DHAD.

Example 1 1 : Construction of Issatchenkia orienta!is Strain with Isobutanol Pathway Genes Integrated into the Genome

[00424] The purpose of this example is to demonstrate that overexpression of Issatchenkia orientalis AFT1-2 (herein referred to as lo__AFT1~2) increases DHAD activity in /. orientalis,

[00425] An /. orientalis strain derived from PTA-6658 (US 2009/0226989) was grown overnight and transformed using the lithium acetate method as described in Gietz, ef a/ (1992, Nucleic Acids Research 20: 1524). The strain was transformed with homologous integration constructs using native /. orientalis promoters to drive protein expression, issatchenkia orientalis strains used are described in Table 25.

[00426] Three strains were used to demonstrate that the overexpression of /. orientalis AFT 1-2 increases DHAD activity in /. orientalis, GEV06155 does not contain the heterologous AFT1-2 expression construct, while both GEV06162 and GEVO8203 have the heterologous AFT1-2 construct integrated into the genome. All three strains were cultured in two different conditions and then tested for DHAD activity.

[00427] In the first condition, cultures were started for each strain (GEV08155,

130

364561 vl/CO GEV06162, and GEVO8203) in 12 mL YP medium (1 % (w/v) yeast extract, 2% (w/V) peptone) containing 5% (w/v) glucose and incubated at 30°C and 250 RPM for 9 h. The OD ₆ oo of the 12 mL cultures was determined and the appropriate volume of each culture was used to inoculate 50 mL of YP medium containing 8% glucose in separate 250 mL baffled flasks to an OD ₆ oo of 0.01 . The flasks were incubated at 30 ^C C and 250 RPM for 18 h. A total of 80 ODeoo of ceils were harvested and the ceil suspension was transferred to 50 mL Falcon tubes. Ceils were pelleted at 3000 rcf for 5 min at 4 ^C C, and washed twice in 2 mL cold, sterile water. The ceil pellets were stored at -80°C until analysis by DHAD assay.

[00428] In the second condition, cultures were inoculated at a starting ODeoo of 0.1 and were incubated at 30°C with 250 rpm shaker speed for 20 h and then the shaker speed was reduced to 75 rpm for an additional 28 h prior to sampling. Ceils were washed twice with cold sterile water and stored at ~SG°C until analysis.

[00429] To determine DHAD activity in whole cell lysates, the frozen cell pellets were thawed on ice and resuspended in 750 pL lysis buffer (100 mM NaP0 ₄ pH 7.0, 5 mM gC and 1 mM DTT). One mL of glass beads (0.5 mm diameter) were added to a 1 .5 mL microcentrifuge tube and the entire ceil suspension for each strain was added to seperate tubes containing glass beads. Yeast cells were lysed using a Retsch MM301 bead beater (Retsch Inc. Newtown, PA), bead beating six times for 1 min each at full speed with 1 min icing in between each bead beating step. The tubes were centrifuged for 10 min at 23,500 xg at 4°C and the supernatant was removed. Supe natants were held on ice until assayed. Yeast lysate protein concentration was determined as described under General Methods.

[00430] DHAD assays were performed in triplicate for each sample, in addition, an assay on a no lysate control with lysis buffer was performed. To assay each sample, 10 pL of lysate in assay buffer was mixed with 90 pL of assay buffer (5 pL of 0.1 M MgSO ₄ , 10 pL of 0.1 M DHIV, and 75 pL 50 mM Tris pH 7.5), and incubated in a thermocycler (Eppendorf, Mastercycler) for 30 min at 30°C, then at 95°C for 5 min. Insoluble material was removed from the samples by centrifugation at 3000 rcf for 5 min. The supernatants were transferred to fresh PGR tubes. 100 pL DNPH reagent (12 mM 2,4 - dinitrophenyi hydrazine, 10 mM citric acid, pH 3.0, in 80% acefonifrile, 20% Mii!iQ H ₂ 0) was added to 50 pL of each sample and 50 pL of MiiiiQ H ₂ 0. Samples were incubated for 30 min at 70°C in a thermocycler.

[00431] Analysis of keto-isovalerate (KiV) was performed on an Agilent 1200 High Performance Liquid Chromatography system equipped with an Eclipse XDB C~18

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364561 yl/CO reverse phase column (Agilent) and a C-18 reverse phase column guard (Phenomenex). Ketoisovaierate was detected using an Agilent 1 100 UV detector (380 nm). The column temperature was 50°C. This method was isocratic with 70% acetonitrile 2.5% phosphoric acid (0.4%), 27.5% water as mobile phase. Flow was set to 3 mL/min. Injection size was 10 pL and run time was 2 min. KIV was quantified on a 3-point linear calibration curve.

[00432] The in vitro DHAD enzymatic activity of iysates from the samples of /, orientals strains were carried out as described above. DHAD activity (U/mg total ceil lysate protein) is reported as averages from biological triplicate samples. In /. orientalis, overexpression of the /. orientalis AFT1-2 gene resulted in an increase in DHAD activity (U/mg total ceil lysate protein). The cultures harvested at 18 h (samples inoculated at 0.01 ) had DHAD activity values as follows: GEV08155 had an activity of 0.039 ± 0.004 U/mg while GEV06162 had an activity of 0.082 ± 0.005 U/mg and GEVO8203 had an activity of 0.080 ± 0.01 1 U/mg. The cultures harvested at 48 h (cultures inoculated at 0.1 ) had DHAD activity values as follows: GEV08155 had an activity of 0.085 ± 0.014 U/mg while GEV06182 had an activity of 0.155 ± 0.020 U/mg and GEVO6203 had an activity of 0.140 ± 0.033 U/mg. Therefore, this example demonstrates that overexpression of lo AFT1-2 increases DHAD activity in /. orientalis.

Example 12: Overexpression of Fe-S Assembly Machinery

[00433] To ascertain the effects of overexpressing a cytosolic 2Fe-2S or 4Fe-4S cluster-containing DHAD with candidate assembly machinery, the following steps, or equivalent steps can be carried out. First, the coding sequence for the open reading frame of the DHAD from spinach or other 2Fe~2S or 4Fe~4S cluster-containing DHAD is cloned into the high-copy (2micron origin) S.cerevisiae expression vector pGV2074, such that expression of the coding sequence is directed by the PGK1 promoter sequence, yielding piasmid pGV2074-1 . Next, the NifU and NifS genes from Entamoeba histolytica or the homologous NIF genes from Lactococcus lactis are successively introduced into the aforementioned vector, eventually yielding a single piasmid (pGV2074-2) where the expression of ail 3 genes is directed by strong constitutive S.cerevisiae promoter sequences. Plasmids pGV2074-1 and pGV2074-2 are transformed into S. cerevisiae strain GEV02244 (relevant genotype, ilv3A) and transformants selected by resistance to Hygromycin B (0.1 g/L). At least 3 individual colonies arising from each transformation are cultured, a ceil lysate

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364561 vl/CO produced, and the DHAD activity present therein measured, all according to previously-described methods.

Example 13: Overexpression of NFS1 and ISD1 1 Increases DHAD Activity and Isobutanol Production

[00434] The purpose of this example is to demonstrate that overexpression of NFS1 and ISD11 in a recombinant yeast strain expressing an isobutanol producing metabolic pathway increases DHAD activity and isobutanol production.

[00435] In this example, NFS1 and ISD11 were cloned downstream of the TEF1 and TDH3 promoters on a p!asmid, pGV2198 (Figure 8), in single and double combinations (Table 28). These piasmids were transformed into a strain of S. cerevisiae, GEVO6014 (Table 27), which contains an isobutanol producing metabolic pathway comprising a cytosolically localized DHAD (the i!vD gene product of L lactis). GEVO6014 was also transformed with an empty vector (pGV2196 without NFS1 and/or ISD11) as a control.

Table 26. Piasmids Containing Specific PromotenGene Combinations Used for NFS1 and/or ISD1 1 Overexpression.

Table 27. Genotype of GEVO8014.

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[pGV2984] [Figure 9]

[00436] Shake flask fermentations were performed with triplicate transformants for each plasmid and sampled at the switch to production (i.e., the end of the growth culture before harvesting the cells for the production phase) and at 48h of production (the end of the fermentation) for DHAD activities and at 24h and 48h of production for isobutanol determination by gas chromatographic analysis and other metabolites and glucose by liquid chromatography.

[00437] The fermentation results with respect to isobutanol production are summarized in Table 28, while the results of DHAD activity assays are shown in Table 29.

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364561 vl/CO Table 28. Fermentation Results for isobutanol Production from GEVO6014 Containing the Indicated Piasmid.

Table 29. DHAD Aciivity Assay Results from GEVO6014 Containing the Indicated Plasmid.

[00438] Figures 10-15 further illustrate the fermentation results for isobutanol production. Expression of NFS1 and ISD11 together in the combination PTEFI- ISD11 PTDH3 ^' -NFS1 (from plasmid pGV3143) in GEVO6014 increased isobutanol volumetric titer and volumetric productivity by 62% and specific isobutanol titer by 73% over the control strain (GEVO6014 containing the empty vector pGV2198) after 48h of production. The NFS1-ISD11 combination PTEFI ^' - NFS1 P TDH3~-1SD11 (from plasmid pGV3144) in GEVO8014 increased specific isobutanol titer over the control strain by 55% after 48h of production. Expression of NFS1 and ISD 11 together in the combination PTEFI ^■ JSD11 P TDH3-N S1 (from plasmid pGV3143 and pGV3144) in GEVO6014 increased isobutanol yield over the control strain (GEVO6014 containing the empty vector pGV2198) as shown in Table 28. In contrast, NFS1 or ISD11 cloned separately behind either promoter did not provide increases in titer, yield, or productivity over the control strain.

[00439] Figure 16 illustrates the DHAD activity assay results. NFS1 and ISD1 1 together in the combination PTEFI :iSD11 P TDH3-NFS1 (from plasmid pGV3143) in GEVO6014 increased DHAD activity at both the switch to production and 48h of production by 79% and 1 15%, respectively, over the control strain containing the empty vector. The combination PTEFI :NFS1 P TDH3-ISD11 (from plasmid pGV3144) in GEVO6014 gave a 70% increase in DHAD activity at the switch to production but no significant increase in DHAD activity at 48h of production compared to the control strain containing the empty vector pGV2198. In contrast, NFS1 or ISD11 cloned separately behind either promoter did not provide increases in DHAD activity at either sampling time.

[00440] Conclusions from Example 13: Overexpression of NFS1 and ISD11 together increased DHAD activity in the S. cerevisiae strain, GEVO8014. Because Nfs1 and Isd1 1 are mitochondrially-localized proteins in yeast and overexpression of NFS1 and ISD11 demonstrated an increase in DHAD activity in GEVO6014, some of the activity increase may have been due to an increase in activity of the mitochondriaiiy-iocaiized yeast Iiv3 DHAD protein. Accordingly, it appears that overexpression of NFS1 and ISD1 1 in yeast biocataiysts can be used to increase the activity of mitochondrially-localized FeS proteins by increased flux through the FeS cluster biogenesis pathway, especially if overexpression of mitochondrially-targeted FeS proteins resulted in incomplete activation by insertion of FeS clusters into the

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364561 vl/CO apo-versions of those proteins in the absence of NFS1 and ISD11 overexpression. Such FeS proteins would include DHAD used for mitochondrialiy-localized pathways or portions of pathways for isoleucine, valine, leucine, pantothenic acid or isobutanol production, or natively mitochondriaily-iocalized proteins such as aconitase or biotin synthase or the mitochondrial respiratory chain Complex I component, or FeS proteins, including heterologous DHADs from other organisms, not natively localized to the yeast mitochondria but engineered to be targeted for mitochondrial localization.

[00441] Interestingly, although Nfs1 and !sd1 1 are mitochondriaily-iocalized proteins in yeast, overexpression of NFS1 and ISD11 demonstrated an increase in isobutanol production in GEVQ8Q14, a strain of S. cerevisiae which contains an isobutanol producing metabolic pathway comprising a cytosoiically localized DHAD (the HvD gene product of L. lactis). This cytosoiically localized DHAD is believed to be a 2Fe2S-ciuster protein. Because both isobutanol production and DHAD activity increased in a recombinant microorganism overexpressing NFS1 and ISD11, much of this DHAD activity increase is probably due to increased activity of the cytosoiic L. lactis DHAD, indicating that overexpression of NFS1 and ISD11 increases the activity of cytosoiic and/or 2Fe2S-cluster proteins. Accordingly, overexpression of NFS1 and ISD11 in yeast biocataiysts can be used to increase activity of cytosolically-localized FeS proteins, containing either 4Fe4S-ciusfers or 2Fe2S- c!usters, and can also be used to increase the activity of 2Fe2S-cluster proteins other than 2Fe2S-ciuster DHADs. Such FeS proteins would include DHAD used for cytosolically-localized pathways or portions of pathways for isobutanol production and cytosoiic pathways or portions of pathways for leucine biosynthesis, for example. This would include FeS proteins that natively remain in the yeast cytosoi when expressed in yeast biocataiysts, either heterologous FeS proteins or native yeast FeS proteins, and/or FeS proteins not natively localized to the yeast cytosoi but engineered to be targeted for cytosoiic localization when expressed in yeast biocataiysts, such as natively mitochondriaily-iocalized FeS proteins, e.g., the yeast DHAD I!v3, engineered to remain in the yeast cytosoi.

[00442] The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood there from as modifications will be obvious to those skilled in the art.

[00443] While the invention has been described in connection with specific

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364561 yl/CO embodiments thereof, it will be understood that It is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[00444] The disclosures, including the claims, figures and/or drawings, of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entireties.

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