WO2013173412A2

WO2013173412A2 - Engineered yeast for production of renewable chemicals

Info

Publication number: WO2013173412A2
Application number: PCT/US2013/041064
Authority: WO
Inventors: Catherine Asleson Dundon; Andrew C. Hawkins; Lynne Albert; Anne SCHULTZ; Justas Jancauskas; Kevin Roberg-Perez
Original assignee: Gevo, Inc.
Priority date: 2012-05-15
Filing date: 2013-05-15
Publication date: 2013-11-21
Also published as: WO2013173412A3

Abstract

The present application relates to recombinant microorganisms comprising biosynthetic pathways and methods of using said recombinant microorganisms to produce various beneficial, renewable metabolites, e.g., isobutanol. In some embodiments, the recombinant yeast microorganisms may be engineered to comprise an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol. In certain exemplary embodiments, said recombinant yeast microorganisms may additionally or independently be engineered to alter the regulation, expression, and/or activity of a Mac1 pathway component.

Description

ENGINEERED YEAST FOR PRODUCTION OF RE EWABLE CHEMICALS CROSS-RErE C TO LATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 61/647,106, filed May 15, 2012, which is herein incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] 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. ¾L» lr S i%JN Ur I tA I r iLfc: U oM ; I S CU L I UmL>ALL Y

[0003] 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_081_01 WO_SeqList_ST25.txt, date recorded: April 19, 2013, file size: 67 kilobytes).

BACKGROUND

[0004] Existing in nature is a class of proteins known as iron-sulfur (Fe~S) cluster containing proteins. In the metabolic reactions catalyzed by these proteins, Fe-S clusters act as cofactors which are essential for activity. When the Fe-S cluster containing proteins are initially produced by the ceil, they lack the Fe-S clusters required for activity and are known as apoproteins. Fe-S clusters are subsequently made via Fe-S cluster biosynthesis, which is a complex process requiring the activities of several proteins. Once the Fe-S cluster is synthesized, it is transferred to the apoprotein to form the functional Fe-S cluster containing nonprotein. A review on Fe-S cluster biosynthesis is provided by Lili and Muhienhoff, 2005, Trends in Bloc. Sci. 30(3): 133-41 .

[0005] One example of a Fe-S cluster containing protein is dihydroxyacid dehydratase (DHAD). DHAD is an enzyme that catalyzes the conversion of 2,3- dihydroxyisovalerate to a-ketoisovalerate and of 2,3-dihydroxy-3-methy!va!erate to 2- keto-3-methylvalerate. 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-ketoisovalerate as part of isobutanoi biosynthetic pathways disclosed in commonly owned US Patent Nos. 8,017,375, 8,017,378, 8,097,440, and 8,133,175. In addition, biosynthetic pathways for the production of 3-methyl-1 - butanoi and 2~methyl-1 ~butanol use DHAD to convert 2,3-dihydroxyisovalerate to a- ketoisovalerate and 2,3-dihydroxy-3-rnethy!va!erate to 2-keto-3-methy!vaierate, respectively. See Atsumi et a!., 2008, Nature 451 (7174): 88-9.

[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 the activity of Fe~S cluster containing proteins, including DHAD.

SUMMARY OF THE INVENTION

[0007] The present inventors have discovered that increasing the expression and/or activity of the transcriptional activator gene MAC1 or homoiogs thereof in a recombinant yeast microorganism improves the activity of Fe-S cluster containing proteins (e.g., DHAD). Thus, the application relates to recombinant yeast cells engineered to provide increased heterologous or native expression and/or activity of the protein encoded by MAC1 {i.e., herein referred to as Mad or Mad p). In general, cells that have an increased expression and/or activity of Mad or homoiogs thereof exhibit an enhanced ability to produce beneficial metabolites such as isobutanoi, 3-methyl-1 -butanol, 2-methyi-l -butanol, valine, isoleucine, leucine, and pantothenic acid.

[0008] One aspect of the application is directed to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins or homoiogs thereof. In one embodiment, the Mad 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: 18, SEQ ID NO: 18, and SEQ ID NO: 20. In another embodiment, one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an exogenous polynucleotide.

[0009] In another aspect, the application is directed to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe~S cluster containing protein, wherein said recombinant yeast microorganism is engineered to express or overexpress one or more polynucleotides encoding one or more constitutiveiy active mutant Mad proteins. In one embodiment, the constitutiveiy active mutant Mad protein or homoiog thereof comprises a mutation at one or more positions corresponding to residues 284 to 279 of the native Saccharomyces cerevisiae Mad protein (SEQ ID NO: 2). In an exemplary embodiment, the constitutiveiy active mutant Mad protein or homoiog thereof comprises a mutation at a position corresponding to the cysteine 271 (i.e., C271 ) residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2). In a specific embodiment, the cysteine 271 residue is replaced with a tyrosine. In another exemplary embodiment, the constitutiveiy active mutant Mad protein or homoiog thereof comprises a mutation at a position corresponding to the histidine 279 (i.e., H279) residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2). In one specific embodiment, the histidine 279 residue is replaced with a glutamine.

[0010] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins or homoiogs thereof regulated by a Mad protein or homoiog thereof. In one embodiment, the proteins regulated by a Mad protein or homoiog thereof are selected from Afg2, Aim25, Axl1 , Cdc20, Cdc80, Coq6, Cn , Ctf19, Ctrl , Ctr3, Ctt1 , Cup1 , Cup9, Das1 , Dbf20, Dpl1 , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun26, Gnd2, Gph1 , His5, Hnm1 , Hos4, Irc7, Isy1 , Kem1 , Kre6, Lsb1 , Lsm5, Mal33, Mdm31 , Mrm2, Mss4, Npc2, Nrd1 , Oac1 , Oms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub Rad50, Reel , Rgm1 , Rpa34, Rpc40, RpM b, Rpi7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr 90, Swd1 , Swh1 , Tgl2, Tma20, Tps2, Trml O, Ubx3, Ubx6, Ume6, Utr1 , Vcx1 , Vhs2, Vik Vip1 , Vic3, Yar009c, Yar069c, Ycr025c, Ydi129w, Ygr137w, Yii1 u2e, Yjl218w, YkiQ63e, Ylh47, YilOS9c, YirG3Sc-a, Ylr123c, Ylr410w-a, Ylr410w-b, Ymr320w, Yn!017c, Yo!079w, Yo!153c, Yor1 1 1w, Yp!251w, Ypr123c, Ypr170c, and Ysp1 , or homologs thereof. In a specific embodiment, Ctrl is targeted for increased expression and/or activity. In another specific embodiment, Fre1 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctrl and Fre1 are targeted for increased expression and/or activity. In yet another specific embodiment, Pho89 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctrl and/or Fre1 and/or Pho89 are targeted for increased expression and/or activity,

[0011] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulate Mad . In one embodiment, the positive regulator of Mad is selected from Ccs1 and Sod1 , or homologs thereof.

[0012] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Mad .

[0013] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a DNA-binding protein activated by copper-replete conditions. In one embodiment, the DNA-binding protein activated by copper-replete conditions is Ace1 , or homologs thereof.

[0014] In each of the aforementioned aspects and embodiments, the Fe-S cluster containing protein may be selected from dihydroxy acid dehydratase (i.e. , DHAD), isopropyimaiate isomerase, sulfite reductase, aconitase, homoaconitase, iipoate synthase, succinate dehydrogenase, NADH ubiquinone oxidoreductase, and ubiquinoi-cytochrome-c reductase. In one embodiment, the Fe-8 duster containing protein is encoded by an endogenous polynucleotide. In another embodiment, the Fe-S cluster containing protein is encoded by an exogenous polynucleotide. In an exemplary embodiment, the Fe-S cluster containing protein is overexpressed.

[0015] In an exemplary embodiment, the Fe-S cluster containing protein is dihydroxy acid dehydratase (i.e., DHAD). Accordingly, an aspect of the application is directed to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins or homoiogs thereof. In one embodiment, the Mad 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: 18, SEQ ID NO: 18, and SEQ ID NO: 20. In another embodiment, one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an exogenous polynucleotide.

[0016] In another aspect, the application is directed to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to express or overexpress one or more polynucleotides encoding one or more constitutively active mutant Mad proteins. In one embodiment, the constitutively active mutant Mad protein or homoiog thereof comprises a mutation at one or more positions corresponding to residues 284 to 279 of the native Saccharomyces cerevisiae Mad protein (SEQ ID NO: 2). In an exemplary embodiment, the constitutively active mutant Mad protein or homoiog thereof comprises a mutation at a position corresponding to the cysteine 271 (i.e., C271 ) residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2). In a specific embodiment, the cysteine 271 residue is replaced with a tyrosine. In another exemplary embodiment, the constitutively active mutant Mad protein or homoiog thereof comprises a mutation at a position corresponding to the histidine 279 (i.e., H279) residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2). In one specific embodiment, the histidine 279 residue is replaced with a giutamine.

[0017] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins or homoiogs thereof regulated by a Mad protein or homolog thereof. In one embodiment, the proteins regulated by a Ma protein or homolog thereof are selected from Afg2, Aim25, Axil , Cdc20, Cdc60, Coq6, Crr1 , Ctf19, Ctrl , Ctr3, Ctt1 , Cup1 , Cup9, Das Dbf20, Dpl1 , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun28, Gnd2, Gph1 , His5, Hnm1 , Hos4, Irc7, !sy1 , Kem1 , Kre8, Lsb1 , Lsm5, Mal33, Mdm31 , Mrm2, Mss4, Npc2, Nrd1 , Oad , Oms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , RadSG, Reel Rgm1 , Rpa34, Rpc40, RpM b, Rpl7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr190, Swd1 , Swh1 , Tg!2, Tma20, Tps2, Trm10, Ubx3, Ubx8, Ume6, Utr1 , Vcx1 , Vhs2, Vik1 , Vip1 , Vtc3, Yar009c, Yar069c, Ycr025c, Ydi129w, Ygr137w, Yil102c, Yjl218w, Yki063c, Yih47, YII059C, Ylr035c~a, Ylr123c, Ylr410w-a, Yir41 Gw-b, Ymr320w, Ynl017c, Yol079w, Yoi153c, Yor1 1 1 w, Ypi251w, Ypr123c, Ypr170c, and Ysp1 , or homoiogs thereof. In a specific embodiment, Ctrl is targeted for increased expression and/or activity. In another specific embodiment, Fre1 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctrl and Fre1 are targeted for increased expression and/or activity. In yet another specific embodiment, Pho89 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctr and/or Fre1 and/or Pho89 are targeted for increased expression and/or activity.

[0018] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulate Mad . In one embodiment, the positive regulator of Mad is selected from Ccs1 and Sod1 , or homoiogs thereof.

[0019] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Ma .

[0020] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a DNA-binding protein activated by copper-replete conditions. In one embodiment, the DNA-binding protein activated by copper-replete conditions is Ace1 , or homologs thereof.

[0021] In one embodiment, the DHAD protein is encoded by an endogenous polynucleotide. In another embodiment, the DHAD protein is encoded by an exogenous polynucleotide. In an exemplary embodiment, the DHAD protein is overexpressed.

[0022] In various embodiments described herein, the DHAD-requiring biosynthetic pathway may be selected from isobutanol, 3-methyl-l -butanoi, 2-methyl- 1 -butanol, valine, isoleucine, leucine, and/or pantothenic acid biosynthetic pathways. In various embodiments described herein, the DHAD enzyme which acts as part of an isobutanol, 3-methyl-l -butanol, 2-methy!-l -butanol, valine, isoleucine, leucine, and/or pantothenic acid biosynthetic pathway may be localized to the cytosoi. In alternative embodiments, the DHAD enzyme which acts as part of an isobutanol, 3- methyl-1 -butanoi, 2-methyi-1 -butanol, valine, isoleucine, 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-l - butanoi, 2-methyi-1 -butanol, valine, isoleucine, leucine, and/or pantothenic acid biosynthetic pathway is localized to the cytosoi and the mitochondria. In yet additional embodiments, the DHAD enzyme which acts as part of an isobutanol, 3- methyl-1 -butanoi, 2-methyi-l -butanol, valine, isoleucine, leucine, and/or pantothenic acid biosynthetic pathway may be overexpressed. In yet additional embodiments, the DHAD enzyme which acts as part of an isobutanol, 3-methyl-l -butanoi, 2-methyi- l -butanol, valine, isoleucine, leucine, and/or pantothenic acid biosynthetic pathway may be encoded by an endogenous polynucleotide (e.g., ILV3). In yet additional embodiments, the DHAD enzyme which acts as part of an isobutanol, 3-methyl-l - butanoi, 2-methyi-l -butanol, valine, isoleucine, leucine, and/or pantothenic acid biosynthetic pathway may be encoded by an exogenous polynucleotide.

[0023] In an exemplary embodiment, the DHAD-requiring biosynthetic pathway is an isobutanol biosynthetic pathway. Accordingly, an aspect of the application is directed to a recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme thai catalyzes a pathway step in the conversion of pyruvate to isobutanoi, and wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins or homoiogs thereof. In one embodiment, the Mad protein is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 18, and SEQ ID NO: 20, In another embodiment, one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an endogenous polynucleotide, !n yet another embodiment, one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an exogenous polynucleotide,

[0024] In another aspect, the application is directed to a recombinant yeast microorganism an isobutanoi producing metabolic pathway, wherein said isobutanoi producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanoi, and wherein said recombinant yeast microorganism is engineered to express or overexpress one or more polynucleotides encoding one or more constitutiveiy active mutant Mad proteins. In one embodiment, the constitutiveiy active mutant Mad protein or homolog thereof comprises a mutation at one or more positions corresponding to residues 264 to 279 of the native Saccharomyces cerevisiae Mad protein (SEQ ID NO: 2). In an exemplary embodiment, the constitutiveiy active mutant Mad protein or homolog thereof comprises a mutation at a position corresponding to the cysteine 271 (i.e., C271 ) residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2). In a specific embodiment, the cysteine 271 residue is replaced with a tyrosine. In another exemplary embodiment, the constitutiveiy active mutant Mad protein or homolog thereof comprises a mutation at a position corresponding to the histidine 279 (i.e., H279) residue of the native 8. cerevisiae Mad protein (SEQ ID NO: 2). In one specific embodiment, the histidine 279 residue is replaced with a gluiamine.

[0025] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising an isobutanoi producing metabolic pathway, wherein said isobutanoi producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanoi, and wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins or homoiogs thereof regulated by a Mad protein or homolog thereof, !n one embodiment, the proteins regulated by a Mad protein or homolog thereof are selected from Afg2, Aim25, Axil , Cdc20, Cdc60, Coq6, Crr1 , Ctf19, Ctrl ., Ctr3, Ctt1 , Cup1 , Cup9, Das1 , Dbf2G, DpM , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun28, Gnd2, Gph His5, Hnm1 , Hos4, !rc7, Isy1 , Kem1 , Kre8, Lsb1 , Lsm5, Mal33, Mdm31 , Mrm2, Mss4, Npc2, Nrd1 , Oad , Oms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , RadSG, Reel Rgm1 , Rpa34, Rpc40, RpM b, Rpl7b, Rpn7, Rrp7, Sdp1 , Sen2, 8nr19G, Swd1 , Swh1 , Tgi2, Tma2G, Tps2, Trm10, Ubx3, Ubx6, Ume6, Utr1 , Vcx1 , Vhs2, Vik1 , Vip1 , Vtc3, YarOG9c, Yar089c., Ycr025c, YdM 29w, Ygr137w, Yil102c, Yji218w, Yki063c, Yih47, YII059C, Ylr035c-a, Yir123c, Ylr410w-a, Ylr410w-b, Ymr32Gw, Ynl017c, Yol079w, YoM 53c, Yor1 1 1w, Ypl251w, Ypr123c, Ypr170c, and Ysp1 , or homoiogs thereof. In a specific embodiment, Ctrl is targeted for increased expression and/or activity. In another specific embodiment, Fre1 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctrl and Fre1 are targeted for increased expression and/or activity. In yet another specific embodiment, Pho89 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctrl and/or Fre1 and/or Pho89 are targeted for increased expression and/or activity.

[0026] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising an isobutanoi producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulate Mad . In one embodiment, the positive regulator of Mad is selected from Ccs1 and Sod1 , or homoiogs thereof.

[0027] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanoi, and wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Mad .

[0028] In yet another aspect, the application is directed to a recombinant yeast microorganism comprising an isobutano! producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a DNA-binding protein activated by copper-replete conditions. In one embodiment, the DNA-binding protein activated by copper-replete conditions is Ace1 , or homologs thereof.

[0029] In various embodiments described herein, the recombinant yeast microorganisms of the application that comprise an isobutanol producing metabolic pathway may be further engineered to reduce or eliminate the expression and/or activity of one or more enzymes selected from a glycerol-3-phosphate dehydrogenase (GPD), a 3-keto acid reductase (3-KAR), or an aldehyde dehydrogenase (ALDH).

[0030] 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, ail of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes. [0031] In one embodiment, one or more of the isobutanoi pathway genes encodes an enzyme that is localized to the cytosol. In one embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least one isobutanoi pathway enzyme iocaiized 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 iocaiized 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.

[0032] 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). in one embodiment, the KARI is an NADH-dependent KARI (NKR). In another embodiment, the ADH is an NADH-dependent ADH. In yet another embodiment, the KARI is an NADH-dependent KARI (NKR) and the ADH is an NADH-dependent ADH.

[0033] As described herein, in preferred embodiments, the recombinant microorganisms of the application are recombinant yeast microorganisms.

[0034] In some embodiments, the recombinant yeast microorganisms may be members of the Saccharomyces ciade, Saccharomyces sensu stricto microorganisms, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre- WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms. [0035] In some embodiments, the recombinant microorganisms may be yeasi recombinant microorganisms of the Saccharomyces clade.

[0036] In some embodiments, the recombinant microorganisms may be Saccharomyces sensu stricto 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.

[0037] 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 Saccharomyces, Kluyveromyces, Pichia, issatchenkia, Hansenuia, or Candida. In additional embodiments, the Crabtree-negative yeast microorganism is selected from Saccharomyces kiuyveri, Kluyveromyces iactis, Kluyveromyces marxianus, Pichia anomaia, Pichia stipitis, Pichia kudriavzevii, Hansenuia anomala, Candida utiiis and Kluyveromyces waitii.

[0038] In some embodiments, the recombinant microorganisms may be Crabtree- positive 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 Schizosaccharomyces. 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, Kluyveromyces thermotolerans, Candida glabrata, Z. bailli, Z rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum,

[0039] In some embodiments, the recombinant microorganisms may be post- WGD (whole genome duplication) yeast recombinant microorganisms. In one embodiment, the post-WGD 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.

[0040] 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 Schizosaccharomyces. In additional embodiments, the pre-WGD yeast is selected from the group consisting of Saccharomyces kiuyveri, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicaiis, Pichia pastoris, Pichia anomala, Pichia stipitis, Issatchenkia orientaiis, issatchenkia occidentaiis, Debaryomyces hansenii, Hansenula anomala, Pachysoien tannophilis, Yarrowia iipolytica, and Schizosaccharomyces pombe.

[0041] 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.

[0042] In another aspect, the present invention provides methods of producing a beneficial metabolite 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 a carbon source until the beneficial metabolite is produced and optionally, recovering the beneficial metabolite. In one embodiment, the microorganism produces the beneficial metabolite from a carbon source at a yield of at least about 5 percent theoretical. In another embodiment, the microorganism produces the beneficial 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 60 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. In various embodiments described herein, the beneficial metabolite may be selected from isobutanol, 3-methyl-1 -butanoi, 2-methyl-1 -butano!, valine, isoieucine, leucine, and/or pantothenic acid. In an exemplary embodiment, the beneficial metabolite is isobutanol. [0043] In one embodiment, the recombinant microorganism converts the carbon source to the beneficial metabolite under aerobic conditions. In another embodiment, the recombinant microorganism converts the carbon source to the beneficial metabolite under microaerobic conditions. In yet another embodiment, the recombinant microorganism converts the carbon source to the beneficial under anaerobic conditions.

BRIEF DESCRIPTION OF DRAWINGS

[0044] Illustrative embodiments of the invention are illustrated in the drawings, in which:

[0045] Figure 1 illustrates an exemplary embodiment of an isobutanol pathway.

[0046] Figure 2 illustrates an exemplary embodiment of an NADH-dependent isobutanol pathway.

[0047] Figure 3, adapted from Serpe ef a/., 1999, J, Biol. Chem. 274(41 ): 2921 1 - 9, illustrates the location of the Mac1 ^UP2 mutation within the Rep Motif (amino acids 264-279) of Mad .

DETAILED DESCRIPTION

[0048] As used herein and in the appended claims, the singular forms "a," "an," and "the" include piurai 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.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

[0050] 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. [0051] The term "microorganism" includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista, The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.

[0052] The term "prokaryotes" is art recognized and refers to ceils which contain no nucleus or other cell organelles. The prokaryotes are generally classified in one of two domains, the Bacteria and the Archaea. The definitive difference between organisms of the Archaea and Bacteria domains is based on fundamental differences in the nucleotide base sequence in the 18S ribosomal RNA.

[0053] The term "Archaea" refers to a categorization of organisms of the division Mendosicutes, typically found in unusual environments and distinguished from the rest of the prokaryotes by several criteria, including the number of ribosomal proteins and the lack of muramic acid in cell walls. On the basis of ssrRNA analysis, the Archaea consist of two phylogenetica!!y-distinct groups: Crenarchaeota and Euryarchaeota. On the basis of their physiology, the Archaea can be organized into three types: methanogens (prokaryotes that produce methane): extreme halophiies (prokaryotes that live at very high concentrations of salt (NaCI); and extreme (hyper) thermophi!es (prokaryotes that live at very high temperatures). Besides the unifying archaeal features that distinguish them from Bacteria (i.e., no murein in eel! wall, ester-linked membrane lipids, etc.), these prokaryotes exhibit unique structural or biochemical attributes which adapt them to their particular habitats. The Crenarchaeota consist mainly of hyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeota contain the methanogens and extreme halophiies.

[0054] "Bacteria", or "eubacteria", refers to a domain of prokaryotic organisms. Bacteria include at least eleven distinct groups as follows: (1 ) Gram-positive (gram-*-) bacteria, of which there are two major subdivisions: (1 ) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g. , Purple photosynthetic +non-photosynthetic Gram-negative bacteria (includes most "common" Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Pianctomyces; (6) Bacteroides, Fiavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non- sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (1 1 ) Thermotoga and Thermosipho thermophiles.

[0055] "Gram-negative bacteria" include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteureila, Brucella, Yersinia, Franciseiia, Haemophilus, Bordeteiia, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0056] "Gram positive bacteria" include cocci, nonsporulating rods, and sporuiating rods. The genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Etysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0057] 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 Tindall, B.J. (2007) The Taxonomic Outline of Bacteria and Archaea. TOBA Release 7.7, March 2007. Michigan State University Board of Trustees, [http://www.taxonomicoutiine.org/]).

[0058] 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.

[0059] 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 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.

[0060] 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 ai, 1989, supra.

[0061] The term "overexpression" refers to an elevated level (e.g., aberrant level) of mRNAs encoding for a protein(s), and/or to elevated levels of protein(s) in cells as compared to similar corresponding unmodified ceils expressing basal levels of mRNAs or having basal levels of proteins. In particular embodiments, mRNA(s) or protein(s) may be overexpressed by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8- foid, 10-fold, 12-fold, 15-fold or more in microorganisms engineered to exhibit increased gene mRNA, protein, and/or activity.

[0062] 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 ceil [e.g. reduced expression). As would be understood by one or ordinary skill in the art, the reduced activity of a protein in a cell may result from decreased concentrations of the protein in the ceil. [0063] The term "wild-type microorganism" describes a celi 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.

[0064] Accordingly, a "parental microorganism" functions as a reference ceil for successive genetic modification events. Each modification event can be accompiished by introducing a nucleic acid molecule in to the reference ceil. 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

[0065] 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.

[0066] 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, a nonsense mutation, an insertion, or a deletion of part or ail of a gene. In addition, in some embodiments of the modified microorganism, a portion of the microorganism genome has been replaced with a heterologous polynucleotide. In some embodiments, the mutations are naturally-occurring. In other embodiments, the mutations are identified and/or enriched through artificial selection pressure. In still other embodiments, the mutations in the microorganism genome are the result of genetic engineering.

[0067] The term "biosynthetic pathway", also referred to as "metabolic pathway", refers to a set of anabolic or cafabolic 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.

[0068] As used herein, the term "isobutanol producing metabolic pathway" refers to an enzyme pathway which produces isobutanol from pyruvate.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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 molecule(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 ceil; and/or (c) the molecule(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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).

[0077] 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 cell dry weight per hour (g/g h).

[0078] The term "yield" is defined as the amount of product obtained per unit weight of raw material 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.

[0079] 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).

[0080] "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.

[0081] 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.

[0082] "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.

[0083] 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."

[0084] 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.

[0085] 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, biofuei precursor, higher alcohol, or higher alcohol precursor.

[0086] 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.

[0087] 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 C0₂ 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 C0₂.

[0088] 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 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 nucieotidic oligomer or oligonucleotide.

[0089] 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, 5'-untranslated region (UTR), and 3'-UTR, as well as the coding sequence.

[0090] 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.

[0091] 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 chromosomes), and PLACs (plant artificial chromosomes), and the like, that are "episomes," that is, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine -conjugated DNA or RNA, a pepfide-conjugafed DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomai 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.

[0092] "Transformation" refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including chemical transformation (e.g. lithium acetate transformation), electroporation, microinjection, biolisfics (or particle bombardment- mediated delivery), or agrobacterium mediated transformation.

[0093] 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 or polypeptides, but can include enzymes composed of a different molecule including polynucleotides.

[0094] 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

[0095] The term "homolog," used with respect to an original polynucleotide or polypeptide of a first family or species, refers to distinct polynucleotides or polypeptides of a second family or species which are determined by functional, structural or genomic analyses to be a polynucleotide or polypeptide of the second family or species which corresponds to the original polynucleotide or polypeptide of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of a polynucleotide or polypeptide can readily be cloned using genetic probes and PGR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.

[0096] A polypeptide has "homology" or is "homologous" to a second polypeptide if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a polypeptide has homology to a second polypeptide if the two polypeptides have "similar" amino acid sequences. (Thus, the terms "homologous polypeptides" or "homologous proteins" are defined to mean that the two polypeptides have similar amino acid sequences).

[0097] The term "analog" or "analogous" refers to polynucleotide or polypeptide sequences 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 Cherrucal Production by Modifying Mad Pathway Components

[0098] The present inventors have discovered that altering the expression and/or activity of Mad pathway components increases Fe-S cluster containing protein activity and contributes, in part, to improved host cell growth under conditions relevant to commercial-scale metabolite production.

[0099] MAC1 encodes a transcription factor ("Metal-binding activator, i.e., "Mad " or "Mad p") that regulates the expression of genes involved in copper homeostasis. Jungmann et a/., 1983, EMBO J 12(13): 5051 -8. In response to low copper levels, Ma p induces expression of the copper transporters Ctr1 p and Ctr3p, the cell- surface metal reductases Frei p and Fre7p, and two additional proteins, YFRQ55W and YJL217W. Graden et aL , 1997, PNAS USA 94(1 1 ): 5550-5; Labbe et ai., 1997, J. Biol. Chem 272(25): 15951 -8; Yamaguchi-lwai et a/., 1997, J. Biol. Chem. 272(28): 1771 1 -8; Pena et ai, 1988, MoL Cell. Biol. 18(5): 2514-23; and Gross et ai, 2000, J. Biol. Chem. 275(41 ): 32310-6. FRE1 is expressed early and FRE2 is expressed late during copper depletion - in Mad p-dep!eted cells, FRE1 expression persists for longer and FRE2 expression begins earlier. Copper is known to inhibit the activity of Mad p. Cells lacking active Ma p are viable, but copper-deficient, respiratory deficient, and sensitive to heat, hydrogen peroxide, cadmium, zinc, and lead. Mad p is known to bind a copper-response element ("CuRE"), TTTGC(T/G)C(A/G), in the promoters of target genes. Labbe et a/., 1997, J. Biol. Chem. 272(25): 15951 -8; and Georgatsou et al., 1999, Yeast 15(7): 573-84. Under norma! conditions, Ma p detects both a minimum and a maximum allowable copper concentration and is thus often referred to as a nutritional copper sensor. Heredia et a!., 2001 , J. Biol. Chem. 278(12): 8793-7. To date, the role that Mad p plays, if any, in regulating iron-sulfur (Fe-S) cluster biosynthesis or the activity of Fe-8 cluster containing proteins has not yet been elucidated.

[00100] Without being bound by any particular theory, it is believed that the improved growth and/or increases in Fe-S cluster containing protein activity described herein results from the altered expression and/or activity of Mad pathway components. Thus, the altered Mad pathway regulation, expression, and/or activity has broad applicability to a variety of biosynthetic pathways comprising a Fe~S cluster containing protein, as Fe-S cluster containing protein activity is often a rate- limiting component of such pathways.

[00101] Accordingly, one aspect of the invention is directed to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein. As used herein, a "biosynthetic pathway requiring a Fe-S cluster containing protein" refers to any metabolic pathway which utilizes a Fe-S cluster containing protein to catalyze a substrate to product conversion necessary for the production of the desired end-product within said biosynthetic pathway.

[00102] In various embodiments described herein, said recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein is engineered to provide increased heterologous or native expression and/or activity of the protein encoded by MAC1 {i.e., herein referred to as Mad or Mad p).

[00103] One aspect of the application is therefore directed to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins or homoiogs thereof, !n one embodiment, the Mad protein comprises SEQ ID NO: 2. Homoiogs of Mad are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, a Mad protein derived from a yeast selected from Ajei!omyces, Arthroderma, Ashbya, Aspergillus, Botsyotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibbereila, Giomereiia, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Peniciiiium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Scierotinia, Sordaria, Talaromyces, Thchoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces may be overexpressed. Sequences for a variety of Mad homoiogs from yeast other than S. cerevisiae are available in the art, e.g., SEO ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20. In certain embodiments, one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an exogenous polynucleotide.

[00104] A person skilled in the art, equipped with this disclosure, will appreciate suitable methods for increasing the expression (i.e. overexpressing) Mad . For instance, in one embodiment, Mad or a homoiog thereof may be overexpressed from a plasmid. In another embodiment, one or more copies of the MAC1 gene or a homoiog thereof is inserted into the chromosome, e.g., 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 MAC1 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.

[00105] Another aspect of the application relates to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to express or overexpress one or more polynucleotides encoding one or more consfitutiveiy active mutant Mad proteins. In one embodiment, the constitutiveiy active mutant Mad protein or homoiog thereof comprises a mutation at one or more positions corresponding to residues 284 to 279 of the native Saccharomyces cerevisiae Mad protein (SEQ ID NO: 2). In an exemplary embodiment, the constitutively active mutant Mad protein or homolog thereof comprises a mutation at a position corresponding to the cysteine 271 (i.e., C271 ) residue of the native S, cerevisiae Mad protein (SEQ ID NO: 2). In a specific embodiment, the cysteine 271 residue is replaced with a tyrosine. In another exemplary embodiment, the constitutively active mutant Mad protein or homolog thereof comprises a mutation at a position corresponding to the histidine 279 (i.e., H279) residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2). In one specific embodiment, the histidine 279 residue is replaced with a g!utamine. The properties of various constitutively active mutant Mad proteins are described by Yarnaguchi-lwai et ai., 1997, J. Biol. Chem. 272(28): 1771 1 -8.

[00106] As described above, homologs of Mad are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, a constitutively active mutant Mad protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibbereiia, Glomerella, Grosmannia, Issatchenksa, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Peniciiiium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticiiiium, Yarrowia or Zygosaccharomyces may be expressed or overexpressed. in certain embodiments, the Mad homolog may be mutated as described above, e.g., be mutated to comprise a mutation at a position corresponding to the C271 or H279 position of Ma (SEQ ID NO: 2).

[00107] As will be understood by one of ordinary skill in the art, modified Mad 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 Mad proteins and homologs thereof, can be obtained by mutating the gene or genes encoding Mad or the homologs of interest by site-directed mutagenesis. Such mutations may include point mutations, deletion mutations and insertionai 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 Mad proteins of the invention. The corresponding cysteine position of Mad homoiogs may be readiiy identified by one skiiled 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 Mad .

[00108] Yet another aspect of the application relates to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins or homoiogs thereof regulated by a Mad protein or homoiog thereof. In one embodiment, the proteins regulated by a Mad protein or homoiog thereof are selected from Afg2, Aim25, Axl1 , Cdc20, Cdc80_s Coq6, Cn , Ctf19, Ctrl , Ctr3, Ctt1 , Cup1 , Cup9, Das1 , Dbf20, Dpl1 , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun26, Gnd2, Gph1 , His5, Hnm1 , Hos4, Irc7, Isy1 , Kem1 , Kre8, Lsb1 , Lsm5, Mal33, Mdm31 , Mrm2, Mss4, Npc2, Nrd1 , Oad , Oms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , Rad50, Reel , Rgm1 , Rpa34, Rpc40, RpU b, Rpi7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr190, Swd1 , Swh1 , Tgl2, Tma20, Tps2, Trm10, Ubx3, Ubx6, Ume6, UtM , Vcx1 , Vhs2, Vik1 , Vip1 , Vtc3, Yar009c, Yar069c, Ycr025c, Ydi129w, Ygr137w, Yii102c, Yji218w, Yki063c, Yih47, YH059c, Yir035c-a, Yir123c, Ylr410w-a, Ylr410w-b, Ymr320w, Ynl017c, Yol079w, Yol153c, Yor1 1 1w, Ypl251 w, Ypr123c, Ypr170c, and Ysp1 , or homoiogs thereof. In a specific embodiment, Ctrl is targeted for increased expression and/or activity. In another specific embodiment, Fre1 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctrl and Fre1 are targeted for increased expression and/or activity. In yet another specific embodiment, Pho89 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctrl and/or Fre1 and/or Pho89 are targeted for increased expression and/or activity, !n certain embodiments, one or more of the polynucleotides encoding said one or more proteins or homoiogs thereof regulated by a Mad protein or homoiog thereof is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said one or more proteins or homoiogs thereof regulated by a Mad protein or homoiog thereof is an exogenous polynucleotide. [00109J Homoiogs of Afg2, Aim25, Axil, Cdc20, Cdc60, Coq6, Crr1, Ctf19, Ctrl, Ctr3, Ctt1, Cup1, Cup9, Das Dbf20, DpM, Dsd1, Ede1, Egd2, Fet4, Fre1, Fre7, Ftr1, Fun26, Gnd2, Gph1, His5, Hnm1, Hos4, !rc7, Isy1, Kem1, Kre8, Lsb1, LsmS, Mal33, Mdm31, Mrm2, ss4, Npc2, Nrd1, Oac Oms1, Pho23, Pho89, Pmp1, Pop8, Pph3, Pre9, PrmS, Prp9, Pub1, RadSO, Reel _., Rgm1, Rpa34, Rpc40, RpMb, Rp!7b, Rpn7, Rrp7, Sdp1, Sen2, Snr190, Swd1, Swh1, Tg!2, Tma20, Tps2, Trm10, Ubx3, Ubx6, Ume8, Uir Vcx1, Vhs2, Vik1, Vip1, Vtc3, Yar009c, Yar069c, Ycr025c, Ydl129w, Ygr137w, Yil102c, Yjl218w, Ykl063c, Y!h47, Yii059c, YirOSSc-a, Ylr123c, Yir410w-a, Ylr410w-b, Ymr320w, Ynl017c, Yoi079w, Yol153c, Yor111w, Ypl251w, Ypr123c, Ypr170c, and Ysp1 are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, a homoiog of Afg2_s Aim25, Axil, Cdc20, Cdc60, Coq6, Crr1, Ctf19, Ctrl, Ctr3, Ctt1, Cup1, Cup9, Das1, Dbf20, DpM, Dsd1, Ede1, Egd2, Fet4, Fre1, Fre7, Ftr1, Fun28, Gnd2, Gph1, His5, Hnm1, Hos4, Irc7, !sy1, Kem1, Kre6, Lsb1, LsmS, Mal33, Mdm31, Mrm2, Mss4, Npc2, Nrd1, Oac1, Oms1, Pho23, Pho89, Pmp1, Pop8, Pph3, Pre9, PrmS, Prp9, Pub1, Rad50, Reel, Rgm1, Rpa34, Rpc40, RpMb, Rpl7b, Rpn7, Rrp7, Sdp1, Sen2, Snr190, Swd1, Swh1, Tgi2, Tma20, Tps2, Trm10, Ubx3, Ubx6, Ume6, Utr1, Vcx1, Vhs2, Vik1, Vip1, Vtc3, Yar009c, Yar069c, Ycr025c, Ydi129w, Ygr137w, Yii102c, Yji218w, Yki063c, Yih47, YI1059C, Yir035c-a, Yir123c, Ylr410w~a, Ylr410w-b, Ymr320w, Yn!017c, Yol079w, Yoi153c, Yor111w, Ypl251w, Ypr123c, Ypr170c, and/or Ysp1 derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botyotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, G!omere!ia, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeha, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarmwia or Zygosaccharomyces may be overexpressed.

[00110] Yet another aspect of the application relates to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulate Mad. In one embodiment, the positive regulator of Ma is selected from Ccs1 and Sod1 , or homo!ogs thereof. In a specific embodiment, Ccs1 is targeted for increased expression and/or activity. In another specific embodiment, Sod1 is targeted for increased expression and/or activity. In yet another specific embodiment, Ccs1 and Sod1 are targeted for increased expression and/or activity. In certain embodiments, one or more of the polynucleotides encoding said positive regulator of Mad is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said positive regulator of Ma is an exogenous polynucleotide.

[00111] Homoiogs of Ccs1 and Sod1 are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, a homoiog of Ccs1 and/or Sod1 derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberel!a, G!omerel!a, Grosmannia, Issatchenkia, Kiuyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Peniciiiium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces,

Schizosaccharomyces, Sclerotinia, Sordaria, Taiaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces may be overexpressed.

[00112] Yet another aspect of the application relates to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Mad .

[00113] Yet another aspect of the application relates to a recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a DNA-binding protein activated by copper-replete conditions. In one embodiment, the DNA-binding protein activated by copper-replete conditions is Ace1 , or homoiogs thereof. See, e.g., Keller ef a/., 2005, Eukaryotic Cell 4(1 1 ): 1863-71 , noting that Ace1 and Mad undergo reciprocal copper metalioregulation in yeast ceils.

[00114] Homoiogs of Ace1 are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, a homoiog of Ace1 derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotsnia, Candida, Chaeiomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Giomere!la, Grosmannia, Issatchenkia, Kiuyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaereila, Neciria, Neosartorya, Neurospora, Paracoccidioides, Peniciliium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticiliium, Yarrowia or Zygosaccharomyces may be disrupt, delete, or mutated.

[00115] In each of the aforementioned aspects and embodiments, the Fe-S cluster containing protein may be selected from dihydroxy acid dehydratase (i.e., DHAD), isopropylma!ate isomerase, sulfite reductase, aconitase, homoaconitase, lipoate synthase, succinate dehydrogenase, NADH ubiquinone oxidoreductase, and ubiquino!-cytochrome-c reductase.

[00116] In one embodiment, the Fe-S duster containing protein is encoded by an endogenous polynucleotide. In another embodiment, the Fe~S cluster containing protein is encoded by an exogenous polynucleotide. In an exemplary embodiment, the Fe-S cluster containing protein is overexpressed.

[00117] In an exemplary embodiment, the Fe-S cluster containing protein is dihydroxy acid dehydratase (i.e., DHAD). Accordingly, an additional aspect of the invention is directed to a recombinant microorganism comprising a DHAD-requiring biosynthetic pathway. As used herein, a "DHAD-requiring biosynthetic pathway" refers to any metabolic pathway which utilizes DHAD to convert 2,3- dihydroxyisovalerate to a-ketoisovalerate or 2,3-dihydroxy-3-methylvalerate to 2- keto-3-methylvalerate. Examples of DHAD-requiring biosynthetic pathways include, but are not limited to, isobutanol, 3-methyl-1 -butanoi, 2-methyi-1 -butanol, valine, isoleucine, leucine, and pantothenic acid (vitamin B5) metabolic pathways. The metabolic pathway may 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 cells. Engineered biosynthetic pathways for synthesis of isobutanol are described in commonly owned US Patent Nos. 8,0 7,375, 8,017,376, 8,097,440, and 8,133,175, all of which are herein incorporated by reference in their entireties for all purposes. Additional DHAD-requiring biosynthetic pathways have been described for the synthesis of valine, leucine, and isoleucine (See, e.g., WO/2001/021772, and McCourt et a/., 2008, Amino Acids 31 : 173-210), pantothenic acid (See, e.g., WO/2001/021772), 3-methyl-1 -butanol (See, e.g., WO/2008/098227, Atsumi et ai, 2008, Nature 451 : 86-89, and Connor et a/., 2008, Αρρί Environ, MicrobioL 74: 5769-5775), and 2-methyi-1 -butanoi (See, e.g., WO/2008/098227, WO/2009/076480, and Atsumi et a!,, 2008, Nature 451 : 86-89).

[00118] As used herein, the terms "DHAD" or "DHAD enzyme" or "dihydroxyacid dehydratase" are used interchangeably herein to refer to an enzyme that catalyzes the conversion of 2,3-dihydroxyisova!erate to ketoisovalerate and/or the conversion of 2,3-dihydroxy-3-methylvalerate to 2-keto-3-mefhylvalerate. DHAD sequences are available from a vast array of microorganisms, including, but not limited to, L iactis, E. coll, S, cerevisiae, B. subti!is, Streptococcus pneumoniae, and Streptococcus mutans, A representative list of DHAD enzymes that can benefit from the methods described herein include, but are not limited to those, disclosed in US Patent Publication No. 2010/0081 154, as well as those disclosed in commonly owned US Patent Publication No. 201 1/0076733 (now US Patent No. 8,232,089), commonly owned US Provisional Application Serial No. 61/619,154, and commonly owned PCT/US12/56225 (published as WO/2013/043801 ). Such DHAD enzymes may be cytosolically localized or mitochondrial!y localized. A representative listing of DHAD enzymes exhibiting cytosolic localization and activity are disclosed in commonly owned US Patent Publication No. 201 1/0076733 (now US Patent No. 8,232,089).

[00119] As described herein, the increased activity of DHAD in a recombinant microorganism is a favorable characteristic for the production of beneficial metabolites including isobutanoi, 3-methyi-1 -butanol, 2-methyi-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 altered regulation, expression, and/or activity of Mad . Thus, in various embodiments described herein, the present invention provides recombinant microorganisms with increased DHAD activity as a result of alterations in Mad regulation, expression, and/or activity. In one embodiment, the alteration in Mad regulation, expression, and/or activity increases the activity of a cytosoiicaily-localized DHAD. In another embodiment, the alteration in Mad regulation, expression, and/or activity increases the activity of a mitochondriaily-iocalized DHAD.

[00120] In one embodiment, the DHAD protein is encoded by an endogenous polynucleotide. In another embodiment, the DHAD protein is encoded by an exogenous polynucleotide. In an exemplary embodiment, the DHAD protein is overexpressed.

[00121] While particularly useful for the biosynthesis of isobutanol, the altered regulation, expression, and/or activity of Mad 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-methyl-1 -butanol, and 3-methyi-1 -butanol.

[00122] Accordingly, another aspect of the application is directed to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins or homologs thereof. In one embodiment, the Mad protein comprises SEQ ID NO: 2. Homologs of Ma are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, a Mad protein derived from a yeast selected from Ajeibmyces, Arthroderma, Ashbya, Aspergillus, Boiryotinia, Candida, Chaetomsum, Ciavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kiuyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Meiarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticiilium, Yarrowia or Zygosaccharomyces may be overexpressed. Sequences for a variety of Mad homologs from yeast other than S. cerevisiae are available in the art, e.g., 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, and SEQ ID NO: 20. In certain embodiments, one or more of the polynucleotides encoding said one or more Mad proteins or homologs thereof is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an exogenous polynucleotide.

[00123] Another aspect of the application relates to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to express or overexpress one or more polynucleotides encoding one or more constitutively active mutant Mad proteins. In one embodiment, the constitutively active mutant Mad protein or homoiog thereof comprises a mutation at one or more positions corresponding to residues 284 to 279 of the native Saccharomyces cerevisiae Mad protein (SEQ ID NO: 2). In an exemplary embodiment, the constitutively active mutant Mad protein or homoiog thereof comprises a mutation at a position corresponding to the cysteine 271 (i.e., C271 ) residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2). In a specific embodiment, the cysteine 271 residue is replaced with a tyrosine. In another exemplary embodiment, the constitutively active mutant Mad protein or homoiog thereof comprises a mutation at a position corresponding to the histidine 279 (i.e., H279) residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2). In one specific embodiment, the histidine 279 residue is replaced with a giutamine.

[00124] Yet another aspect of the application relates to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins or homoiogs thereof regulated by a Mad protein or homoiog thereof. In one embodiment, the proteins regulated by a Mad protein or homoiog thereof are selected from Afg2, Aim25, Axil , Cdc20, Cdc60, Coq6, Crr1 , Ctf19, Ctrl , Ctr3, Ctti , Cupi , Cup9, Dasi , Dbf20, Dpl1 , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun26, Gnd2, Gph1 , His5, Hnm1 , Hos4, !rc7, Isy1 , Kem1 , Kre8, Lsb1 , Lsm5, Mal33, Mdm31 , Mrm2, Mss4, Npc2, Nrd1 , Oad , Qms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , RadSG, Reel Rgm1 , Rpa34, Rpc40, RpM b, Rpi7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr190, Swd1 , Swh1 , Tgl2, Tma20, Tps2, Trmi O, Ubx3, Ubx8, Ume8, Utr1 , Vcx1 , Vhs2, Vik1 , Vipi , Vtc3, Yar009c, Yar069c, Ycr025c, Ydi129w, Ygr137w, Yil102c, Yjl218w, Yki063c, Yih47, YII059C, Ylr035c~a, Ylr123c, Ylr410w-a, Yir410w-b, Ymr320w, Ynl017c, Yol079w, Yoi153c, Yor1 1 1 w, Ypi251w, Ypr123c, Ypr170c, and Ysp1 , or homoiogs thereof. In a specific embodiment, Ctrl is targeted for increased expression and/or activity. In another specific embodiment, Fre1 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctrl and Fre1 are targeted for increased expression and/or activity. In yet another specific embodiment, Pho89 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctrl and/or Fre1 and/or Pho89 are targeted for increased expression and/or activity. In certain embodiments, one or more of the polynucleotides encoding said one or more proteins or homoiogs thereof regulated by a Mad protein or homolog thereof is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said one or more proteins or homoiogs thereof regulated by a Mad protein or homolog thereof is an exogenous polynucleotide.

[00125] Yet another aspect of the application relates to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulate Mad . In one embodiment, the positive regulator of Mad is selected from Ccs1 and Sod1 , or homoiogs thereof. In a specific embodiment, Ccs1 is targeted for increased expression and/or activity. In another specific embodiment, Sod1 is targeted for increased expression and/or activity. In yet another specific embodiment, Ccs1 and Sod1 are targeted for increased expression and/or activity. In certain embodiments, one or more of the polynucleotides encoding said positive regulator of Mad is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said positive regulator of Mad is an exogenous polynucleotide.

[00126] Yet another aspect of the application relates to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Mad .

[00127] Yet another aspect of the application relates to a recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a DNA-binding protein activated by copper-replete conditions. In one embodiment, the DNA-binding protein activated by copper-replete conditions is Ace1 , or homoiogs thereof. Isobutanoi Producing Recombinant Yeast Microorganisms

[00128] In certain exemplary embodiments, the DHAD-requiring biosynthetic pathway is an isobutanoi biosynthetic pathway, i.e., an isobutanoi producing metabolic pathway. Accordingly, the present application relates to a recombinant yeast microorganism comprising an engineered isobutanoi producing metabolic pathway. In recent years, yeast cells have been engineered to produce increased quantities of isobutanoi, an important commodity chemical and biofuel candidate (See, e.g., commonly owned patent application publications, US 2009/0226991 (now US Patent No. 8,017,375), US 2010/0143997, US 201 1 /0020889, US 201 1/0076733 (now US Patent No. 8,232,089), US 201 1/0201090, and WO 2010/075504).

[00129] As described herein, the present invention 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:

1 . 2 pyruvate→ acetoiactate + COa

2. acetoiactate + NAD(P)H→ 2,3-dihydroxyisovaierate + NAD(P)⁺

3. 2,3-dihydroxyisovaierate→ alpha-ketoisovalerate

4. alpha-ketoisovaierate→ isobutyraldehyde + CO2

5. isobutyraldehyde +NAD(P)H --> isobutanoi + NADP

[00130] In one embodiment, these reactions are carried out by the enzymes 1 ) Acetoiactate 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.

[00131] Alternative pathways for the production of isobutanoi in yeast have been described in WO/2007/050671 and in Dickinson et a/., 1998, J Biol Chem 273:25751 -6. These and other isobutanoi producing metabolic pathways are within the scope of the present application. In one embodiment, the isobutanoi 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.

[00132] As described above, the present inventors have observed increases in growth and DHAD activity in recombinant yeast microorganisms comprising altered Mad regulation, expression, and/or activity. Accordingly, an aspect of the application is directed to a recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins or homologs thereof. In one embodiment, the Mad protein comprises SEQ ID NO: 2. Homologs of Mad are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, a Mad protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetorniurn, Clavispora, Coccidioides, Debaryomyces, Gibbereiia, Giomereiia, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderornyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaereila, Nectria, Neosartorya, Neurospora, Paracoccidioides, Peniciiiium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces,

Schizosaccha myces, Scierotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces may be overexpressed. Sequences for a variety of Mad homologs from yeast other than S. cerevisiae are available in the art, e.g., SEQ 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: 18, SEQ ID NO: 18, and SEQ ID NO: 20. !n certain embodiments, one or more of the polynucleotides encoding said one or more Mad proteins or homologs thereof is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said one or more Mad proteins or homologs thereof is an exogenous polynucleotide.

[00133] Another aspect of the application relates to a recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanoi, and wherein said recombinant yeast microorganism is engineered to express or overexpress one or more polynucleotides encoding one or more constitutively active mutant Mad proteins. In one embodiment, the constitutively active mutant Mad protein or homolog thereof comprises a mutation at one or more positions corresponding to residues 264 to 279 of the native Saccharomyces cerevisiae Mad protein (SEQ ID NO: 2). In an exemplary embodiment, the constitutively active mutant Mad protein or homolog thereof comprises a mutation at a position corresponding to the cysteine 271 (i.e., C271 ) residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2). !n a specific embodiment, the cysteine 271 residue is replaced with a tyrosine. In another exemplary embodiment, the constitutively active mutant Mad protein or homolog thereof comprises a mutation at a position corresponding to the histidine 279 (i.e., H279) residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2). In one specific embodiment, the histidine 279 residue is replaced with a g!utamine.

[00134] Yet another aspect of the application relates to a recombinant yeast microorganism comprising an isobutanoi producing metabolic pathway, wherein said isobutanoi producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanoi, and wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins or homologs thereof regulated by a Mad protein or homolog thereof. In one embodiment, the proteins regulated by a Mad protein or homolog thereof are selected from Afg2, Aim25, Axli , Cdc20, Cdc60, Coq6, Cn , Ctf19, Ctrl , Ctr3, Ctt1 , Cup1 , Cup9, Das Dbf20, DpM , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun28, Gnd2, Gph1 , His5, Hnm1 , Hos4, Irc7, Isy1 , Kem1 , Kre6, Lsb1 , Lsm5, Mal33, Mdm31 , Mrm2, Mss4, Npc2, Nrdi , Oad , Oms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , RadSG, Reel , Rgm1 , Rpa34, Rpc40, RpM b, Rpl7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr190, Swd1 , Swh1 , Tgi2, Tma20, Tps2, Trml O, Ubx3, Ubx6, Ume6, Utr1 , Vcx1 , Vhs2, Vik1 , Vip1 , Vtc3, Yar009c, Yar069c, Ycr025c, Ydi129w, Ygr137w, Yil102c, Yji218w, Yki063c, Ylh47, YII059C, Ylr035c-a, Yir123c, Ylr410w-a, Ylr410w-b, Ymr320w, Ynl017c, Yol079w, Yoi153c, Yor1 1 w, Ypi251w, Ypr123c, Ypr170c, and Ysp1 , or homologs thereof. In a specific embodiment, Ctrl is targeted for increased expression and/or activity. In another specific embodiment, Fre1 is targeted for increased expression and/or activity. In yet another specific embodiment, Ctrl and Fre1 are targeted for increased expression and/or activity. In yet another specific embodiment, Pho89 is targeted for increased expression and/or activity, in yet another specific embodiment, Ctrl and/or Fre1 and/or Pho89 are targeted for increased expression and/or activity. In certain embodiments, one or more of the polynucleotides encoding said one or more proteins or homoiogs thereof regulated by a Mad protein or homolog thereof is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said one or more proteins or homoiogs thereof regulated by a Mad protein or homolog thereof is an exogenous polynucleotide.

[00135] Yet another aspect of the application relates to a recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanoi producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanoi, and wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulate Mad . In one embodiment, the positive regulator of Mad is selected from Ccs1 and Sod1 , or homoiogs thereof. In a specific embodiment, Ccs1 is targeted for increased expression and/or activity. In another specific embodiment, Sod1 is targeted for increased expression and/or activity, in yet another specific embodiment, Ccs1 and Sod1 are targeted for increased expression and/or activity. In certain embodiments, one or more of the polynucleotides encoding said positive regulator of Mad is an endogenous polynucleotide. In yet another embodiment, one or more of the polynucleotides encoding said positive regulator of Mad is an exogenous polynucleotide.

[00136] Yet another aspect of the application relates to a recombinant yeast microorganism comprising an isobutanoi producing metabolic pathway, wherein said isobutanoi producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanoi, and wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Mad .

[00137] Yet another aspect of the application relates to a recombinant yeast microorganism comprising an isobutanoi producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanoi, and wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a DNA-binding protein activated by copper-replete conditions. In one embodiment, the DNA-binding protein activated by copper-replete conditions is Ace1 , or homoiogs thereof.

[00138] In one embodiment, the yeast microorganism comprising an isobutanoi producing metabolic pathway has reduced or no pyruvate decarboxylase (PDC) activity. PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is then reduced to ethanoi by ADH via an oxidation of NADH to NAD+. Ethanoi production is the main pathway to oxidize the NADH from glycolysis. Deletion, disruption, or mutation of this pathway increases the pyruvate and the reducing equivalents (NADH) available for an isobutanoi biosynthetic pathway. Accordingly, deletion, disruption, or mutation of one or more genes encoding for pyruvate decarboxylase and/or a positive transcriptional regulator thereof can further increase the yield of isobutanoi. In one embodiment, said pyruvate decarboxylase gene targeted for disruption, deletion, or mutation is selected from the group consisting of PDC1, PDC5, and PDC6, or homoiogs or variants thereof. In another embodiment, all three of PDC1, PDC5, and PDC6 are targeted for disruption, deletion, or mutation. In yet another embodiment, a positive transcriptional regulator of the PDC1, PDC5, and/or PDC6 is targeted for disruption, deletion or mutation. In one embodiment, said positive transcriptional regulator is PDC2, or homoiogs or variants thereof.

[00139] As is understood by those skilled in the art, there are several additional mechanisms available for reducing or disrupting the activity of a protein encoded by PDC1, PDC5, PDC6, and/or PDC2, including, but not limited to, the use of a regulated promoter, use of a weak constitutive promoter, disruption of one of the two copies of the gene in a diploid yeast, disruption of both copies of the gene in a diploid yeast, expression of an anti-sense nucleic acid, expression of an siRNA, over expression of a negative regulator of the endogenous promoter, alteration of the activity of an endogenous or heterologous gene, use of a heterologous gene with lower specific activity, the like or combinations thereof

[00140] As described herein, strains that naturally produce low levels of pyruvate decarboxylase can also have applicability for producing increased levels of isobutanol. As would be understood by one skilled in the art equipped with the instant disclosure, strains that naturally produce low levels of pyruvate decarboxylase may inherently exhibit low or undetectable levels of pyruvate decarboxylase activity, a trait which may be favorable for the production of isobutanol,

[00141] In various embodiments described herein, the recombinant microorganism comprises an engineered 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.

[00142] 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 cyiosoL !n yet another embodiment, the recombinant microorganisms comprise an isobutano! producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol. !n yet another embodiment, the recombinant microorganisms comprise an isobutano! 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. Isobutanol producing metabolic pathways in which one or more genes are localized to the cytosol are described in commonly owned and co-pending publication, US 201 1/0078733, which is herein incorporated by reference in its entirety for ail purposes.

[00143] As is understood in the art, a variety of organisms can serve as sources for the isobutano! pathway enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including V. spp. stipstis, Torulaspora pretoriensis, issatchenkia orientalis, Schizosaccharomyces spp., including S. pom he, Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustilago spp. Sources of genes from anaerobic fungi include, but not limited to, Piromyces spp., Orpinomyces spp., or Neocailimastix 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., Slackia spp., Lactococcus spp., Enterobacter spp., Streptococcus spp., Salmonella spp., Bacteroides spp., Methanococcus spp., Eryth hacter spp., Sphingomonas spp., Sphingohium spp., and Novosphingobium spp.

[00144] 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.

[00145] For example, acetolactafe synthases capable of converting pyruvate to acefoiactate may be derived from a variety of sources {e.g., bacterial, yeast, Archaea, etc.), including B. subiiiis (GenBank Accession No. Q04789.3), L lactis (GenBank Accession No. NP 267340.1 ), S. mutans (GenBank Accession No. P 721805.1 ), K. pneumoniae (GenBank Accession No. ZP 06014957.1 ), C. glutamicum (GenBank Accession No. P42463.1 ), E. cloacae (GenBank Accession No. YP_...00361361 1 .1 ), M. maripaludis (GenBank Accession No. ABX01060.1 ), M. grisea (GenBank Accession No. AAB81248.1 ), T. stipitatus (GenBank Accession No. XP_002485976.1 ), or S. cerevisiae ILV2 (GenBank Accession No. NP_013826.1 ). Additional acetolactate synthases capable of converting pyruvate to acetoiactate are described in commonly owned US Publication No. 201 1/0076733 (now US Patent No. 8,232,089), which is herein incorporated by reference in its entirety. A review article characterizing the biosynthesis of acetoiactate from pyruvate via the activity of acetoiactate synthases is provided by Chipman et a/., 1998, Biochimica et Biophysica Acta 1385: 401 -19, which is herein incorporated by reference in its entirety. Chipman et a/, provide an alignment and consensus for the sequences of a representative number of acetoiactate synthases. Motifs shared in common between the majority of acetoiactate synthases include:

SGPG(A/C/V)(T/S)N (SEQ ID NO: 21 ),

GX(P/A)GX(V/A/T) (SEQ ID NO: 22),

GX(Q/G)(T./A)(L/M)G(Y/F/W)(A/G)X(P/G)(VV./A)AX(G/T)(A/V) (SEQ ID NO: 23), and

GD(G/A)(G/S/C)F (SEQ ID NO: 24)

motifs at amino acid positions corresponding to the 163-169, 240-245, 521 -535, and 549-553 residues, respectively, of the S. cerevisiae ILV2, Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit acetoiactate synthase activity.

[00146] Ketol-acid reductoisomerases capable of converting acetoiactate to 2,3- dihydroxyisova!erate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including E. coii (GenBank Accession No. EGB30597.1 ), L. lactis (GenBank Accession No. YP 003353710.1 ), S. exigua (GenBank Accession No. ZPJ36160130.1 ), C. curiam (GenBank Accession No. YP_003151266.1 ), Shewanella sp. (GenBank Accession No. YP 732498.1 ), V. fischeri (GenBank Accession No. YP_20591 1 .1 ), M. maripaludis (GenBank Accession No. YPJ301097443.1 ), B. subtilis (GenBank Accession No. CAB14789), S. pombe (GenBank Accession No. NP_001018845), B. thetaiotamicron (GenBank Accession No. NP_810987), or S. cerevisiae ILV5 (GenBank Accession No. NP_013459.1 ). Additional ketol-acid reductoisomerases capable of converting acetoiactate to 2,3- dihydroxyisova!erate are described in commonly owned US Publication No. 201 1 /0076733 (now US Patent No. 8,232,089), which is herein incorporated by reference in its entirety. An alignment and consensus for the sequences of a representative number of ketoi-acid reductoisomerases is provided in commonly owned and co-pending US Publication No. 2010/0143997, which is herein incorporated by reference in its entirety. Motifs shared in common between the majority of ketol-acid reductoisomerases include:

G(Y/C/W)GXQ(G/A) (SEQ ID NO: 25),

(F/Y/L)(S/A)HG(F/L) (SEQ ID NO: 26),

V(V/!/F)(M/L/A)(A/^'C)PK (SEQ ID NO: 27),

D(L/I)XGE(Q/R)XXLXG (SEQ ID NO: 28), and

S(D/N T)TA(E/Q/R)XG (SEQ ID NO: 29)

motifs at amino acid positions corresponding to the 89-94, 175-179, 194-200, 262- 272, and 459-465 residues, respectively, of the £. coil ketoi-acid reductoisomerase encoded by HvC. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit ketoi-acid reductoisomerase activity.

[00147] To date, all known, naturally existing ketoi-acid reductoisomerases are known to use NADPH as a cofactor. In certain embodiments, a ketoi-acid reductoisomerase which has been engineered to used NADH as a cofactor may be utilized to mediate the conversion of acetoiactate to 2,3-dihydroxyisovaierate. Engineered NADH-dependent KARI enzymes ("NKRs") and methods of generating such NKRs are disclosed in commonly owned and co-pending US Publication No. 2010/0143997.

[00148] In accordance with the invention, any number of mutations can be made to a KARI enzyme, and in a preferred aspect, multiple mutations can be made to a KARI enzyme to result in an increased ability to utilize NADH for the conversion of acetoiactate to 2,3-dihydroxyisovaierate. Such mutations include point mutations, frame shift mutations, deletions, and insertions, with one or more (e.g. , one, two, three, four, five or more, etc.) point mutations preferred.

[00149] Mutations may be introduced into naturally existing KARI enzymes to create NKRs using any methodology known to those skilled in the art. Mutations may be introduced randomly by, for example, conducting a PGR reaction in the presence of manganese as a divalent metal ion cofactor. Alternatively, oligonucleotide directed mutagenesis may be used to create the NKRs which allows for all possible classes of base pair changes at any determined site along the encoding DNA molecule. In general, this technique involves annealing an oligonucleotide complementary (except for one or more mismatches) to a single stranded nucleotide sequence coding for the KARI enzyme of interest. The mismatched oligonucleotide is then extended by DNA polymerase, generating a double-stranded DNA molecule which contains the desired change in sequence in one strand. The changes in sequence can, for example, result in the deletion, substitution, or insertion of an amino acid. The double-stranded polynucleotide can then be inserted into an appropriate expression vector, and a mutant or modified polypeptide can thus be produced. The above-described oligonucleotide directed mutagenesis can, for example, be carried out via PGR.

[00150] Dihydroxy acid dehydratases capable of converting 2,3- dihydroxyisovalerate to α-ketoisovaierate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including E, cols (GenBank Accession No. YP__028248.1 ), L. iactis (GenBank Accession No. NP_267379.1 ), S. mutans (GenBank Accession No. NP__722414.1 ), M. stadtmanae (GenBank Accession No. YP_ 448586.1 ), M. tractuosa (GenBank Accession No. YP_004053736.1 ), Eubacterium SCB49 (GenBank Accession No. ZPJ31890126.1 ), G. forsetti (GenBank Accession No. YP 862145,1 ), Y. lipolytica (GenBank Accession No. XP__502180.2), N. crassa (GenBank Accession No. XP__963045.1 ), or S. cerevisiae ILV3 (GenBank Accession No. NP__012550.1 ). Additional dihydroxy acid dehydratases capable of 2,3-dihydroxyisovalerate to α-ketoisovalerate are described in commonly owned US Publication No. 201 1/0076733 (now US Patent No. 8,232,089). Motifs shared in common between the majority of dihydroxy acid dehydratases include:

SLXSRXXIA (SEQ ID NO: 30),

CDKXXPG (SEQ ID NO: 31 ),

GXCXGXXTAN (SEQ ID NO: 32),

GGSTN (SEQ ID NO: 33),

GPXGXPGMRXE (SEQ ID NO: 34),

ALXTDGRXSG (SEQ ID NO: 35), and

GHXXPEA (SEQ ID NO: 36) motifs at amino acid positions corresponding to the 93-101 , 122-128, 193-202, 276- 280, 482-491 , 509-518, and 526-532 residues, respectively, of the E, coii di hydroxy acid dehydratase encoded by HvD. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit dibydroxy acid dehydratase activity.

[00151] 2-keto-acid decarboxylases capable of converting α-ketoisova!erate to isobutyra!dehyde may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including L. lactis kivD (GenBank Accession No. YP 003353820,1 ), E. cloacae (GenBank Accession No. P23234.1 ), M. smegmatis (GenBank Accession No. A0R480.1 ), M. tuberculosis (GenBank Accession No. 053865.1 ), M. avium (GenBank Accession No. Q742Q2.1 , A. brasilense (GenBank Accession No. P51852.1 ), L lactis kdcA (GenBank Accession No. AAS49166.1 ), S. epidermidis (GenBank Accession No. NP_765765.1 ), M, caseo!yticus (GenBank Accession No. YP_002560734.1 ), B. megaterium (GenBank Accession No. YPJ)Q3561644.1 ), S. cerevisiae ARO10 (GenBank Accession No. NPJ310668.1 ), or S. cerevisiae THI3 (GenBank Accession No. CAA98646.1 ). Additional 2-keto-acid decarboxylases capable of converting a-ketoisovalerate to isobutyraldehyde are described in commonly owned US Publication No. 201 1/0076733 (now US Patent No. 8,232,089). Motifs shared in common between the majority of 2-keto-acid decarboxylases include:

FG(V/I)(P/S)G(D/E)(Y/F) (SEQ ID NO: 37),

(T/V)T(F/Y)G(V/A)G(E/A)(L/F)(S/N) (SEQ ID NO: 38),

N(G/A)(L/i/V)AG(S/A)(Y/F)AE (SEQ ID NO: 39),

(V/!)(L/!/V)X!(V/T/S)G (SEQ ID NO: 40), and

GDG(S/A)(L/F/A)Q(L/M)T (SEQ ID NO: 41 ) motifs at amino acid positions corresponding to the 21 -27, 70-78, 81 -89, 93-98, and 428-435 residues, respectively, of the L. lactis 2-keto-acid decarboxylase encoded by kivD, Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 2-keto-acid decarboxylase activity.

[00152] Alcohol dehydrogenases capable of converting isobutyraldehyde to isobutanoi may be derived from a variety of sources (e.g. , bacterial, yeast, Archaea, etc.), including L lactis (GenBank Accession No. YP__003354381 ), B. cereus (GenBank Accession No. YP 001374103.1 ), N, meningitidis (GenBank Accession No. CBA03965.1 ), S. sanguinis (GenBank Accession No, YP _.001035842.1 ), L. brevis (GenBank Accession No. YP_794451 .1 ), B. thuringiensis (GenBank Accession No. ZP 04101989.1 ), P, acidiiactici (GenBank Accession No. ZP_06197454.1 ), β. subtiiis (GenBank Accession No. EHA31 1 15.1 ), N. crassa (GenBank Accession No. CAB91241 .1 ) or S. cerevisiae ADH6 (GenBank Accession No. NP_014051 .1 ). Additional alcohol dehydrogenases capable of converting isobutyra!dehyde to isobutanol are described in commonly owned US Publication Nos. 201 1/0076733 (now US Patent No. 8,232,089) and 201 1/0201072. Motifs shared in common between the majority of alcohol dehydrogenases include:

C(H/G)(T/S)D(L/I)H (SEQ !D NO: 42),

GHEXXGXV (SEQ ID NO: 43),

(L/V)(Q/K/E)(V/I/K)G(D/Q)(R/H)(V/A) (SEQ ID NO: 44),

CXXCXXC (SEQ ID NO: 45),

(C/A)(A/G/D)(G/A)XT(T/V) (SEQ ID NO: 46), and

G(L/A/C)G(G/P)(L/I/V)G (SEQ ID NO: 47)

motifs at amino acid positions corresponding to the 39-44, 59-66, 76-82, 91 -97, 147- 152, and 171 -176 residues, respectively, of the L. lactis alcohol dehydrogenase encoded by adhA, Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit alcohol dehydrogenase activity.

[00153] In an exemplary embodiment, pathway steps 2 and 5 of the isobutanol pathway may be carried out by KARl and ADH enzymes that utilize NADH (rather than NADPH) as a cofactor. It has been found previously that utilization of NADH- dependent KARl (NKR) and ADH enzymes to catalyze pathway steps 2 and 5, respectively, surprisingly enables production of isobutanol at theoretical yield and/or under anaerobic conditions. See, e.g., commonly owned and co-pending patent publication US 2010/0143997. An example of an NADH-dependent isobutanol pathway is illustrated in Figure 2. Thus, in one embodiment, the recombinant microorganisms of the present invention may use an NKR to catalyze the conversion of acetoiacfate to produce 2,3-dihydroxyisovaierate. In another embodiment, the recombinant microorganisms of the present invention may use an NADH-dependent ADH to catalyze the conversion of isobutyraidehyde to produce isobutanol. In yet another embodiment, the recombinant microorganisms of the present invention may use both an NKR to catalyze the conversion of acetoiactate to produce 2,3- dihydroxyisovaierate, and an NADH-dependent ADH to catalyze the conversion of isobutyraidehyde to produce isobutanol.

[00154] In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutanol. 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-isovaierate. 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.

[00155] 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.

The M icroorgan ism i General

[00156] As described herein, the recombinant microorganisms of the present invention can express a plurality of heterologous and/or native enzymes involved in pathways for the production of a pyruvate-derived metabolite (e.g. , isobutanol).

[00157] As described herein, "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 and/or extracellular metabolite. As described herein, the introduction of genetic material into and/or the modification of the expression of native genes in a parental microorganism results in a new or modified ability to produce a desired pyruvate- derived metabolite (e.g. , isobutanol) 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 a pyruvate-derived metabolite (e.g. , isobutanoi) and may also include additional elements for the expression and/or regulation of expression of these genes, e.g. , promoter sequences.

[00158] In addition to the introduction of a genetic materia! into a host or parental microorganism, an engineered or modified microorganism can also include the 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, to improve the flux of a metabolite down a desired pathway, and/or to reduce the production of by-products).

[00159] 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~ketoisovalerate), 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.

[00160] 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 polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art. [00161] 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.

[00162] 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 84 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, in a process sometimes called "codon optimization" or "controlling for species codon bias."

[00163] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray ef a/., 1989, Nuci Acids Res. 17: 477-508) can be prepared_., for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coii commonly use UAA as the stop codon (Dalphin et a/., 1998, 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. 8,015,891 , and the references cited therein.

[00164] Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure. The native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabo!ic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure,

[00165] In addition, homologs of enzymes useful for generating a pyruvate-derived metabolite (e.g. , isobutanol) are encompassed by the microorganisms and methods provided herein.

[00166] 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 typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[00167] When "homologous" is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g. , charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (See, e.g., Pearson W.R., 1994, Methods in Mol Biol 25: 385-89).

[00168] The following six groups each contain amino acids that are conservative substitutions for one another: 1 ) Serine (S), Threonine (T); 2) Aspar ic 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 8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[00169] 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 2009/0228991 . 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 U.S. Pat. No. 8,017,375.

[00170] It is understood that a range of microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of a desired pyruvate- derived metabolite (e.g., isobutanol). In various embodiments, microorganisms may be selected from yeast microorganisms. Yeast microorganisms for the production of a desired pyruvate-derived metabolite (e.g., isobutanol) may be selected based on certain characteristics:

[00171] One characteristic may include the property that the microorganism is selected to convert various carbon sources into a desired pyruvate-derived metabolite (e.g., isobutanol). 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 U.S. Pat. No. 8,017,375. Accordingly, in one embodiment, the recombinant microorganism herein disclosed can convert a variety of carbon sources, including but not limited to glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof, to one or more pyruvate-derived metabolites (e.g., isobutanol).

[00172] The recombinant microorganism may thus further include a pathway for the production of a desired pyruvate-derived metabolite (e.g., isobutanol) 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 xylulose via a xylitol dehydrogenase (XDH) enzyme. The xylulose is then phosphorylated via a xylu!okinase (XK) enzyme. This pathway operates inefficiently in yeast species because it introduces a redox imbalance in the cell. The xylose-to-xylitol step uses primarily NADPH as a cofactor (generating NADP+), whereas the xylitol-to-xyiuiose 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.

[00173] 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 ai, US2008/0234364, which is herein incorporated by reference in its entirety. In an embodiment according to this aspect, the exogenous XI gene is operativeiy linked to promoter and terminator sequences that are functional in the yeast ceil. 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 xyiuiokinase (XK) gene operativeiy linked to promoter and terminator sequences that are functional in the yeast ceil. In one embodiment, the xyiuiokinase (XK) gene is overexpressed.

[00174] In one embodiment, the yeast microorganism has reduced or no pyruvate decarboxylase (PDC) activity. PDC catalyzes the decarboxylation of pyruvate to acetaidehyde, which is then reduced to ethanoi by ADH via an oxidation of NADH to NAD+. Ethanoi production is the main pathway to oxidize the NADH from glycolysis. Deletion, disruption, or mutation of this pathway increases the pyruvate and the reducing equivalents (NADH) available for a biosynthetic pathway which uses pyruvate as the starting material and/or as an intermediate. Accordingly, deletion, disruption, or mutation of one or more genes encoding for pyruvate decarboxylase and/or a positive transcriptional regulator thereof can further increase the yield of the desired pyruvate-derived metabolite {e.g., isobutanoi). In one embodiment, said pyruvate decarboxylase gene targeted for disruption, deletion, or mutation is selected from the group consisting of PDC1, PDC5, and PDC8, or homologs or variants thereof. In another embodiment, ail three of PDC1, PDC5, and PDC6 are targeted for disruption, deletion, or mutation. In yet another embodiment, a positive transcriptional regulator of the PDC1, PDC5, and/or PDC6 is targeted for disruption, deletion or mutation. In one embodiment, said positive transcriptional regulator is PDC2, or homologs or variants thereof.

[00175] As is understood by those skilled in the art, there are several additional mechanisms available for reducing or disrupting the activity of a protein encoded by PDC1, PDC5, PDC6, and/or PDC2, including, but not limited to, the use of a regulated promoter, use of a weak constitutive promoter, disruption of one of the two copies of the gene in a diploid yeast, disruption of both copies of the gene in a diploid yeast, expression of an anti-sense nucleic acid, expression of an siRNA, over expression of a negative regulator of the endogenous promoter, alteration of the activity of an endogenous or heterologous gene, use of a heterologous gene with lower specific activity, the like or combinations thereof. Yeast strains with reduced PDC activity are described in commonly owned U.S. Pat. No. 8.017,375, as well as commonly owned and co-pending US Patent Publication No. 201 1/0183392.

[00176] In another embodiment, the microorganism has reduced glycerol-3- phosphate dehydrogenase (GPD) activity. GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+. Glycerol is then produced from G3P by Glycerol-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 production of a pyruvate-derived metabolite (e.g., isobutanoi). Thus, disruption, deletion, or mutation of the genes encoding for glyceroi-3-phosphate dehydrogenases can further increase the yield of the desired pyruvate-derived metabolite (e.g., isobutanoi). Yeast strains with reduced GPD activity are described in commonly owned and co-pending US Patent Publication Nos. 201 1/0020889 and 201 1/0183392.

[00177] In yet another embodiment, the microorganism has reduced 3-keto acid reductase (3-KAR) activity. 3-KARs catalyze the conversion of 3-keto acids (e.g., aceto!actate) to 3-hydroxyacids (e.g., DH2MB). Yeast strains with reduced 3-KAR activity are described in commonly owned U.S. Pat. Nos. 8,133,715, 8,153,415, and 8,158,404, which are herein incorporated by reference in their entireties.

[00178] In yet another embodiment, the microorganism has reduced aldehyde dehydrogenase (ALDH) activity. Aldehyde dehydrogenases catalyze the conversion of aldehydes (e.g., isobutyraldehyde) to acid by-products (e.g., isobutyrate). Yeast strains with reduced ALDH activity are described in commonly owned U.S. Pat. Nos. 8,133,715, 8,153,415, and 8,158,404, which are herein incorporated by reference in their entireties.

[00179] In yet another embodiment, the microorganism has increased activator of ferrous transport (AFT) activity. Increased AFT activity has been demonstrated to improve the activity of DHAD and concomitantly improve the production of the beneficial metabolites in recombinant microorganisms comprising a DHAD-requiring biosynthetic pathway. See, e.g., commonly owned U.S. Pat. Nos. 8,017,378 and 8,071 ,358, which are herein incorporated by reference in its entirety for all purposes. The microorganisms of the present application may be engineered to have increased AFT activity via the overexpression of one or more AFT polynucleotides and/or via the expression of one or more polynucleotides encoding one or more constitutively active AFT polypeptides. In some embodiments, the AFT polynucleotide to be overexpressed is a polynucleotide encoding a constitutively active AFT polypeptide.

[00180] In one embodiment, the yeast microorganisms may be selected from the "Saccharomyces Yeast Ciade", as described in commonly owned U.S. Pat. No. 8,017,375.

[00181] 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. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids derived from these species (Masneuf et a!., 1998, Yeast 7: 61 - 72).

[00182] An ancient whole genome duplication (WGD) event occurred during the evolution of the hemiascomycete yeast and was discovered using comparative genomic tools (Kellis et a/., 2004, Nature 428: 617-24; Dujon et ai, 2004, Nature 430:35-44; Langkjaer et al., 2003, Nature 428: 848-52; Wolfe et al., 1997, Nature 387: 708-13). Using this major evolutionary event, yeast 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).

[00183] 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. casteili, and C. glabrata.

[00184] 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. waitii, K, lactis, C. tropicalis, P, pastoris, P. anomala, P. stipitis, I, orientalis, I, occidentaiis, I, scutulata, D. hansenii, H. anomala, Y. lipolytlca, and S. pombe.

[00185] A yeast microorganism may be either Crabtree-negative or Crabtree- positive as described in described in commonly owned U.S. Pat. No. 8,017,375. In one embodiment the yeast microorganism may be selected from yeast with a Crabtree-negative phenotype including but not limited to the following genera: Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida. Crabtree-negative species include but are not limited to: 8. kluyveri, K. lactis, K. marxianus, P. anomala, P. stipitis, I. orientalis, I. occidentaiis, I. scutulata, H. anomala, and C. utiiis. In another embodiment, the yeast microorganism may be selected from 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. casteili, K. thermotolerans, C. glabrata, Z bailli, Z, rouxii, D. hansenii, P. pastorius, and S. pombe.

[00186] Another characteristic may include the property that the microorganism is that it is non-fermenting. In other words, it cannot metabolize a carbon source anaerobically 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, the main pathway to oxidize the NADH from glycolysis is through the production of ethanoi. Ethanol 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 isobutanol 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 pyruvate-derived metabolites such as isobutanol. Accordingly, deletion of one or more PDC genes may increase yield and productivity of a desired pyruvate-derived metabolite (e.g., isobutanol).

[00187] 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, Rhodotoru!a, Myxozyma, or Candida. In a specific embodiment, the non-fermenting yeast is C. xestobii.

Methods in General Identification of Enzyme Homoloqs

[00188] Any method can be used to identify genes that encode for enzymes that are homologous to the genes described herein. Generally, genes that are homologous or similar to the enzymes described herein may be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar genes and/or homologous or similar enzymes will have functional, structural, or genetic similarities. [[0000118899]] TTeecchhnniiqquueess kknnoowwnn ttoo tthhoossee sskkiilllleedd iinn tthhee aarrtt mmaayy bbee ssuuiittaabbllee ttoo iiddeennttiiffyy aaddddiittiioonnaall hhoommoollooggoouuss ggeenneess aanndd hhoommoollooggoouuss eennzzyymmeess.. GGeenneerraallllyy,, aannaallooggoouuss ggeenneess aanndd//oorr aannaallooggoouuss eennzzyymmeess ccaann bbee iiddeennttiiffiieedd bbyy ffuunnccttiioonnaall aannaallyyssiiss aanndd wwiillll hhaavvee ffuunnccttiioonnaall ssiimmiillaarriittiieess.. TTeecchhnniiqquueess kknnoowwnn ttoo tthhoossee sskkiilllleedd iinn tthhee aarrtt mmaayy bbee ssuuiittaabbllee ttoo iiddeennttiiffyy aannaallooggoouuss ggeenneess aanndd aannaallooggoouuss eennzzyymmeess.. FFoorr eexxaammppllee,, ttoo iiddeennttiiffyy hhoommoollooggoouuss oorr aannaallooggoouuss ggeenneess,, pprrootteeiinnss,, oorr eennzzyymmeess,, tteecchhnniiqquueess mmaayy iinncclluuddee,, bbuutt nnoott lliimmiitteedd ttoo,, cclloonniinngg aa ggeennee bbyy PPGGRR uussiinngg pprriimmeerrss bbaasseedd oonn aa ppuubblliisshheedd sseeqquueennccee ooff aa ggeennee//eennzzyymmee oorr bbyy ddeeggeenneerraattee PPGGRR uussiinngg ddeeggeenneerraattee pprriimmeerrss ddeessiiggnneedd ttoo aammpplliiffyy aa ccoonnsseerrvveedd rreeggiioonn aammoonngg kkeettooll--aacciidd rreedduuccttooiissoommeerraassee ggeenneess.. FFuurrtthheerr,, oonnee sskkiilllleedd iinn tthhee aarrtt ccaann uussee tteecchhnniiqquueess ttoo iiddeennttiiffyy hhoommoollooggoouuss oorr aannaallooggoouuss ggeenneess,, pprrootteeiinnss,, oorr eennzzyymmeess wwiitthh ffuunnccttiioonnaall hhoommoollooggyy oorr ssiimmiillaarriittyy.. TTeecchhnniiqquueess iinncclluuddee eexxaammiinniinngg aa cceellll oorr cceellll ccuullttuurree ffoorr tthhee ccaattaallyyttiicc aaccttiivviittyy ooff aann eennzzyymmee tthhrroouugghh iinn vviittrroo eennzzyymmee aassssaayyss ffoorr ssaaiidd aaccttiivviittyy ((ee..gg.. aass ddeessccrriibbeedd hheerreeiinn oorr iinn KKiirriittaannii,, KK.. BBrraanncchheedd--CChhaaiinn AAmmiinnoo AAcciiddss MMeetthhooddss EEnnzzyymmooiiooggyy,, 11997700)),, tthheenn iissoollaattiinngg tthhee eennzzyymmee wwiitthh ssaaiidd aaccttiivviittyy tthhrroouugghh ppuurriiffiiccaattiioonn,, ddeetteerrmmiinniinngg tthhee pprrootteeiinn sseeqquueennccee ooff tthhee eennzzyymmee tthhrroouugghh tteecchhnniiqquueess ssuucchh aass EEddmmaann ddeeggrraaddaattiioonn,, ddeessiiggnn ooff PPGGRR pprriimmeerrss ttoo tthhee lliikkeellyy nnuucclleeiicc aacciidd sseeqquueennccee,, aammpplliiffiiccaattiioonn ooff ssaaiidd DDNNAA sseeqquueennccee tthhrroouugghh PPGGRR,, aanndd cclloonniinngg ooff ssaaiidd nnuucciieeiicc aacciidd sseeqquueennccee.. TToo iiddeennttiiffyy hhoommoollooggoouuss oorr ssiimmiillaarr ggeenneess aanndd//oorr hhoommoollooggoouuss oorr ssiimmiillaarr eennzzyymmeess,, aannaallooggoouuss ggeenneess aanndd//oorr aannaallooggoouuss eennzzyymmeess oorr pprrootteeiinnss,, tteecchhnniiqquueess aallssoo iinncclluuddee ccoommppaarriissoonn ooff ddaattaa ccoonncceerrnniinngg aa ccaannddiiddaattee ggeennee oorr eennzzyymmee wwiitthh ddaattaabbaasseess ssuucchh aass BBRREENNDDAA,, KKEEGGGG,, oorr MeettaaCCYYCC.. TThhee ccaannddiiddaattee ggeennee oorr eennzzyymmee mmaayy bbee iiddeennttiiffiieedd wwiitthhiinn tthhee aabboovvee mmeennttiioonneedd ddaattaabbaasseess iinn aaccccoorrddaannccee wwiitthh tthhee tteeaacchhiinnggss hheerreeiinn..

[00190] In various embodiments, the endogenous nucieic acid or polypeptide identified herein is the S. cerevisiae version of the nucleic acid or polypeptide (e.g., Mad , Afg2, Aim25, Axl1 , Cdc20, Cdc60, Coq8, Crr1 , Ctf19, Ctrl , Ctr3, Ctt1 , Cup1 , Cup9, Das1 , Dbf20, DpM , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun26, Gnd2, Gph1 , His5, Hnm1 , Hos4, Irc7, !sy1 , Kem1 , Kre8, Lsb1 , LsrrsS, Mai33, Mdm31 , Mrm2, Mss4, Npc2, Nrd1 , Oad , Oms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , Rad50, Reel , Rgm Rpa34, Rpc40, RpU b, Rpl7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr190, Swd1 , Swh1 , Tgi2, Tma20, Tps2, Trm10, Ubx3, Ubx6, Ume6, Utr1 , Vcx1 , Vhs2, Vik1 , Vip1 , Vtc3, Yar009c, Yar069c, Ycr025c, Ydi129w, Ygr137w, YiM 02c, Yjl218w, Ykl063c, Y!h47, Yl!059c, YirQ35c~a, Ylr123c, Ylr410w-a, Ylr410w-b, Ymr320w, Yni017c, Yoi079w, Yoi153c, Yor1 1 1w, Ypl251 w, Ypr123c, Ypr170c, Ysp1 , Ccs1 , Sod1 , Ace1 , etc.) Any meihod can be used to identify genes that encode for the endogenous polypeptide of interest in a variety of yeast strains. Generally, genes that are homologous or similar to the endogenous polypeptide of interest can be identified by functional, structural, and/or genetic analysis. Homologous or similar polypeptides will generally have functional, structural, or genetic similarities.

[00191] The chromosomal location of the genes encoding endogenous S. cerevisiae polypeptides (e.g., Mad , Afg2, Aim25, Axl1 , Cdc20, Cdc80, Coq6, Crr1 , Ctf19, Ctrl , Ctr3, Ctt1 , Cup1 , Cup9, Das1 , Dbf20, DpM , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun26, Gnd2, Gph1 , His5, Hnm1 , Hos4, Irc7, Isy1 , Kem1 , Kre8, Lsb1 , Lsm5, Mal33, Mdm31 , Mrm2, Mss4, Npc2, Nrd1 , Oad , Oms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , Rad50, Reel , Rgm1 , Rpa34, Rpc40, RpM b, Rpl7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr190, Swd1 , Swh1 , Tgi2, Tma20, Tps2, Trml O, Ubx3, Ubx6, Ume8, Utr1 , Vcx1 , Vhs2, Vik1 , Vip1 , Vtc3, Yar009c, Yar089c, Ycr025c, Ydl129w, Ygr137w, Yil102c, Yji218w, Ykl063c, Ylh47, YII059c, Ylr035c-a, Yir123c, Ylr410w~a, Yir410w-b, Ymr320w, Ynl017c, Yol079w, Yoi153c, Yor1 1 1w, Ypi251w, Ypr123c, Ypr170c, Ysp1 , Ccs1 , Sod1 , Ace1 , etc.) may be syntenic to chromosomes in many related yeast [Byrne, K.R and K. H. Wolfe (2005) "The Yeast Gene Order Browser: combining cu rated homology and syntenic context reveals gene fate in polyploid species." Genome Res. 15(10):1456-61 . Scannel!, D. R., K. P. Byrne, J. L. Gordon, S. Wong, and K. H. Wolfe (2006) "Multiple rounds of speciation associated with reciprocal gene loss in polyploidy yeasts." Nature 440: 341 -5. Scanneli, D. R., A. C. Frank, G. C. Conant, K. P. Byrne, M. Woolfit, and K. H. Wolfe (2007)" Independent sorting-out of thousands of duplicated gene pairs in two yeast species descended from a whole-genome duplication." Proc Natl Acad Sci U S A 104: 8397-402]. Using this syntenic relationship, species-specific versions of these genes are readily identified in a variety of yeast, including but not limited to, Ashbya gossypii, Candida g!abrata, Kluyveromyces iactis, K!uyveromyces polyspora, Kluyveromyces thermotolerans, Kluyveromyces waiiii, Saccharomyces kluyveri, Saccharomyces castelli, Saccharomyces bayanus, and Zygosaccharomyces rouxli, [00192] As will be understood by one skilled in the art, this technique is therefore additionally suitable for the identification homologous e.g., Mad , Afg2, Aim25, AxM , Cdc20, Cdc60, Coq6, Crr1 , Ctf19, Ctrl , Ctr3, Ctt1 , Cup1 , Cup9, Das1 , Dbf20, Dpl1 , Dsd1 , Ede1 , Egd2, Fet4, Fre Fre7, Ftr1 , Fun26, Gnd2, Gph His5, Hnm1 , Hos4, Irc7, !syl , Kem1 , Kre8, Lsb1 , Lsm5, Ma!33, Mdm31 , Mrm2, Mss4, Npc2, Nrd1 , Oac Oms1 , Pho23, PhoS9, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , Rad50, Reel , Rgm1 , Rpa34, Rpc40, RpM b, Rpl7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr19G, Swd1 , Swh1 , Tgl2, 101320, Tps2, Trm10, Ubx3, Ubx8, Ume6, Utr1 , Vcx1 , Vhs2, Vik1 , Vip1 , Vic3, Yar009c, Yar069c, Ycr025c, Ydl129w, Ygr137w, Yil102c, Yjl218w, Yki063c, Ylh47, YII059c, Ylr035c-a, Yir123c, Ylr410w-a, Ylr410w-b, Ymr320w, Yni017c, Yoi079w, Yoi153c, Yos 1 1 w, Yp!251w, Ypr123c, Ypr170c, Ysp1 , Ccs1 , Sod1 , and Acel polypeptides in yeast other than S. cerevisiae.

Genetic Insertions and Deletions

[00193] 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., Gie z et a/., 1992, Nuc Acids Res. 27: 69-74; !to et aL, 1983, J. Bacteriol. 153: 183-8; and Becker et al., 1991 , Methods in Enzymo!ogy 194: 182-7.

[00194] 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 gene to be integrated (internal 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 interna! module replacing the chromosoma! 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).

[00195] 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_S bar, ble, hph, and kan. The recombinogenic sequences can be chosen at will, depending on the desired integration site suitable for the desired application.

[00196] 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).

[00197] 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-fluoro-orotic acid) containing medium and selecting for FOA resistant colonies (Boeke et al., 1984, Mol. Gen. Genet 197: 345-47).

[00198] The exogenous nucleic acid molecule contained within a yeast cell 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 cell or maintained in an episomal state that can stably be passed on ("inherited") to daughter ceils. Such extra-chromosomal genetic elements (such as plasmids, mitochondrial genome, etc.) can additionally contain selection markers that ensure the presence of such genetic elements in daughter cells. Moreover, the yeast ceils can be stably 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.

Reduction of Enzymatic Activity

[00199] Yeast microorganisms within the scope of the invention may have reduced enzymatic activity such as reduced PDC, GPD, ALDH, or 3-KAR activity. The term "reduced" as used herein with respect to a particular polypeptide activity refers to a lower level of polypeptide activity than that measured in a comparable yeast ceil of the same species. The term reduced also refers to the elimination of polypeptide activity as compared to a comparable yeast cell of the same species. Thus, yeast ceils lacking activity for an endogenous PDC, GPD, ALDH, or 3-KAR are considered to have reduced activity for PDC, GPD, ALDH, or 3-KAR since most, if not ail, comparable yeast strains have at least some activity for PDC, GPD, ALDH, or 3- KAR. Such reduced PDC, GPD, ALDH, or 3-KAR activities can be the result of lower PDC, GPD, ALDH, or 3-KAR concentration (e.g., via reduced expression), lower specific activity of the PDC, GPD, ALDH, or 3-KAR, or a combination thereof. Many different methods can be used to make yeast having reduced PDC, GPD, ALDH, or 3-KAR activity. For example, a yeast cell can be engineered to have a disrupted PDC-, GPD-, ALDH-, or 3-KAR-encoding locus using common mutagenesis or knock-out technology. See, e.g. , Methods in Yeast Genetics (1997 edition), Adams, Gottschiing, Kaiser, and Stems, Cold Spring Harbor Press (1998). In addition, a yeast ceil can be engineered to partially or completely remove the coding sequence for a particular PDC, GPD, ALDH, or 3-KAR. Furthermore, the promoter sequence and/or associated regulatory elements can be mutated, disrupted, or deleted to reduce the expression of a PDC, GPD, ALDH, or 3-KAR. Moreover, certain point-mutation(s) can be introduced which results in a PDC, GPD, ALDH, or 3-KAR 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 PDC, GPD, ALDH, or 3-KAR activities.

[00200] Alternatively, antisense technology can be used to reduce PDC, GPD, ALDH, or 3-KAR activity. For example, yeasts can be engineered to contain a cDNA that encodes an antisense molecule that prevents a PDC, GPD, ALDH, or 3-KAR 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 moiecule cleaves RNA.

Overexpression of Heterologous Genes

[00201] 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 Kiuyveromyces and Saccharomyces are well known {see, e.g., U.S. Pat. Nos. 4,859,598 and 4,943,529, for Kiuyveromyces and, e.g., Gellissen ef a/., 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, b!e, hph, or kan.

[00202] 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.

[00203] As described herein, any yeast within the scope of the disclosure can be identified by selection techniques specific to the particular polypeptide {e.g. an isobutanoi pathway 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 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 cell 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 ceil with a vector encoding acetoiactate ssyynntthhaassee aanndd ddeetteeccttiinngg iinnccrreeaasseedd aacceettoollaaccttaattee ccoonncceennttrraattiioonnss ccoommppaarreedd ttoo aa cceeiill wwiitthhoouutt tthhee vveeccttoorr iinnddiiccaatteess tthhaatt tthhee vveeccttoorr iiss bbootthh pprreesseenntt aanndd tthhaatt tthhee ggeennee pprroodduucctt iiss aaccttiivvee.. MMeetthhooddss ffoorr ddeetteeccttiinngg ssppeecciiffiicc eennzzyymmaattiicc aaccttiivviittiieess oorr tthhee pprreesseennccee ooff ppaarrttiiccuullaarr pprroodduuccttss aarree wweellll kknnoowwnn ttoo tthhoossee sskkiilllleedd iinn tthhee aartrt.. FFoorr eexxaammppllee,, tthhee pprreesseennccee ooff aacceettoollaaccttaattee ccaann bbee ddeetteerrmmiinneedd aass ddeessccrriibbeedd bbyy HHuuggeennhhooiittzz aanndd SSttaarrrreennbbuurrgg,, 11999922,, AAppppll.. MMiiccrroo.. BBlloott,. 3388::1177--2222,,

[00204] Yeast microorganisms of the invention may be further engineered to have increased activity of enzymes (e.g. , increased activity of enzymes involved in an isobutanoi producing metabolic pathway). 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 ceil 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 isobutanoi.

[00205] 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 K_M 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 Yeast Microorganisms for Production of Renewable Chemicals

[00206] 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.

[00207] In one aspect, the present invention provides a method of producing a beneficial metabolite from a recombinant yeast microorganism described herein. In one embodiment, the recombinant yeast microorganism comprises a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to alter the regulation, expression, and/or activity of a Mad pathway component. In one embodiment, the recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins as described herein. In another embodiment, the recombinant yeast microorganism is engineered to express or overexpress one or more polynucleotides encoding one or more constitutively active mutant Mad proteins as described herein. In yet another embodiment, the recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins regulated by a Mad protein, e.g., Ctrl and/or Fre1 as described herein. In yet another embodiment, the recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulates Mad , e.g., Ccs1 and/or Sod1 . In yet another embodiment, the recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Mad . In yet another aspect, the recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a DNA- binding protein activated by copper-replete conditions, e.g., Ace1 .

[00208] In an exemplary embodiment, the Fe-S cluster containing protein is DHAD and the beneficial metabolite is selected from isobutanol, 3-methy!-1 -butanol, 2- methyl-1 -butanoi, valine, isoleucine, leucine, and pantothenic acid.

[00209] In a further exemplary embodiment, the beneficial metabolite is isobutanol. Thus, in a related aspect, the present invention provides a method of producing isobutanol from a recombinant yeast microorganism described herein. In one embodiment, the recombinant yeast microorganism comprises an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said recombinant yeast microorganism is engineered to alter the regulation, expression, and/or activity of a Mad pathway component. In one embodiment, the recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins as described herein. In another embodiment, the recombinant yeast microorganism is engineered to express or overexpress one or more polynucleotides encoding one or more constitutively active mutant Mad proteins as described herein. In yet another embodiment, the recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins regulated by a Mad protein, e.g., Ctrl and/or Fre1 as described herein. In yet another embodiment, the recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulates Mad , e.g., Ccs1 and/or Sod1 . !n yet another embodiment, the recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Ma . In yet another aspect, the recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a DNA- binding protein activated by copper-replete conditions, e.g. , Ace1 .

[00210] In a method to produce a beneficial metabolite (e.g., isobutanol) from a carbon source, the recombinant yeast microorganism is cultured in an appropriate culture medium containing a carbon source. In certain embodiments, the method further includes isolating the beneficial metabolite (e.g., isobutanol) from the culture medium. For example, a beneficial metabolite (e.g., isobutanol) 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.

[00211] In certain exemplary embodiments, the beneficial metabolite is selected from isobutanol , 3-methyl-1 -butanoi, 2-methyl-1 -butanol, valine, iso!eucine, leucine, and pantothenic acid.

[00212] In one embodiment, the recombinant microorganism may produce the beneficial metabolite (e.g., isobutanol) from a carbon source at a yield of at least 5 percent theoretical. In another embodiment, the microorganism may produce the beneficial metabolite (e.g. , isobutanol) 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 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% theoretical. In a specific embodiment, the pyruvate-derived metabolite is isobutanol.

Distillers Dried Grains Comprising Spent Yeas! Biocataiysts

[00213] In an economic fermentation process, as many of the products of the fermentation as possible, including the co-products that contain biocataiyst cell material, should have value. Insoluble material produced during fermentations using grain feedstocks, like corn, is frequently sold as protein and vitamin rich animal feed called distillers dried grains (DDG). See, e.g., commonly owned U.S. Publication No. 2009/0215137, which is herein incorporated by reference in its entirety for all purposes. As used herein, the term "DDG" generally refers to the solids remaining after a fermentation, usually consisting of unconsumed feedstock solids, remaining nutrients, protein, fiber, and oil, as well as spent yeast biocatalysts or cell debris therefrom that are recovered by further processing from the fermentation, usually by a solids separation step such as centrifugation.

[00214] Distillers dried grains may also include soluble residual material from the fermentation, or syrup, and are then referred to as "distillers dried grains and solubles" (DDGS). Use of DDG or DDGS as animal feed is an economical use of the spent biocataiyst following an industrial scale fermentation process.

[00215] Accordingly, in one aspect, the present invention provides an animal feed product comprised of DDG derived from a fermentation process for the production of a beneficial metabolite (e.g., isobutanol), wherein said DDG comprise a spent yeast biocataiyst of the present invention. In an exemplary embodiment, said spent yeast biocataiyst has been engineered to comprise an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol. In certain embodiments, said spent yeast biocataiyst has additionally or independently been engineered to alter the regulation, expression, and/or activity of a Mad pathway component. In one embodiment, the spent yeast biocataiyst has been engineered to overexpress one or more polynucleotides encoding one or more Ma proteins as described herein. In another embodiment, the spent yeast biocataiyst has been engineered to express or overexpress one or more polynucleotides encoding one or more constitutively active mutant Mad proteins as described herein. In yet another embodiment, the spent yeast biocatalyst has been engineered to overexpress one or more polynucleotides encoding one or more proteins regulated by a Mad protein, e.g., Ctrl and/or Fre1 and/or Pho89 as described herein. In yet another embodiment, the spent yeast biocatalyst has been engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulates Ma , e.g., Ccs1 and/or Sod1 . in yet another embodiment, the spent yeast biocatalyst has been engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Ma . In yet another aspect, the recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a DNA- binding protein activated by copper-replete conditions, e.g., Ace1 .

[00216] In certain additional embodiments, the DDG comprising a spent yeast biocatalyst of the present invention comprise at least one additional product selected from the group consisting of unconsumed feedstock solids, nutrients, proteins, fibers, and oils.

[00217] In another aspect, the present invention provides a method for producing DDG derived from a fermentation process using a yeast biocatalyst (e.g., a recombinant yeast microorganism of the present invention), said method comprising: (a) cultivating said yeast biocatalyst in a fermentation medium comprising at least one carbon source; (b) harvesting insoluble material derived from the fermentation process, said insoluble material comprising said yeast biocatalyst; and (c) drying said insoluble material comprising said yeast biocatalyst to produce the DDG.

[00218] In certain additional embodiments, the method further comprises step (d) of adding soluble residual material from the fermentation process to said DDG to produce DDGS. In some embodiments, said DDGS comprise at least one additional product selected from the group consisting of unconsumed feedstock solids, nutrients, proteins, fibers, and oils.

[00219] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of ail 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.

EXAMPLES Example 1 : Evolution of Yeast fviuiant with Enhanced Growth and Isobutanol Production

[00220] The following example illustrates the evolution strategy by which a mutant yeast strain with enhanced growth and isobutanol production was obtained.

[00221] In this example, the daily serial transfer of yeast strains in Balch tubes with limited head space was used to generate evolved strains with an increased growth rate under low aeration condtions. Strains described in the examples disclosed herein are listed in Table 1 .

Table 1. Genotype of Strains Disclosed in Examples.

[00222] In this experiment, GEVO9094 (derived from GEVO7046) was inoculated into Balch tubes with a total volume of 13mL YPD (YPD = 1 % w/v yeast extract, 2% w/v peptone, and 2% w/v glucose) + 0.3g/L Uracil + 0.2 g/L G418 in 27rnL Balch tubes. The tubes were capped, sealed, and incubated at 33°C. The tubes were shaken at 250 RPM. The change in ODeoo (ΔΟϋβοο) was calculated by determining the difference between OD measurements taken approximately 18 hrs apart.

[00223] High throughput screening was used to identify mutants of GEVO9094 with increased growth rates and increased isobutanol titers. An isolate was considered to be a positive mutant if the value (AOD₆oo or iBuOH titer) was greater than the control average plus two standard deviations. Amongst the positive mutants identified was GEVG9682, which exhibited increased growth and isobutanoi titers as compared to the parent strain, GEVO9094 (Table 2).

Table 2. Growth and Isobutanoi Production in GEV09682 vs. GEVO9094.

[00224] Shake flask fermentations were also conducted to compare the performance of GEV09682 with GEVO9094. As shown in Table 3, growth rates and isobutanoi titers were increased in GEV09882 as compared to GEVO9094.

Table 3. Shake Flask Fermentation Data Comparing Growth and Isobutanoi Production in GEV09682 vs. GEVO9094.

Example 2: Analysis of DHAD Activity in GEVQ9682

[00225] The following example illustrates that GEV09682 has increased specific DHAD activity as compared to GEVO7046 from which it was derived.

[00226] In this example, specific DHAD activity of yeast cell lysates was measured. To measure DHAD activity, each sample was diluted in DHAD assay buffer (50 mM Tris pH 8, 5 mM MgS0₄) to a 1 :10 and a 1 :40 to 1 :100 dilution. 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. 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 2,3-dihydroxyisova!erate, i.e. , DHIV). Samples were put in a thermocyc!er at 35°C for 30 min followed by 5 minutes of incubation at 95°C. Samples were cooled to 4°C on the thermocycier and centrifuged at 3000 rcf for 5 minutes. Finally, 75 pL of supernatant was transferred to new PGR tubes and submitted for analysis by Liquid Chromatography. DHAD activity units were calculated as μΜ KIV produced/min/mg total ceil lysate protein in the assay. [00227] Tabie 4 illustrates the specific DHAD activity of GEV09682 as compared to GEVO7046 at 24 hours and 58 hours.

Table 4. DHAD specific activity in GEV09682 vs. GEVO7048.

[00228] As Tabie 4 shows, DHAD activity in GEV09882 is increased by more than 2-fold at 24 hours and by approximately 3-fold at 58 hours. Based on the positive results demonstrated in Examples 1 and 2, GEV09682 was subjected to additional analysis via genome sequencing as described in Example 3.

Example 3: Genome Sequencing of GEVQ9682

[00229] The following example demonstrates the identification of a MAC? mutation in GEV09682.

[00230] To identify relevant mutations that occurred during the evolutionary engineering of GEV09882, sequencing of GEV09882 as well as progenitor strain GEVO7046 was performed. Approximately 90 Mb of sequence data was generated for each strain and compared to the published S. cerevisiae reference strain EF4. These data were used to identify mutations linked to the improved growth, improved DHAD activity, and improved isobutanoi production in GEV09882.

[00231] Sequencing revealed that GEV09882 harbors a mutation at a codon that encodes the 271 amino acid of Mad p. Specifically, a G to A transition was identified in the MAC1 coding sequence, resulting in an amino acid change of cysteine to tyrosine at position 271 (i.e., C271 -> Y). This mutation has previously been identified in MAC1. See, e.g. , Serpe et ai , 1999, J. Bio!. Chem. 274(41 ): 2921 1 -9. The mutation, known as "UP2", i.e. , Mac1 ^UP2, results in a gain-of-function copper uptake phenotype which removes the ability of copper to inhibit Mad activity. See, e.g. , Figure 3, adapted from Serpe et ai. A similar gain-of-function mutation, known as "UP1 ", has also been characterized and occurs at the H279 residue (i.e., H279 ~> Q). See, e.g., Serpe et ai, 1999, J. Biol, Chem. 274(41 ): 2921 1 -9. Given the associated increases in growth, DHAD activity, and isobutanoi production, the Mad "UP"-mufation identified in GEV09882 implicates modification of the Mad regulatory pathway as a means to achieving commercially viable productivities, yields, and titers in a recombinant yeast microorganism. Accordingly, given the guidance provided herein, those skilled in the art will appreciate that any host modification which increases Mad activity or the activity of a protein regulated Mad can be made to improve growth and/or product yield in a recombinant yeast microorganism of interest.

[00232] 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.

[00233] While the invention has been described in connection with specific 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.

[00234] 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.

Claims

WHAT IS CLAI ED US:

1 . A recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S duster containing protein, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins or homologs thereof.

2. The recombinant yeast microorganism of claim 1 , wherein said one or more of the polynucleotides encoding said one or more Mad proteins or homologs thereof is an endogenous polynucleotide.

3. The recombinant yeast microorganism of claim 1 , wherein said one or more of the polynucleotides encoding said one or more Mad proteins or homologs thereof is an exogenous polynucleotide.

4. The recombinant yeast microorganism of any of the preceding claims, wherein said Mad protein is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEO ID NO: 8, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20.

5. A recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered express one or more polynucleotides encoding one or more constitutively active mutant Mad proteins.

6. The recombinant yeast microorganism of claim 5, wherein said constitutively active mutant Mad protein or homolog thereof comprises a mutation at one or more positions corresponding to residues 284 to 279 of the native Saccharomyces cerevisiae Mad protein (SEQ ID NO: 2).

7. The recombinant yeast microorganism of ciaim 6, wherein said constitutively active mutant Mad protein or homolog thereof comprises a mutation at a position corresponding to the cysteine 271 residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2).

8. The recombinant yeast microorganism of claim 7, wherein said cysteine 271 residue is replaced with a tyrosine residue.

9. The recombinant yeast microorganism of claim 6, wherein said constitutively active mutant Mad protein or homoiog thereof comprises a mutation at a position corresponding to the histidine 279 residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2).

10. The recombinant yeast microorganism of claim 9, wherein said histidine 279 residue is replaced with a glutamine.

1 1 . The recombinant yeast microorganism of any of claims 5-10, wherein said one or more polynucleotides encoding one or more constitutively active mutant Ma proteins is overexpressed.

12. A recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins or homologs thereof regulated by a Mad protein or homoiog thereof.

13. The recombinant yeast microorganism of claim 12, wherein said one or more polynucleotides is an endogenous polynucleotide.

14. The recombinant yeast microorganism of claim 12, wherein said one or more polynucleotides is an exogenous polynucleotide.

15. he recombinant yeast microorganism of claim 12, wherein said protein regulated by a Mad protein or homoiog thereof is selected from Afg2, Aim25, Axil , Cdc20, Cdc60, Coq6, Cn , Ctf19, Ctrl , Ctr3, Ctt1 , Cup1 , Cup9, Das Dbf20, Dpi1 , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun28, Gnd2, Gph1 , His5, Hnm1 , Hos4, !rc7, Isy1 , Kem1 , Kre6, Lsb1 , Lsm5, Mal33, Mdm31 , Mrm2, Mss4, Npc2, Nrd1 , Oaci , Oms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , RadSO, Reel , Rgm1 , Rpa34, Rpc40, RpM b, Rpl7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr190, Swd1 , Swh1 , Tgi2, Tma20, Tps2, Trm10, Ubx3, Ubx6, Ume6, Utr1 , Vcx1 , Vhs2, Vik1 , Vip1 , Vtc3, Yar009c, Yar069c, Ycr025c, Ydi129w, Ygr137w, Yil102c, Yji218w, Yki063c, Ylh47, YilG59c, YirG35c~a, Yir123c, Ylr410w~a, Ylr410w-b, Ymr320w, Yn!G17c, Yol079w, Yoi153c, Yor1 1 1 w, Ypl251w, Ypr123c, Ypr170c, and Ysp1 , or hornologs thereof.

18. The recombinant yeast microorganism of claim 15, wherein said protein regulated by a Mad protein is Ctrl .

17. The recombinant yeast microorganism of claim 15, wherein said protein regulated by a Mad protein is Fre1 .

18. The recombinant yeast microorganism of claim 15, wherein said protein regulated by a Mad is Pho89.

19. A recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulate Ma .

20. The recombinant yeast microorganism of claim 19, wherein the positive regulator of Ma is Ccs1 .

21 . The recombinant yeast microorganism of claim 19, wherein the positive regulator of Mad is Sod1 .

22. A recombinant yeast microorganism comprising a biosynthetic pathway requiring a Fe-S cluster containing protein, wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Mad .

23. The recombinant yeast microorganism of any of the preceding claims, wherein said Fe-S cluster containing protein is selected from dihydroxy acid dehydratase (DHAD), isopropyimaiate isomerase, sulfite reductase, aconitase, homoaconitase, iipoate synthase, succinate dehydrogenase, NADH ubiquinone oxidoreductase, and ubiquino!-cytochrome-c reductase.

24. The recombinant yeast microorganism of any of the preceding claims, wherein said Fe-S cluster containing protein is DHAD.

25. The recombinant yeast microorganism of any of the preceding claims, wherein said Fe-S cluster containing protein is encoded by an endogenous polynucleotide.

26. The recombinant yeast microorganism of any of the preceding claims, wherein said Fe-S cluster containing protein is encoded by an exogenous polynucleotide.

27. The recombinant yeast microorganism of any of the preceding claims, wherein said Fe-S cluster containing protein is overexpressed.

28. A recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins or homoiogs thereof.

29. The recombinant yeast microorganism of claim 28, wherein said one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an endogenous polynucleotide.

30. The recombinant yeast microorganism of claim 28, wherein said one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an exogenous polynucleotide.

31 . The recombinant yeast microorganism of any of claims 28-30, wherein said Mad 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, and SEQ ID NO: 20.

32. A recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered express one or more polynucleotides encoding one or more constitutiveiy active mutant Mad proteins.

33. The recombinant yeast microorganism of claim 32, wherein said constitutively active mutant Mad protein or homoiog thereof comprises a mutation at one or more positions corresponding to residues 284 to 279 of the native Saccharomyces cerevisiae Mad protein (SEQ ID NO: 2).

34. The recombinant yeast microorganism of claim 33, wherein said constitutively active mutant Mad protein or homoiog thereof comprises a mutation at a position corresponding to the cysteine 271 residue of the native S. cerevisiae Mad protein (SEQ !D NO: 2).

35. The recombinant yeast microorganism of claim 34, wherein said cysteine 271 residue is replaced with a tyrosine residue.

36. The recombinant yeast microorganism of claim 33, wherein said constitutively active mutant Mad protein or homoiog thereof comprises a mutation at a position corresponding to the histidine 279 residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2).

37. The recombinant yeast microorganism of claim 36, wherein said histidine 279 residue is replaced with a g!utamine.

38. The recombinant yeast microorganism of any of claims 32-37, wherein said one or more polynucleotides encoding one or more constitutively active mutant Mad proteins is overexpressed.

39. A recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins or homoiogs thereof regulated by a Mad protein or homoiog thereof.

40. The recombinant yeast microorganism of claim 39, wherein said one or more polynucleotides is an endogenous polynucleotide.

41 . The recombinant yeast microorganism of claim 39, wherein said one or more polynucleotides is an exogenous polynucleotide.

42. The recombinant yeast microorganism of claim 39, wherein said protein regulated by a Mad protein or homolog thereof is selected from Afg2, Aim25, AxM , Cdc20, Cdc60, Coq6, Crr1 , Ctf19, Ctrl , Ctr3, Ctt1 , Cup1 , Cup9, Das1 , Dbf20, Dpl1 , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun26, Gnd2, Gph1 , His5, Hnm1 , Hos4, Irc7, !sy1 , Kem1 , Kre8, Lsb1 , Lsm5, Mal33, Mdm31 , Mrm2, Mss4, Npc2, Nrd Oac1 , Oms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , Rad50, Reel , Rgm1 , Rpa34, Rpc40, RpM b, Rpl7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr190, Swd1 , Swh1 , Tgl2, Tma20, Tps2, Trml Q, Ubx3, Ubx6, Ume8, Utr1 , Vcx1 , Vhs2, Vik1 , Vip1 , Vtc3, Yar009c, Yar069c, Ycr025c, Ydi129w, Ygr137w, Yil102c, Yjl218w, Ykl063c, Yih47, YII059C, Ylr035c-a, Yir123c, Ylr410w~a, Ylr410w-b, Ymr320w, Yn!017c, Yol079w, Yoi153c, Yor1 1 1 w, Ypl251w, Ypr123c, Ypr170c, and Ysp1 , or homologs thereof.

43. The recombinant yeast microorganism of claim 42, wherein said protein regulated by a Mad protein is Ctrl .

44. The recombinant yeast microorganism of claim 42, wherein said protein regulated by a Mad protein is Fre1 .

45. he recombinant yeast microorganism of claim 42, wherein said protein regulated by a Mad is Pho89.

46. A recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulate Ma .

47. The recombinant yeast microorganism of claim 48, wherein the positive regulator of Mad is Ccs1 .

48. The recombinant yeast microorganism of claim 48, wherein the positive regulator of Mad is Sod1 .

49, A recombinant yeast microorganism comprising a DHAD-requiring biosynthetic pathway, wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Mad .

50, The recombinant yeast microorganism of any of claims 28-49, wherein said DHAD is encoded by an endogenous polynucleotide.

51 .The recombinant yeast microorganism of any of claims 28-49, wherein said DHAD is encoded by an exogenous polynucleotide.

52. The recombinant yeast microorganism of any of claims 28-51 , wherein said DHAD is overexpressed.

53. The recombinant yeast microorganism of any of claims 28-52, wherein said DHAD is localized to the cytosol.

54. he recombinant yeast microorganism of any of claims 28-52, wherein said DHAD is localized to the mitochondria.

55. The recombinant yeast microorganism of any of claims 28-54, wherein said DHAD- requiring biosynthetic pathway is a biosynthetic pathway for the production of a metabolite selected from isobutanol, 3-methyi-l -butanoI, 2-methyi-l -butanol, valine, isoleucine, leucine, and/or pantothenic acid.

56. A recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Mad proteins or homoiogs thereof.

57. The recombinant yeast microorganism of claim 58, wherein said one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an endogenous polynucleotide,

58. The recombinant yeast microorganism of claim 56, wherein said one or more of the polynucleotides encoding said one or more Mad proteins or homoiogs thereof is an exogenous polynucleotide.

59. The recombinant yeast microorganism of any of claims 56-58, wherein said Mad 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 18, and SEQ ID NO: 20.

60. A recombinant yeast microorganism comprising an isobufanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said recombinant yeast microorganism is engineered express one or more polynucleotides encoding one or more constitutively active mutant Mad proteins.

61 . The recombinant yeast microorganism of claim 60, wherein said constitutively active mutant Mad protein or homolog thereof comprises a mutation at one or more positions corresponding to residues 284 to 279 of the native Saccharomyces cerevisiae Mad protein (SEQ ID NO: 2).

62. The recombinant yeast microorganism of claim 61 , wherein said constitutively active mutant Mad protein or homolog thereof comprises a mutation at a position corresponding to the cysteine 271 residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2).

63. The recombinant yeast microorganism of claim 62, wherein said cysteine 271 residue is replaced with a tyrosine residue.

64. The recombinant yeast microorganism of claim 61 , wherein said constitutively active mutant Mad protein or homoiog thereof comprises a mutation at a position corresponding to the histidine 279 residue of the native S. cerevisiae Mad protein (SEQ ID NO: 2).

65. The recombinant yeast microorganism of claim 64, wherein said histidine 279 residue is replaced with a glutamine.

66. The recombinant yeast microorganism of any of claims 60-65, wherein said one or more polynucleotides encoding one or more constitutively active mutant Ma proteins is overexpressed.

67. A recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins or homoiogs thereof regulated by a Mad protein or homoiog thereof.

68. The recombinant yeast microorganism of claim 67, wherein said one or more polynucleotides is an endogenous polynucleotide.

69. The recombinant yeast microorganism of claim 67, wherein said one or more polynucleotides is an exogenous polynucleotide.

70. The recombinant yeast microorganism of claim 67, wherein said protein regulated by a Mad protein or homoiog thereof is selected from Afg2, Aim25, Ax!1 , Cdc20, Cdc60, Coq6, Crr1 , Ctf19, Ctrl , Ctr3, Ctt1 , Cup1 , Cup9, Das1 , Dbf20, Dpi1 , Dsd1 , Ede1 , Egd2, Fet4, Fre1 , Fre7, Ftr1 , Fun26, Gnd2, Gph1 , His5, Hnm1 , Hos4, !rc7, !syl , Kem1 , Kre6, Lsb1 , Lsm5, Mal33, Mdm31 , Mrm2, Mss4, Npc2, Nrd Oad , Oms1 , Pho23, Pho89, Pmp1 , Pop8, Pph3, Pre9, Prm5, Prp9, Pub1 , Rad50, Reel , Rgm1 , Rpa34, Rpc40, RpM b, Rpl7b, Rpn7, Rrp7, Sdp1 , Sen2, Snr190, Swd Swh1 , Tgl2, Tma20, Tps2, Trm10, Ubx3, Ubx6, Ume6, Utr1 , Vcx1 , Vhs2, Vik1 , Vip1 , Vtc3, Yar009c, Yar069c, Ycr025c, Ydi129w, Ygr137w, Yil102c, Yjl218w, Yki063c, Yih47, YII059c, YirG35c-a, Ylr123c, Ylr410w-a, Yir410w-b, Ymr320w, Ynl017c, YolQ79w, Yol153c, Yor1 1 1w, Ypl251w, Ypr123c, Ypr170c, and Ysp1 , or homologs thereof.

71 .The recombinant yeast microorganism of claim 70, wherein said protein regulated by a Mad protein is Ctrl .

72. The recombinant yeast microorganism of claim 70, wherein said protein regulated by a Mad protein is Fre1 .

73. he recombinant yeast microorganism of claim 70, wherein said protein regulated by a Mad is Pho89.

74. A recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more proteins which positively regulate Ma .

75. The recombinant yeast microorganism of claim 74, wherein the positive regulator of Mad is Ccs1 .

76. The recombinant yeast microorganism of claim 74, wherein the positive regulator of Mad is Sod1 .

77. A recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said recombinant yeast microorganism is engineered to disrupt, delete, or mutate one or more endogenous polynucleotides encoding a negative regulator of Mad .

78. The recombinant yeast microorganism of any of claims 58-77, wherein said enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol is selected from an acetolactate synthase, ketoi-acid reductoisomerase, dihydroxy-acid dehydratase, 2-keto-acid decarboxylase, and an alcohol dehydrogenase.

79. The recombinant yeast microorganism of claim 78, wherein each of said acetolactate synthase, ketoi-acid reductoisomerase, dihydroxy-acid dehydratase, 2-keto-acid decarboxylase, and an alcohol dehydrogenase is encoded by an exogenous gene.

80. The recombinant yeast microorganism of any of claims 78-79, wherein said ketoi- acid reductoisomerase is an NADH-dependent ketoi-acid reductoisomerase.

81 . The recombinant yeast microorganism of any of claims 78-79, wherein said alcohol dehydrogenase is an NADH-dependent alcohol dehydrogenase.

82. The recombinant yeast microorganism of any of the preceding claims, wherein said recombinant yeast microorganism is engineered to reduce pyruvate decarboxylase (PDC) activity.

83. The recombinant yeast microorganism of any of the preceding claims, wherein said recombinant yeast microorganism is engineered to reduce giycerol-3-phosphate dehydrogenase (GPD) activity.

84. The recombinant yeast microorganism of any of the preceding claims, wherein said recombinant yeast microorganism is engineered to reduce 3-keto acid reductase (3- KAR) activity.

85. The recombinant yeast microorganism of any of the preceding claims, wherein said recombinant yeast microorganism is engineered to reduce aldehyde dehydrogenase (ALDH) activity.

86. The recombinant yeast microorganism of any of the preceding claims, wherein said recombinant yeast microorganism is engineered to increase the activator of ferrous transport (AFT) activity.

87. The recombinant yeast microorganism of claim 88, wherein said recombinant yeast microorganism is engineered to overexpress one or more polynucleotides encoding one or more Aft proteins,

88. The recombinant yeast microorganism of claim 86, wherein said recombinant yeast microorganism is engineered to express one or more polynucleotides encoding one or more constitutively active Aft proteins.

89. A method of producing a beneficial metabolite, said method comprising:

(a) providing a recombinant yeast microorganism according to any of the preceding claims; and

(b) cultivating said recombinant yeast microorganism in a culture medium containing a feedstock providing a carbon source, until the beneficial metabolite is produced,

90. The method of claim 89, wherein said metabolite is selected from isobutanoi, 3~ methyl-1 -butanoi, 2-methy!-1 -butanol, valine, isoieucine, leucine, and pantothenic acid,

91 . The method of claim 90, wherein said metabolite is isobutanoi.