Attorlley Docket No. GEV(}«{}41/{JSUS (310142-2128)
`
`PA+'\'l."TENVT
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`ILS. PATENT AND TRADE1\’L~’&RK OFFICE
`PRO IXYSIO.-W.‘~‘£L JPPL-{CA ?'I£}N {.‘D!"5ER SHEET
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`The .‘£nve.m.ion was made by an agency oftheUni’, ontrsutt with an agent‘ {if the United States Gnvcrnmeni.
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`SECSNATURE
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`: 5%‘:‘£“"‘*"""-
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`Dated: /
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`'I'YPED er PRINTEYI N.-§§‘e‘IE:
`303209 s=}.-‘DC
`
`Paul Vwiciunzm
`
`I{l:€GI3‘l‘R.-'\TI(31\‘ N0.
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`GL2-$2
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`BUTAMAX 10 10
`
`

`
`(iYTC3SC.lLiC’ALLY ACTIVE E}iHYDRDXYAC!D DEHYDRATASES
`
`Afty. Docket No. GEVO~O41i03US
`
`SPEGFSCATZON
`
`T0 WHOM IT MAY CONCERN:
`
`Be it known that we, with names, residences, and cit.izenshi;:s5 iisted.
`invented {he inventions described in the foiiawing specificaiion aniitied:
`
`taebwé, have
`
`CYTOS{J¥_!CALLY ACTIVE ENHYDROXYACID DEHY{}RATASES
`
`Jun Urano
`
`Residence: Aurora, Coioradc
`
`Citizenship: USA
`
`Catherine Asieson Dundon
`
`Resiciemez Engiewood, Cokzvradta
`
`Citizenship: USA
`
`Peter Meinhaid
`
`Residence: Denver, Coioradu
`
`Citizenship: Germany
`
`Ranny Faidiman
`
`Residence: Denver, Cclgrada
`
`Citizenship: USA
`
`Aristcss Ari$t§d0u
`
`Reskience: Highiamis Ranch, Comrade
`
`Citizenship: Cyprus
`
`Andrew Hawkins
`
`Residence: Parker, Coiorado
`
`Citizenship: USA
`
`Thomag Buelter
`
`Residence: Denver, Colorado
`
`Citizenship: Germany
`
`Matthew Peters
`
`Residence: Highiands Ranch, Colorada
`
`Citizenship: USA
`
`Doug Lies
`
`Residence: Parker,Cok3rad0
`
`10313;-1 \»‘1.,-"DC
`
`

`
`Atty. Dackei N0. GEVD~G4‘i!'03US
`
`C1iti’zenshE;3: USA
`
`Stephanie Porterficheinman
`
`Residence: Conifer, Comrade
`
`Citizenship: USA
`
`Christapher Smith
`
`Residence:
`
`Eng¥ew:3o<:*, Comrade
`
`Citizenship: USA
`
`Melissa Dray
`Residence: Aurora, Comrada
`
`Citizenship: USA
`
`1:33:52 viimc
`
`Page 2 of 144
`
`

`
`GYTOSOLiCA.LLY ACTNE [3’!HYDRC}XY.A(3iB DEHYBRATASES
`
`TECHNICAL FiEi..D
`
`Atty. Docket NC}. GE’VC}-{)4'iffl3US
`
`Recombinant microorganisms and metheds ef preducing such ergenisms
`{G081}
`are provided. Aise provided are methods of preducing metaboiites that are biefueis
`by contacting a suitable substrate with recembinent micreerganisms and enzymatic
`preparations therefrom.
`
`BACKGRDUND
`
`Biotueis have s tong history ranging back to the beginning ef the 20th
`{G002}
`century. As eeriy as 390$, Rudoif Diesel demonstrated at the Worid Exhibition in
`Paris, France, an engine running on peanut eii.
`Soon thereafter, Henry Ferd
`demonstrated his Model T running an etherioi derived irem corn. Petmieum-derived
`fueis dispiaeed biofueis in the 19303 and ”iQ4{is due to increased supply, and
`efficiency at a tower cost.
`{U003} Market fiuetuetisns in the 1970s coupled to the decrease in US on
`preductien led to an increase in crude sit prices and a renewed interest in biofueis.
`Today, many interest grasps,
`inciuding poiiey makers,
`industry pienners-, aware
`citizens. and the finenciei community, are interested in substituting petroleum-
`derived fueis with biomass-—derived biefueis. The ieading motivations tor deveieping
`biefueis are of eccmemicai, peiiti.c:ei_, and environmental nature.
`[0004]
`One is the threat at ‘peak eii’, the point at which the consumption rate of
`crude nit exceeds the suppiy rate, thus ieading to significentiy increased fuei cost
`resuits in an increased demand for aiternetive fueis.
`in addition, instabiiity in the
`Middle East and other oii—:'ich regions has increased the demand for domesticaiiy
`produced biofueis. Aise, environmentei concerns reiating to the possibiiity at carbon
`»dio:<ide reiated ciirnate change is an important sociai and ethicsi driving force which
`is starting to resuit in government reguietions and pniiczies such as caps on carbon
`dioxide emissions from automebiies, taxes on carbon dioxide emissions, and tax
`incentives for the use of biefueis.
`
`Ethenbi is the most abundant fermentetiveiy preduced. fuei today but has
`{G005}
`severe! drawbacks when cdmmred to gesoiine. Butane}, in eemparisen, has severei
`advantages over ethane! as a fuel:
`it can be made item the same feedstociis as
`etinanoi but, uniike ethenei, it is sempetibie with gssoiine at any ratio and can aiso be
`used as a pure met in existing combustien engines without modifications. Uniike
`ethanei, butanoi does not absnrb water and can thus be stared and distributed in the
`existing pet.i'oc,hemicai infrastructure. Due ts its higher energy content which is ciese
`to that at gasoiine, the met economy (mites per gaiien) is better than that of ethenei.
`Also, butanekgeseiine biends have iewer vapor pressure than ethennkgasoiine
`biends. which is impertsnt in reducing evaperative hydreserbcm emissions.
`[BN6]
`isotnutenei has the same advantages as butenoi with the addiiienei
`advantage at having a higher octane number due to its branched carbon chain.
`isobutanoi is ei.-so usefui as a commodity ishemitsai and is aiso a piecurser ta MTBE.
`
`103182 v1;’DC
`
`Page 3 of i4-4
`
`

`
`Atty. Docket No. {3EV’C3—Ci4’!I'i33US
`
`ieooutenoi can he produced in microorganisms expressing e heteroiogoue rnetaboiio
`pathway, but these microorganisms are not of cornmemiai reievence due to their
`inherent low performance characteristics, which inciude iow productivity,
`tow titer,
`tow yieid, and the requirement for oxygen during the fermentation process.
`{DOW}
`The present
`inventors have overcome these probiems by cieveiooing
`meiehoiioaiiy
`engineered microorgeni-sine
`that
`exhibit
`increased
`isobutenoi
`productivity. titer, endior yieid.
`
`SUMMARY OF THE iN\i'ENTiC.iN
`dihydroxyacid
`cytosoiioaiiy active
`provides
`£00118}
`The present
`invention
`dehydrataee {DHAEZH enzymes and recombinant microorganisms composing said
`cytosoiieeiiy active DHAB enzymes.
`in some embodiments, said recombinant
`microorganisms may further comprise one or more edditionai enzymes cataiyzing a
`reaction in an isotiiitanoi producing metaboiic pathway. As described herein, the
`recombinant microorganisms of the present invention are usefui for the production of
`severe! heneiiciai meteboiites, inciuding, but not iimited to isohutenoi.
`[(30093
`in e first aspect, the invention provides oytosoiicaiiy active dihydroxyaoid
`dehydrateae {DHAi.'J) enzymes. These eytoeoiicatiy active DHAD enzymes wiii
`generaiiy exhibit the ahiiity to convert 2,3«dihydro:a:yieovaierate to iretoisovaierete in
`the cytosoi. The eytoeoiiceiiy active DHAD enzymes of the present invention, as
`described herein, can include modified or alternative dihydroxyacid dehydrataee
`(Di-—iAD} enzymes, wherein said DHAE) enzymes exhibit increased cytosoiic: activity
`as compared to the parental or native DHAD enzyme.
`[(30101
`in various embodiments described herein, the DHAD enzymes may be
`deiriveo from a orokaryotic organism.
`in one embodiment, the prokaryotit: organism
`is a bacteria! organism.
`in another embodiment,
`the iaecteriei organism is
`Lectoicoccue faetisi.
`in a specific embodiment, the DHAB enzyme from L.
`testis
`comprises the amino acid sequence of SEQ iii) NC): 9.
`in another embodiment, the
`bacteriei organism is Escherichia coli,
`in a specific embodiment, the DHAE3 enzyme
`from E. coir‘ comprises the amino acid sequence of SEQ it} NC): 129.
`[0811]
`in aiteriiative embodiments described herein, the DHAD enzyme may be
`derived from a eukaryotic organism in one embodiment. the eukaryotio organism is
`e tungei organism.
`in an exemplary embodiment, the fungei organism is Pironiyces
`Sp. E2.
`in another embodiment, the eukeryotic: organism is a yeast organism, such
`as S. ceravisiee.
`in another embodiment, the etikeryotio organism is eeiected from
`the group consisting of the genera Enamoeizia and Giardie.
`[3012]
`in some embodiments, the invention provides modified or mutated DHAD
`enzymes, wherein said Di-IAB enzymes exhibit
`increased cytosoiit: activity as
`compared to their narentai DHAD enzymes,
`in another embodiment, the invention
`provides modified or mutated DHAD enzymes, wherein said DHAD enzymes exhibit
`increased cytoeolio activity as conipaireci
`to the DHAD enzyme comprised by the
`amino acid sequence of SEQ ii.) NO: ‘ii.
`{D013}
`in some embodiments, the invention provides modified or mutated DHAD
`enzymes have one or more amino acid deletions at
`the N-termintis.
`in one
`embodiment, said modified or mutated DHAD enzyme has at ieest about it) amino
`
`103132 mac
`
`Page 4 of ‘Merit
`
`

`
`Atty. Docket No, GEVQ—U4‘iz"03U‘S
`
`in another embodiment, said modified or mutated
`acid deletions at the N—terminus.
`DHAD enzyme has at ieest about ‘it, ‘$2., 13, 14, '35, 16. 17, i8, ’iQ, 28, 21, 22, 23,
`24, 25. St} or amino acid deietions at the N-terminus.
`in a specific embodiment, said
`modified or mutated DHAD has 19 amino acid deietions at the N—terminus.
`In
`another specific embodiment, said modified or mutated DHAD has 23» amino acid
`deietions at the N-terrninus.
`
`in further embodiments, the invention provides DHAD enzymes comprising
`{0014}
`the amino acid sequence P(ifL))()O{GX(i!L)><tt_ (SEQ: 1D NC): 19), wherein X is any
`amino acid, and wherein said DHAD enzymes exhibit the ebiiity to convert 2,3-
`ciihydroxyisoveierate to tzetoisovaierete in the cytosoi,
`[0915]
`in additions! embodiments;
`the invention provides DHAD enzymes
`oomorisirig the amino acid sequence CPGXGXC {SEQ iD NO: ‘i23), wherein X is
`any amino acid, and wherein said DHAD enzymes exhibit the ebiiity to convert 2,3-
`dihydroxyisovaierete to ketoisoveierete in the oytosoii
`{OMB}
`in another embodiment, the invention provides DHAD enzymes comprising
`the amino acid sequence CF‘GXG{Ai’S}C (SEQ ID NO: 124}, wherein: X is any amino
`‘acid, and wherein said DHAE) enzymes exhibit
`the ability to convert 2,3-
`dinydroxyieoveierate to ketoisovaierate in the cytosoi.
`{@017}
`in yet another embodiment,
`the invention provides DHAD enzymes
`comprising the amino acid sequence CX>O(P£3>(C—l‘sX€3 {SEQ ii) NO: 125}, wherein X
`is any amino eoici, end wherein said DHAE) enzymes exhibit the ability to convert 2,3-
`dihyciroxyisovaierete to ketoisovaierate in the cytosoi.
`{(1018}
`in some embodiments, the DHAD enzymes of the present invention exhibit
`a property foided iron~suifur oiuster domain endlor redox active domain in the
`cytosoi.
`in one embodiment, the DHAD enzymes oornprise a mutated or modified
`ironwsuifur ciuster domain andfor redox active domain.
`recombinant
`provides
`{OO‘iQI
`in
`another
`aspect,
`the
`present
`invention
`microorganisms
`comprising a cytosoiiceiiy active DHAD enzyme‘
`in one
`embodiment,
`the invention provides recombinant microorganisms comi:-«rising e
`DHAD enzyme derived from a proiteryotic organism, wherein said Di-iAi3 enzyme
`exhibits activity in the cytosoi.
`in one embodiment, the DHAD enzyme is derived
`from a becteriei organism.
`in at specific embodiment. the DHAD enztyme is derived
`from L. radio and comprises the amino acid sequence of SEQ ID NO: Q.
`in another
`embodiment,
`the invention provides recombinant microorganisms comprising a
`DHAD enzyme derived from a euiizeryotic: organism, wherein said DHAD enzyme
`exhibits activity in the oytosoi.
`in one embodiment, the DHAD enzyme is derived
`from a tungai organism.
`in en eiternetive embodiment, the DHAD enzyme is derived
`from a yeast organism.
`{ease}
`in one embodiment, the invention: provides recombinant microorganisms
`comprising a modified or mutated DHAD enzyme, wherein said DHAB enzyme
`exhibits increased cytosoiic activity as compared to the perentei DHAD enzyme.
`in
`another
`embodiment,
`the
`invention
`provides
`recombinant microorganisms
`comprising a modified or mutated DHAD enzyme, wherein said DHAD enzyme
`exhibits increased oytosoiio activity as compared to the BHAD enzyme comprised by
`the amino acid sequence of SEQ iii) NO: 11.
`
`103132 VIEDC
`
`Page 5 of 144
`
`

`
`Atty. Docket No. (3EVO~{)4’il03US
`
`recombinant
`provides
`invention
`the
`embociiment,
`another
`in
`{£3021}
`microorganisms comprising at BHAE} enzyme comprising the amino acid sequence
`P(ifL.)XX>{GX{iiL)Xii_ (SEQ it‘) NO: 19}, wherein X is any amino acid, and wherein
`said DHAD enzyme exhibits the abiiity to convert. 2,3»dihyciroxyisoveierate to
`ketoisoveierete in the cytosoi.
`recombinant
`provides
`invention
`the
`£0022]
`in
`some
`embodiments,
`microorganisms comprising a DHAD enzyme fused to a peptide tag, whereby said
`DHAD enzyme exhibits increased cytosoiic iocaiization sridfor cytosoiic DHAD
`activity as compared to the parentai microorganism.
`in one embodiment, the peptide
`tag is non—cieavabie.
`in another embodiment, the peptide tag is fused at the N~
`terminus of the DHAD enzyme.
`in another embodiment, the peptide tag is fused at
`the C—terminus of the DHAD enzyme.
`in certain embodiments, the peptide tag may
`be seiected from the group consisting oi ubiouitin, ubiquitin«-iike (USU proteins, myc,
`HA—tag, green fluorescent protein (G FF’), and the rrieitose binding protein (MBP).
`[(30233
`in
`various
`embodiments
`described
`herein,
`the
`recombinant
`microorganisms may further comprise a nucieic acid encoding a chaoerone protein,
`wherein said cheperone protein assists the ioiciing of a protein exhibiting cytosoiic
`activity.
`in a preferred embodiment, the protein exhibiting cytosoiic activity is Di~iAfD.
`in one embodiment, the cheperorie may be a native protein.
`in soother embodiment,
`the chaoerone protein may be an exogenous protein.
`in some embodiments. the
`chaperone protein may be seiecteci from the group consisting oi‘: ehdopiasmic
`reticuium oxidoreduciin 1 (Eroi, accession no. NP__D‘i3576.‘i), inciuding variants of
`Eroi that have been suitabiy aitered to reduce or prevent its normei iocsiizetion to
`the ehciopiesmic reticulum;
`thioredoxins {which inciudes Tr>r‘i, accession no.
`i\iP*0'i 3144.1; and Tr;~::2, accession no. i\iP_O‘i ‘i?25.‘i),. thioredoxin reduct-ese (Trri,
`accession no. NP*010640.“i); giuterecioxins (which includes God, accession no.
`NP_0G9895.1; Grx2,
`accession
`no. NF{_0‘i080‘i.1;
`Grx:3,
`accession
`no.
`NP__i3’ii338S.’i: Grxcti, accession no. NPHG1 101.1; Grxb, accession no. i\iF’M}3’i526i5.i;
`Grxfi, accession no. NPMG1G274.t; Grxii, accession no. NP__00957i3.1; Grxfi,
`accession
`no. NP__0’i34€:8.1);
`giutathione
`reouotese Girt
`(accession
`no.
`hiP“_0’i 5234.1}; and dare”? (accession no. NP__G1t49?.“i), inciuding variants of Jae‘?
`that have been suitebiy aitered to reduce or prevent
`its normsi mitochondriei
`iorcaiization; Hsoiii, Hsofifi, GroEL., and iI.—3roE8 and hornoiogs or veriahts thereof.
`{D324}
`in some embodiments,
`the recombinant. microorganisms may further
`comprise one or more genes encoding an iron~suif1.ir cluster essembiy protein.
`in
`one embodiment, the iron-suifur ciuster sssembiy protein encoding genes may be
`derived from prokaryotic organisms.
`in one embociiment, the iron—suifur cluster
`assembiy protein encoding genes are derived from a bacteria! organism, ihciuding,
`but not
`iimiteo in Escherichia coir’,
`t..
`lactic, Heiicobecier pyiori, and Enfernoebe
`iiistoiyrice.
`in specific embodiments,
`the ioecteriaiiy derived iron-suifur ciuster
`assembly protein encoding genes are selected from the group consisting of cyei’,
`i.scS, {sci}, iscfir. hscfi, nsc,-ii, fdx, isux, si.rfA, sufB, suftfi, soft), sufS, sufff, eobc, and
`hornoiogs or variants thereof.
`{(3025}
`in another embodiment, the iron—suifur ciuster assembiy protein encoding
`genes may be derived from euiteryotic organisms, inoiuciing, but not iimited to yeasts
`
`123132 VIXDC
`
`Page 8 of 144
`
`

`
`Atty. Docket No. GEVCi—O4’i;’03US
`
`in one embodiment, the iron—suifur ciuster protein encoding genes are
`and pients.
`derived from a yeast organism, inoiuding, but not iimited to 3. cerevieiee.
`in specific
`embodiments,
`the yeast derived genes encoding ironeuifur oiuster ass-embiy
`proteins are
`seiected from the group consisting of Ciidi
`(accession no.
`NP~_G‘i2263.'i), Nbo35 (accession no.
`.NP_O’i 1424.1), Nari
`(accession no.
`NP__Oi«<i’i 59.1 ), Ciai
`(accession no. NP_0‘I 0553.1), and homoiogs or variants
`thereof. in 3 further embodiment, the iromsuifur oiuster aeeembiy protein encoding
`genes may be derived from piant nuciear genes which encode proteins trensiooeted
`to ohiorooiast or piant genes found in the chioropiast genome itse-ii.
`{G026}
`in some embodiments, one or more genes encoding an iron-suifur oiuster
`essernbiy protein may be mutated or modified to remove a eignai peptide, whereby
`iooaiizetion of the product of said one or more genes to the mitochondria or other
`subceiiuier compartment is prevented.
`in certain embodiments, it may be preferable
`to overexoress one or more genes encoding an ii‘Oi'i-SLt-ifLi!’ duster essembiy protein.
`{t3(!2?]
`in certain embodiments described herein, it may be deairebie to reduce or
`eiirninate the aotivity andlor proteins ieveis of one or more ironeuitur ciuster
`containing oytoeoiio proteins.
`in a specific embodiment,
`the ironeuifur oiuster
`containing oytosoiio protein is 3—isoprpyimeiate dehydretase {Lento}.
`in one
`embodiment, the recombinant microorganism comprises a mutation in the LE‘iJ'i
`gene resuiting in the reduction of Leutp protein ieveie.
`in another embodiment, the
`recombinant microorganism comprises a partiei deietion in the LEU1 gene resuiting
`in the reduction of Leuip protein ieveis,
`in another embodirnent, the recombinant
`microorganism comprises a oomoiete deietion in the LEM! gene reeuiting in the
`reduction of Letrip protein ieveis.
`in another embodiment.
`the recombinant
`microorganism comprises a modification of the reguietory region associated with the
`LEU1 gene resuiting in the reduction of Leuio protein ieveis.
`in yet another
`embodiment,
`the recombinant microorganism comprises a modification of e
`traneoriptionei regoiator for the LEU1 gene resulting in the reduction of Leuip protein
`ieveis.
`
`it may be desirabie to inoreese
`in certain embodiments described herein,
`[D0281
`the ieveis of iron within the yeast cytosoi and mitochondria, such that this iron is
`more aveiiabie for the production of iron—suifur t:iuster—-containing proteins in the
`oytosoi. Thus, in certain embodiments, the recombinant microorganism may further
`been engineered to overexpress one or more genes eeieoted from the group
`consisting of AFT1, AFT2, GRX3, and GRX4, or homoiogs thereof.
`in alternative
`embodiments, the microorganism may be engineered to deiete anti/‘or attenuate one
`or more genes eeieoied from the group consisting of GRX3 and GRX4, or homoioge
`thereof.
`
`it may be desirebie to reduce
`in various embodiments described herein,
`{0029}
`the concentration of reactive oxygen species (ROS) in said oytosoi. as DHAD
`enzymes may be susoeptibie to inactivation by R03.
`Thus,
`the recombinant
`microorganisms oi the present invention may further be engineered to express one
`or more proteins in the oytosoi that reduce the concentration of reactive oxygen
`species (ROS) in said oytosoi, The proteins to be expressed in the cytosoi for
`reducing the concentration of reactive oxygen species in the oytoeoi may be seiected
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`from ostaisses, suoeroxide oiismuteses,. meteiiothioneins, and methionine suiohoxide
`reduotsses.
`in 3 specific emhooimenit, said oeteiese may be encoded by one of
`more of the genes seieoted from the group consisting of the E. coli genes kez‘G and
`itetE, the S. cerewsiee genes CTT1 and CTA1, or homoiogs thereof.
`in another
`specific embodiment, said superoxicie ciismutese is encodes by one of more of the
`genes seiected from the group consisting of the E. ooif genes sodflt, sao'B, sooc, the
`S’. oerevisiee genes SOD‘? and 30132, or homoiogs thereof.
`in another specific
`embodiment, said meteiiothionein is encoded by one of more of the genes seieoteo
`from the group consisting of the S. cerevisiee CUP‘?-ii‘ and CUP‘!-2 genes or
`homoiogs thereof.
`in another specific embodiment, said msteiiothionein is encoded
`by one or more genes seieoteo from the group consisting of the Myoobaoteritim
`tuberouiosis Mym?" gene and the Synechoooctzus PCC 7942 Smte gene or
`homoiogs thereof.
`in another specific embodiment, said methionine suiphoxide
`reductase is encoded by one or more genes seieoteo from the group consisting of
`the S. Cerevisiae genes MX.Fi’i end MXR2, or homoiogs thereof.
`{D039}
`in some embodiments,
`it may be ciesirabie to increase the level of
`evaiiehie giutathione in the cytosoi, which is essentiai for F-e8 oiuster biogenesis.
`Thus,
`the recombinant microorganisms of the present invention may further be
`engineered to express one or more enzymes that increase the ievei of eveiiatziie
`giutathione in the cytosoi. The proteins to he expressed to inorease the ievei of
`avaiiebie giutethione in the oytosoi can be seiected from giutereooxin, giutathione
`reduotase, and giutethione synthese.
`in a specific embodiment, said giutereooxin is
`encoded by one of more of the genes seieoteo from the group the S. cerevisiee
`genes GRX2, GRJM, GRX6, and GRX7, or homoiogs thereof.
`in another specific
`embodiment, said giutathione redostase is encoded by the S. ceremisiee genes
`Gt.Ri or homoiogs thereof.
`in another specific embodiment, said giutethione
`synthsse is encoded by one of more of the genes seieoted from the group the 8.
`cerevisiee genes (SSH? and GSH2, or homoiogs thereof.
`in some embodiments,
`two enzymes are expressed in and targeted to the cytosoi of yeast to increase the
`ievei of svaiiabie giutathione in the oytosoi.
`in one embodiment, the enzymes are
`enzymes are y—giutamyi cysteine synthase and giutethione synthase.
`in a specific:
`embodiment, said giutathione synthase is encoded by one of more of the genes
`seieoted from the group the S. oerevisiee genes SSH? and GSH2‘, or homoiogs
`thereof.
`
`it may be desirehie to overexpress one or more
`in some embodiment.s,
`[@0313
`cytosoiic: functionei components of the thiorecioxin system, as overexoression of the
`essentiai oytosoiio functionei oomoonents of the thioredoxin system is can increase
`the amount of bioaveiiahie cytosotio thiorecioxin, resulting in a significant increase in
`ceiiuiar redox buffering potentisi and concomitant inorease in stebie, active oytosoiii:
`FeS otusters and DHAO activity.
`in one embodiment, the ftinotionsi components of
`the thioreitioxiri system may be seieoted from a thioredoxin and a thiorecioxin
`redustsse.
`in a specific embodiment, said thiorecioxin is encoded by the S,
`cerevisise TRXi and TRX2 genes or homologs thereof.
`in another soeoifio
`emboointent, saint tnioredoxin redoctsse is encoded by S. cerevisise TRR1 gene or
`homoiogs thereof.
`in eociitionai embodiments, the reooinhinent microorganism may
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`in one
`further be engineered to overexoress» the mitochondriei thioredoxin system.
`embodiment, the mitochondria! thioredoxin system is comprised of the mitoohondrisi
`thioredoxin end mitooiiondriai thioredoxin reduotese.
`in a specific embodiment, said
`mitochondrial thioredoxin is encoded by the S. oerevisiee TRX3 gene or nornoiogs
`thereof.
`in another specific; embodiment, said mitochondriei thioredoxin reduciase is
`encoded by the S. cerevisiee TRR2 gene or homoiogs thereof.
`{(1032}
`in various embodiments described herein, it may be desirsitiie to engineer
`the recombinant microorganism to overexoress one or more mitoohondriai export
`proteins.
`in a specific. embodiment, said mitochondria! export protein may be
`seieotsd from the group consisting of the S. oerevisiee ATM1, the S. oerevisiee
`ERVL and the S. oereirisise BA T1, or homoiogs thereof.
`{0033}
`in addition, the present invention provides recombinant microorganisms
`that have further been engineered to increase the inner mitoohondriei membrane
`eiestrioai potentisi,
`1.”x’~i«‘M.
`in one embodiment,
`this
`is ecoompiished vie
`overexpression of en ATPIADP carrier protein, wherein said overexpression
`increases ATP4‘ import into the mitochonoriai matrix in exchange for ADP3".
`in a
`specific embodiment, said ATVPIADP carrier protein is encoded by the S. oerevisiee
`AAGL AAC2, endior AAG3 genes or homoiogs thereof.
`in another embodiment, the
`inner mitoohondrisi membrane eieotrioei potential, AWN is increased vie a mutation in
`the rnitoohondriei ATP synthese oornpiex that increases ATP hydroiysis activity.
`in 3
`specific embodiment, said mutation is so ATP’:-1’ii suppressor mutation or e
`oorresponding mutation in a homoiogous protein.
`it may further be desirable to
`{(1034}
`in various embodiments described herein,
`engineer the recombinant microorganism to express one or more enzymes in the
`cytosoi that reduce the concentration of reactive nitrogen species (RN53) endior nitric
`oxide (NO) in said oytosoi.
`in one embodiment, said one or more enzymes are
`seieoted from the group consisting of nitric oxide reduotases and gIutsthione—S-
`nitrosothioi reduotsse.
`in a specific embodiment, said nitric oxide rsductaise is
`encoded by one of more of the genes selected from the group consisting of the E.
`coir‘ gene norv and the Fusarfum oxysoorum gene P~45DdN!R, or homoiogs thereof.
`in another specific embodiment, said giutsthione~S~nitrosothioi reduotase is encoded
`by the S. oerevisiee gene SPA?‘ or hornoiogs thereof.
`in one embodiment, said
`Qiutethione—S—nitrosothioi
`reductese gene SF/-it
`is overexpressed.
`in another
`specific embodiment, said one or more enzymes is encoded by a gene selected from
`the group consisting of the E. col? gene yifE, the Stapiiyioooocus eursus gene scdxi,
`and Neisserfa gonorriioeee gene o'nrN, or homoiogs thereof.
`[0035]
`Aiso provided herein are recombinant microorganisms that demonstrate
`increased the ieveis of suifumconiaining compounds within yeast coils, inoiuding the
`amino acid cysteine, such that this sulfur is more avaiiabie for the production of iron~
`suitor ciuster—oonteining proteins in the yeast oytosoi.
`in one embodiment, the
`recombinant microorganism has loeen engineered to overexpress one or more of the
`genes selected from the S. serevisiee genes MET1, MET2, MET3, MET5, METB,
`ii/iET”i0, MET?-4,.
`iieiE’f1d, MET? 7, HOME, HOMS, HOM6, CV83, CYS4, sum, and
`SUL2, or homoiogs thereof. The recombinant microorganism may edlditionaiiy or
`optioneily eiso overexpress one or more of the genes seieoied from the S. ce.revi'sfae
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`Atty, Docket No. €3EVO~{Ii4‘i!'O3US
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`genes YGT’i, MUP1, {5AP?‘, AGP1, GNPT, BAP1, BAP2, TAT1, and TAT2, or
`homoioge thereof’.
`{M336}
`in various embodiments described herein, the recombinant microorganism
`may exhibit at ieast about 5 percent greater dihydroxyacici dehydratase (DHAD)
`activity in the cytosoi as compared to the parents! microorganism.
`in another
`embodiment, the recombinant microorganism may exhibit at ieast about 10 percent,
`at ieest about ‘£5 percent, about least about 20 percent, at toast about 25 percent, at
`ieast about 30 percent, at ieast about 35 percent, at tees’: about 40 percent, at ieast
`about 45 percent, at ieast about 50 percent, at toast about 55 percent, at ieast about
`60 percent, at ieast about 65 percent, at ieest about ?'0 percent, at ieest about T5
`percent, at ieest about 30 percent, at ieast about '30!) percent, at ieast about 200
`percent, or at ieast about 509 percent greater dihydroxyacid dehydretase (EJHAD)
`activity in the cytosoi as compared to the pereritai microorganism.
`{DOW}
`in certain embodiments described herein,
`it may be desirsbie to further
`overexpress so additionai enzyme that converts 2,3—dihydroxyisoveierate to
`ketoisovaierete in the cytosoi.
`in a specific embodiment,
`the enzyme may be
`seiected
`from the
`group
`consisting
`of
`3-isopropyimeiate
`(Leutp)
`and
`imioazoiegiyceroi—phosphate dehydrogenase (i-iissp).
`recombinant
`the
`herein,
`£0038]
`in
`various
`embodiments
`described
`microorganisms may be further engineered to express an isobutanoi producing
`rneteboiic pathway comprising at ieest one exogenous gene that cataiyzee a step in
`the conversion of pyruvate to isooutsnoi.
`in one embodiment, the recombinant
`microorganism may be engineered to express an isobutanoi producing metabolic
`psitnwayi comprising at least two exogenous genes.
`in another embodiment, the
`recombinant microorganism may be engineered to express an isobutenoi producing
`metaboiic pathway comprising at
`toast
`three exogenous genes.
`in another
`embodiment, the recornbinant microorganism may be engineered to express an
`isobutanoi producing metaboiic pathway comprising at toast four exogenous genes.
`in another ernbodiment,
`the recombinant microorganism may be engineered to
`express an isobutanoi producing metaboiic pathway comprising five exogenous
`genes.
`
`the isobutsnoi pathway
`in various embodiments described herein,
`{(3039}
`enzymeis} is/‘are seiecteci” from the group consisting of acetoiactate syntbese (ALE),
`i<etoi~acic| reciuciioieomerase {i<AFti). ciihydroxyecid debydretase (DHAD), .2~i<eto-acid
`decarboxylese (KEVD), and ieobutyraidehyde dehydrogenase (iDi-ti).
`in a preferred
`embodiment, said ciihydroxyacid oehyoirstase (DHAD)
`is a cytosoiicaiiy active
`(DHAD) enzyme.
`
`recombinant
`the
`herein,
`oiesoribeti
`embodiments
`various
`in
`{{}{i4i1i}
`microorganisms may be engineered to express native genes that catalyze a step in
`the conversion of pvruvate to isobutanoi.
`in one embodiment. the recombinant
`microorganism is engineered to increase the activity of a native metaboiic pathway
`gene for conversion of pyruvate to isobutanoi.
`in another embodiment.
`the
`recombinant microorganism is turtner engineered to inciucie at ieast one enzyme
`encoded by a heteroiogous gene and at iesst one enzyme encoded by a native
`gene.
`in yet another embodiment,
`the recombinant microorganism comprises a
`
`19313:: mac
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`
`reduction in the activity of a native metabolic pathway as compared to a parental
`microorganism.
`[0041]
`in various embodiments described herein, one or more of the enzymes
`e cataiyzzing the conversion of pyruvate to isobutanoi isfare localized in the cytosoi.
`in
`a preferred embodiment, the enzyme is diltydroxyacid dehydratase (DHAD).
`{oil-42}_
`in some embodiments,
`the present
`invention provides
`recombinant
`microorganisms that have been engineered to express a heteroiogous metabolic
`pathway for conversion of pyruvate to isobutanoi.
`in another ernbodliment,
`the
`recombinant microorganism further comprises a pathway for the fermentation of
`isobotanoi from a pentose sugar:
`in one embodiment, the pentose sugar is xyiose.
`in one embodiment,
`the recombinant microorganism is engineered to express a
`functions! xylose isornerase (Xi).
`in another embodiment,
`the recombinant
`microorganism further comprises a deletion or disruption of a native gene encoding
`for an enzyme that catalyzes the conversion of xyiose to xylitol.
`in one embodiment,
`the native gene is xylose reductase (XR).
`in another embodiment, the native gene is
`xylitoi dehydrogenaae (XDH).
`in yet another embodiment, both native genes are
`deleted or disrupted-
`in yet another embodiment, the recombinant microorganism is
`engineered to express a xyiulose kinese enzyme.
`recombinant
`[{}843}
`in another aspect,
`the present
`invention provides a
`microorganism engineered to include reduced pyruvate dscarboxyiase {PEG} activity
`as compared to a parental microorganism.
`in one embodiment, PDC activity is
`eliminated. PDQ catatyzss the decarboxylation of pyruvate to acetaldehyds, which is
`reduced to ethane! by aloohoi denydrogenases via the oxida