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`INVENTOR(S)
`Family Name or Sumame
`sansssnsanse
`
`_
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`ARISTUDOU
`
`
`Residence
`
`
`|ecity andeitherStateorForeign Country)
`
`
`
`
`ASLESON-DUNDON
`Catherine
`
`
`MEINHOLD
`Denver, Colorade
`
`|_FeLowAN
`Denver, Colorado
`
`
`
`
`Stephanie
`
`
`Christopher
`Englewood, Colorado
`
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`
`
`
`
`TITLE OF THE INVENTION (500 characters max):
`
`ISOBUTANOL PRODUCTION
` METHODS AND COMPOSITIONS FOR INCREASING DIHYDROXYACID DEHYDRATASE ACTIVITY AND
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`TYPED or PRINTED NAME PaulWickman
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`ceeREGISTRATION NO, 61,242
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`TELEPHONE 202-842-78001/07US340142-2104Docket Number, GEVO-04
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`

`

`METHODS AND COMPOSITIONS FOR INCREASING DIHYDROXYACID
`DEHYDRATASE ACTIVITY AND ISOBUTANOL PRODUCTION
`
`Atty. Docket No. GEVO-041/07US
`
`SPECIFICATION
`
`TO WHOM If MAY CONCERN:
`
`Be it known that we, with names, residences, and citizenships listed below, have
`invented the inventions described in the following specification entitled:
`
`FOR INCREASING DIHYDROXYACID
`METHODS AND COMPOSITIONS
`DEHYDRATASE ACTIVITY AND ISOBUTANOL PRODUCTION
`
`Jun Urano
`Residence: Aurora, Colorado
`Citizenship: USA
`
`Catherine Asieson Dundon
`Residence: Englewood, Colorado
`Citizenship: USA
`
`Peter Meinhold
`
`Residence:
`Citizenship:
`
`Denver, Colorade
`Germany
`
`Renny Feldman
`Residence:
`Citizenship:
`
`Aristos Aristiday
`
`Denver, Colorado
`USA
`
`Residence:
`Citizenship:
`
`Highlands Ranch, Colorado
`Cyprus
`
`Andrew Hawkins
`
`Residence:
`Citizenship:
`
`Parker, Colorado |
`USA
`
`Thomas Buelter
`
`Residence:
`Citizenship:
`
`Denver, Colorado
`Germany
`
`Matthew Peters
`
`Residence:
`Cithzenship:
`
`Highlands Ranch, Colorado
`USA
`
`2IS87S vSfDr
`
`

`

`Ally. Docket No. GEVO-0417/07US
`
`Doug Lies
`Residence: Parker, Colorado
`Citizenship: USA
`
`Stephanie Porter-Scheinman
`Residence: Conifer, Colorado
`Citizenship: USA
`
`Christopher Smith
`Residence: Englewood, Colorado
`Citizenship: USA
`
`Melissa Dey
`Residence: Aurora, Colorado
`Citizenship: USA
`
`Lynne Leach
`Residence: Golden, Colorado
`Citizenship: USA
`
`LLRs vEsOo
`
`Page 2 of 179
`
`

`

`METHODS AND COMPOSITIONS FOR INCREASING DIHYDROXYACID
`DEHYDRATASE ACTIVITY AND ISOBUTANOL PRODUCTION
`
`Atty. Docket No. GEVO-0414/07US
`
`TECHNICAL FIELD
`
`Recombinant microorganisms and methads of producing such organisms
`fa004]
`are provided. Also provided are methods of producing metabolites that are biofuels
`by contacting a suitable substrate with recombinant microorganisms and enzymatic
`preparations therefrom.
`
`BACKGROUND
`
`Biofuels have a long history ranging back to the beginning of the 20th
`[0002]
`century. As early as 1900, Rudolf Diesel demonstrated at the World Exhibition in
`Paris, France, an engine running on peanut oll.
`Soon thereafter, Henry Ford
`demonstrated his Mode! T running on ethanol derived from corn. Petroleurm-derived
`fuels displaced biofuels in the 1930s and 1940s due to increased supply, and
`efficiency at a lower cost.
`[6003] Market fluctuations in the 1970s coupled to the decrease in US oil
`production jed to an increase in crude ol] prices and a renewed interest in biofuels.
`Today, many interest groups,
`including policy makers,
`industry planners, aware
`citizens, and the financial community, are interested in substituting petroleum-
`derived fuels with biomass-derived biofuels. The leading motivations for developing
`biofuels are of economical, political, and environmental nature.
`[0004]
`One is the threat of ‘peak ail’, the point at which the consumption rate of
`crude oil exceeds the supply rate, thus leading to significantly increased fuel cost
`results in an increased demand for alternative fuels.
`In addition,
`instability in the
`Middle East and other oil-rich regions has increased the demand for domestically
`produced biofuels. Also, environmental concerns relating to the possibility of carbon
`dioxide related climate change is an important social and ethical driving force which
`is starting to result in government regulations and policies such as caps on carbon
`dioxide emissions from automobiles, taxes on carbon dioxide emissions, and tax
`incentives for the use of biofuels.
`[O005]
`Ethanol is the most abundant fermentatively produced fuel today but has
`several drawbacks when compared fo gasoline. Butanol, in comparison, has several!
`advantages over ethanol as a fuel:
`it can be made from the same feedstocks as
`ethanol but, unlike ethanol, it is compatible with gasoline at any ratio and can also be
`used as a pure fuel in existing combustion engines without modifications. Unlike
`ethanol, butanol dees not absorb water and can thus be stored anddistributed in the
`existing petrochemical infrastructure. Due to its higher energy content which Is close
`to that of gasoline, the fuel economy (miles per gallon) is better than that of ethanol.
`Also, butanol-gasoline blends have lower vapor pressure than ethancl-gasoline
`blends, which is important in reducing evaporative hydrocarbon emissions.
`[OG06]
`isobutanol has the same advantages as butanol with the additional
`advantage of having a higher octane number due to its branched carbon chain.
`
`LESB7S vE/Oc
`
`Page 3 of 179
`
`

`

`Alty. Docket No. GEVO-041/07US
`
`Isobutanol is also useful as a commodity chemical and is also a precursor to MTBE.
`Isobutanol can be produced in microorganisms expressing a heterologous metabolic
`pathway, but existing microorganisms are not of commercial relevance due to their
`inherent low performance characteristics, which include low productivity,
`low titer,
`lowyield, and the requirement for oxygen during the fermentation process.
`[0007]
`The present
`inventors have overcome these problems by developing
`metabolically
`engineered microorganisms
`that
`exhibit
`increased
`isobufano!
`productivity, titer, and/or yield.
`
`SUMMARYOF THE INVENTION
`dihydroxyacid
`active
`cytosolically
`provides
`[0008]
`The
`present
`invention
`dehydratase (DHAD) enzymes and recombinant microorganisms comprising said
`cytosolically active DHAD enzymes.
`in some embodiments, said recombinant
`microorganisms may further comprise one or more additional enzymes catalyzing a
`reaction in an isobutanol producing metabolic pathway. As described herein, the
`recombinant microorganisms of the present invention are useful for the production of
`several beneficial metabolites, including, but not limited to isobutanol.
`[6009]
`In a first aspect, the Invention provides cytosolically active dihydroxyacid
`dehydratase (DHAD) enzymes. These cytosolically active DHAD enzymes will
`generally exhibit the ability to convert 2,3-dihydroxyisovalerate to ketoisovalerate in
`the cytosol. The cytosolically active DHAD enzymes of the present Invention, as
`described herein, can include modified or alternative dihydroxyacid dehydratase
`(DHAD) enzymes, wherein said DHAD enzymes exhibit increased cytosolic activity
`as compared to the parental or native DHAD enzyme.
`[0010]
`In various embodiments described herein, the DHAD enzymes may be
`derived fram a prokaryotic organism.
`in one embodiment, the prokaryotic organism
`is @ bacterial organism.
`in another embodiment,
`the bacterial organism is
`Lactococcus factis.
`in a specific embodiment,
`the DHAD enzyme from L. factis
`comprises the amino acid sequence of SEQ [ID NO: 9.
`In another embodiment, the
`bacterial organism is Escherichia coli.
`In a specific embodiment, the DHAD enzyme
`from E. coff comprises the amino acid sequence of SEQ ID NO: 43.
`{0011]
`In alternative embodiments described herein, the DHAD enzyme may be
`derived from a eukaryotic organism.
`In one embodiment, the eukaryotic organism is
`a fungal arganism.
`In an exemplary embodiment, the fungal organism is Piromyces
`sp. E2.
`in another embodiment, the eukaryotic organism is a yeast organism, such
`as S. cerevisiae.
`In another embodiment, the eukaryotic organism is selected from
`the group consisting of the genera Enamoehba and Giardia.
`f0012}
`in some embodiments, the invention provides modified or mutated DHAD
`enzymes, wherein said DHAD enzymes exhibit
`increased cytosolic activity as
`compared to their parental DHAD enzymes.
`In another embodiment, the invention
`provides modified or mutated DHAD enzymes, wherein said DHAD enzymes exhibit
`increased cytosolic activity as compared to the DHAD enzyme camprised by the
`amino acid sequence of SEQ ID NO: 11.
`{0013}
`In some embodiments, the invention provides modified or mutated DHAD
`enzymes have one or more amino acid deletions at
`the N-terminus.
`In one
`
`LiB875 v8/pc
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`Page 4 of 179
`
`

`

`Atty. Docket No. GEVO-O4-4/07US
`
`embodiment, said modified or mufated DHAD enzyme hasat least about 10 amino
`acid deletions at the N-terminus.
`In another embodiment, said modified or mutated
`DHAD enzyme has at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
`24, 25, 30 or amino acid deletions at the N-ferminus.
`in a specific embodiment, said
`modified or mutated DHAD has 19 amino acid deletions at the N-terminus.
`In
`another specific embodiment, said modified or mutated DHAD has 23 amino acid
`deistions at the N-terminus.
`[0074]=In further embodiments, the invention provides DHAD enzymes comprising
`the amino acid sequence PU/L)XXXGX(UL)XIL (SEQ ID NO: 19), wherein X is any
`amino acid, and wherein said DHAD enzymes exhibit the ability to convert 2,3-
`dihydroxyisovalerate to ketoisovalerate in the cytosol.
`[0015]
`in additional embodiments,
`the invention provides DHAD enzymes
`comprising the arnino acid sequence CPGXGXC (SEQ ID NO: 37), wherein X is any
`amino acid, and wherein said DHAD enzymes exhibit the ability to convert 2,3-
`dihydroxyisovalerate to kefoisovalerate in the cytosol.
`[0016]=in another embodiment, the invention provides DHAD enzymes comprising
`the amino acid sequence CPGXG(A/S)C (SEQ ID NO: 38), wherein X is any amino
`acid, and wherein said DHAD enzymes exhibit
`the ability to convert 2,3-
`dinydroxyisovalerate to ketolsovalerate in the cytosol.
`[0017]
`In yet another embodiment,
`the invention provides DHAD enzymes
`comprising the amino acid sequence CXXXPGXGXC (SEQ ID NO: 39), wherein X is
`any amino acid, and wherein said DHAD enzymes exhibit the ability to convert 2,3-
`dihydroxyisovalerate to ketoisovalerate in the cytosol.
`[0018]
`In some embodiments, the DHAD enzymes of the present invention exhibit
`@ properly folded iron-sulfur cluster domain and/or redox active domain in the
`cytosol.
`In one embodiment, the DHAD enzymes comprise a mutated or modified
`iron-sulfur cluster domain and/or redox active domain.
`recombinant
`provides
`[0019]
`In
`another
`aspect,
`the
`present
`invention
`microorganisms comprising a cytosolically active DHAD enzyme.
`in one
`embodiment,
`the invention provides recombinant microorganisms comprising a
`DHAD enzyme derived from a prokaryotic organism, wherein said DHAD enzyme
`exhibits activity in the cytasol.
`[n one ernbodiment, the DHAD enzyme is derived
`from a bacterial organism.
`In a specific embodiment, the DHAD enzyme is derived
`from L. lactis and comprises the amino acid sequence of SEQ 1D NO: 9.
`[n another
`embodiment,
`the invention provides recombinant microorganisms comprising a
`DHAD enzyme derived from a eukaryotic organism, wherein said DHAD enzyme
`exhibits activity in the cytosol.
`In one embodiment, the DHAD enzyme is derived
`from a fungal organism.
`In an alternative embodiment, the DHAD enzymeis derived
`from a yeast organism.
`[0020]
`In one embodiment, the invention provides recombinant microorganisms
`comprising a modified or mutated DHAD enzyme, wherein said DHAD enzyme
`exhibits increased cytosolic activity as compared to the parental DHAD enzyme.
`In
`another
`embodiment,
`the
`invention
`provides
`recombinant microorganisms
`comprising a modified or mutated DHAD enzyme, wherein said DHAD enzyme
`
`LIGK7S vE/OC
`
`Page 5 of 179
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`

`

`Atty. Docket No. GEVO-041/07US
`
`exhibits increased cytosolic activity as compared to the DHAD enzyme comprised by
`the amino acid sequence of SEQ ID NO: 11.
`recombinant
`provides
`invention
`{00271}
`In
`another
`embodiment,
`the
`microorganisms comprising a DHAD enzyme comprising the amino acid sequence
`PU/AL)XXXGX(U/LIXIL (SEQ ID NO: 19), wherein X is any amino acid, and wherein
`said DHAD enzyme exhibits the ability to convert 2,3-dihydroxyisovalerate to
`ketoisovalerate in the cytosol.
`recombinant
`provides
`invention
`the
`{0022}
`in
`some
`embodiments,
`mucroorganisms comprising a DHAD enzyme fused to a peptide tag, whereby said
`DHAD enzyme exhibits increased cytosolic localization and/or cytosolic DHAD
`activity as compared to the parental microorganism.
`{[n one embodiment, the peptide
`tag is non-cleavable.
`In another embodiment, the peptide fag 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 selected from the group consisting of ubiquitin, ubiquitin-like (UBL) proteins, myc,
`HA-tag, green fluorescent protein (GFP}, and the maltose binding protein (MBP).
`[6023]
`in
`various
`embodiments
`described
`herein,
`the
`recombinant
`microorganisms may further comprise a nucleic acid encoding a chaperone protein,
`wherein said chaperone protein assists the folding of a protein exhibiting cytosolic
`activity.
`In a preferred ambodiment, the protein exhibiting cytosolic activity is DHAD.
`In one embodiment, the chaperone may be a native protein.
`in another embodiment,
`the chaperoneprotein may be an exogenous protein.
`In some embadiments, the
`chaperane protein may be selected from the group consisting of: endoplasmic
`reticulum oxidoreductin 1 (Ero1, accession no. NP_013576.1),
`including variants of
`Erot that have been suitably altered to reduce or prevent its normal localization to
`the endoplasmic reticulum;
`thioredoxins {which includes Trxt, accession no.
`NP_0713144.1; and Trx2, accession no. NP_011725.1), thioredoxin reductase (Trr1,
`accession no. NP_010640.1); glutaredoxins (which includes Grx1, accession no.
`NP_009895.1; Grx2,
`accession
`no. NP_010801.1; Grx3,
`accession
`no.
`NP_010383.1; Grx4, accession no. NP_01101.1; Grx5, accession no. NP_015266.1;
`Grx6, accession no. NP_010274.1; Grx7, accession no. NP_008570.1; Gnd,
`accession
`no. NP_013468.1};
`glutathione
`reductase Girt
`(accession
`no.
`NP_015234.1); and Jaci (accession no. NP_011497.1), including variants of Jaci
`that have been suitably altered to reduce or prevent
`its normal mitochondrial
`localization; Hsp10, Hsp60, GroEL, and GroES and homologs or variants thereof.
`{0024]
`In same embodiments,
`the recombinant microorganisms may further
`comprise one or more genes encoding an iron-sulfur cluster assembly protein.
`In
`one embodiment, the iron-sulfur cluster assembly protein encoding genes may be
`derived from prokaryotic organisms.
`In one embodiment, the iron-sulfur cluster
`assembly protein encoding genes are derived from a bacterial organism, including,
`but not limited ta Escherichia coli, L.
`lactis, Helicobacter pylori, and Entamoeba
`histolytica.
`in specific embodiments,
`the bacterially derived iron-sulfur cluster
`assembly protein encoding genes are selected from the group consisting of cyay,
`iscsS, iscuU, iscA, fiscB, hscA, fdx, isuX, sufA, sufB, sufC, sufD, sufS, sufE, apbC, and
`homologs or variants thereof.
`
`RA3875 val
`
`Page 6 of 179
`
`

`

`Atty. Docket No. GEVO-047/07US
`
`In another embodiment, the iron-sulfur cluster assembly protein encoding
`[0025]
`genes may be derived from eukaryotic organisms, including, but not imited to yeasts
`and plants.
`In one embodiment, the iron-sulfur cluster protein encoding genes are
`derived from a yeast organism, including, but not limited to S. cerevisiae.
`In specific
`embodiments,
`the yeast derived genes encoding iron-sulfur cluster assembly
`proteins
`are
`selected from the group consisting of Cfd1
`(accession no.
`NP_012263.1}, Nbp35 (accession no. NP_011424.1}), Nari
`(accession no.
`NP_014159.1), Ciat
`(accession no. NP_010553.1), and homologs or variants
`thereof. In a further embodiment, the iron-sulfur cluster assembly protein encoding
`genes may be derived from plant nuclear genes which encode proteins translocated
`to chloroplast or plant genes found in the chloroplast genomeitself.
`{0026}
`In some embodiments, one or more genes encoding an iron-sulfur cluster
`assembly protein may be mutated or modified to remove a signal peptide, whereby
`localization of the product of said one or more genes to the mitochondria or other
`subcellular compartment is prevented.
`In certain embodiments, it may be preferable
`fo overexpress one ar more genes encoding an iron-sulfur cluster assembly protein.
`[0027]=In certain embodiments described herein, it may be desirable to reduce ar
`eliminate the activity and/or proteins levels of one or more iron-sulfur cluster
`containing cytosolic proteins.
`in a specific embodiment,
`the iron-sulfur cluster
`containing cyfosolic protein is 3-isopropyimalate dehydratase (Leutp}.
`In one
`embodiment, the recombinant microorganism comprises a mutation in the LEU?
`gene resulting in the reduction of Leutp protein levels.
`In another embodiment, the
`recombinant microorganism comprises apartial deletion in the LEU? gene resulting
`in the reduction of Leuip protein levels.
`In another embodiment, the recombinant
`microorganism comprises a complete deletion in the LEU? gene resulting in the
`reduction of Leutp protein levels.
`In another embodiment,
`the recombinant
`microorganism comprises a modification of the regulatory region associated with the
`LEUT gene resulting in the reduction of Leutp protein levels.
`in yet another
`embodiment,
`the recombinant microorganism comprises a modification of a
`transcriptional regulator for the LEU? gene resulting in the reduction of Leutp protein
`levels.
`|
`{0028}
`In certain embodiments described herein, it may be desirable to increase
`the levels of iran within the yeast cytosol and/or mitochondria, such that more iron is
`available for iron-sulfur cluster-containing proteins, such as DHAD,in the cytosol or
`mitochondria. Thus, in certain embodiments, the recombinant microorganism may
`be engineered to overexpress one or more genes selected from the group consisting
`of AFTT, AFT2, GRX3, and GRX4, or homologs thereof,
`{0028}
`In one embodiment,
`the present
`invention provides a recombinant
`microorganism for producing isobutanol, wherein said recombinant microorganism
`comprises an isobutanol producing metabolic pathway, and wherein the expression
`of AFT? or a homolog thereof is increased.
`In another embodiment, the present
`invention provides a recombinant microorganism for producing isobutanol, wherein
`said recombinant microorganism comprises an isobutanol producing metabolic
`pathway, and wherein the expression of AFT2 or a homolog thereof is increased.
`In
`yet
`another
`embodiment,
`the present
`invention
`provides
`a
`recombinant
`
`143875 vR/0C
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`Page 7of 179
`
`

`

`Atty. Docket No. GEVO-041/07US
`
`microorganism fer producing isobutano!l, wherein said recombinant microorganism
`comprises an isobutanol producing metabolic pathway, and wherein the expression
`of AFT? and AFT2 or homologs thersofis increased.
`{0030]
`[n one embodiment,
`the present
`invention provides a recombinant
`microorganism for producing isobutanol, wherein said recombinant micrcorganism
`comprises an isobutanol producing metabalic pathway, and wherein the activity of
`Aft] or a homolog thereof is increased.
`In another embodiment,
`the present
`invention provides a recombinant microorganism for producing isobutanol, wherein
`said recombinant microorganism comprises an isobutanol producing metabolic
`pathway, and wherein the activity of Aft2 ar a homolog thereof is increased.
`fn yet
`another embodiment, the present invention provides a recombinant microorganism
`for producing isobutanol, wherein said recombinant microorganism comprises an
`isobutanol producing metabolic pathway, and wherein the activity of Aft and Aff2 or
`homolog thereofs is increased.
`{0031}
`[In alternative embodiments, the recombinant microorganism comprising an
`isobutanol producing metabolic pathway may be engineered to delete, reduce,
`and/or attenuate one or more genes selected from the group consisting of AFT,
`AFT2, GRX3, and GRX4 and homologs thereof.
`it may be desirable to reduce
`[0832]
`in various embodiments described herein,
`the concentration of reactive oxygen species (ROS) in said cytosol, as DHAD
`enzymes may be susceptible to inactivation by ROS.
`Thus,
`the recombinant
`microorganisms of the present invention may further be engineered to express one
`or more proteins in the cytosol that reduce the concentration of reactive oxygen
`species (ROS) in said cytosol. The proteins to be expressed in the cytosol for
`reducing the concentration of reactive oxygen species in the cytosol may be selected
`from catalases, superoxide dismutases, metallothioneins, and methionine sulphoxide
`reductases.
`In a specific embodiment, said catalase may be ancoded by one of
`more of the genes selected from the group consisting of the E. coff genes kafG and
`katE, the S. cerevisiae genes CTT? and CTA, or homologs thereof. In another
`specific embodiment, said superoxide dismutase is encoded by one of more of the
`genes selected from the group consisting of the E. coli genes sodA, sodB, sodC, the
`S. cerevisiae genes SOD? and SOD2, or homologs thereof.
`In another specific
`embodiment, said metallothionein is encoded by one of more of the genes selected
`from the group consisting of the S. cerevisiae CUP1-1 and CUP1-2 genes or
`homologs thereof.
`In another specific embodiment, said metallothionein is encoded
`by one or more genes selected from the group consisting of the Mycobacterium
`tuberculosis MymT gene and the Synechococcus PCC 7942 SmtA gene or
`nomologs thereof.
`In another specific embodiment, said methionine sulphoxide
`reductase is encoded by one or more genes selected from the group consisting of
`the S. cerevisiae genes MXRT and MXR2, or homologs thereof.
`[0033]
`In some embodiments,
`it may be desirable to increase the level of
`available glutathione in the cytosol, which is essential for FeS cluster biogenesis.
`Thus,
`the recombinant microorganisms of the present invention may further be
`engineered fo express one or more enzymes that increase the level of available
`glutathione in the cytosol. The proteins to be expressed to increase the Jevel of
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`available glutathione in the cytosol can be selected fromm glutaredoxin, glutathione
`reductase, and glutathione synthase.
`In a specific embodiment, said glutaredoxin is
`encoded by one of more of the genes selected from the group the S. cerevisiae
`genes GRX2, GRX4, GRX6, and GRX7, or homologs thereof.
`In another specific
`embodiment, said glutathione reductase is encoded by the S. cerevisiae genes
`GLR1 or homologs thereof.
`In another specific embodiment, said glutathione
`synthase is encoded by one of more of the genes selected from the group the S.
`cerevisiae genes GSH1 and GSH2, or homologs thereof.
`In some embodiments,
`two enzymes are expressed in and targeted to the cytoso! of yeast to increase the
`level of available glutathione in the cytosol.
`In one embodiment, the enzymes are
`enzymes are y-glutamyl cysteine synthase and glutathione synthase.
`In a specific
`embodiment, said glutathione synthase is encoded by one of more of the genes
`selected fram the group the S. cerevisiae genes GSH1 and GSH2, or homologs
`thereof,
`In some embodiments, if may be desirable to overexpress one or more
`{0034}
`cytosolic functional components of the thioredoxin system, as overexpression of the
`essential cytosolic functional components of the thioredoxin system is can increase
`the amount of bioavailable cytosolic thioredoxin, resulting in a significant increase in
`cellular redox buffering potential and concomitant increase in stable, active cytosolic
`FeS clusters and DHAD activity.
`In one embodiment, the functional components of
`the thioredoxin system may be selected from a thioredoxin and a thioredoxin
`reductase.
`In a specific embodiment, said thioredoxin is encoded by the S.
`cerevisiae FRXT and TRX2 genes or homologs thereof.
`[n another specific
`embodiment, said thioredoxin reductase is encoded by S. cerevisiae TRR? gene or
`homologs thereof.
`In additional embodiments, the recombinant microorganism may
`further be engineered to overexpress the mitochondrial thioredoxin system.
`in one
`embodiment, the mitochondrial thioredoxin system is comprised of the mitochondrial
`thioredoxin and mitochondrial thioredoxin reductase.
`in a specific embodiment, said
`mitochondrial thioredoxin is encoded by the S. cerevisiae TRX3 gene or homologs
`thereof.
`in another specific embodiment, said mitochondrial thioredoxin reductase is
`encaded by the S. cerevisiae TRR2 gene or homologs thereof.
`[0035]
`in various embodiments described herein, it may be desirable to engineer
`the recombinant microorganism to overexpress one or more mitochondrial export
`proteins.
`In a specific embodiment, said mitochondrial export protein may be
`selected frorn the group consisting of the S. cerevisiae ATMT, the S. cerevisiae
`ERV1, and the S. cerevisiae BATT, or hamologs thereof.
`[0036]
`In addition, the present invention provides recombinant microorganisms
`that have further been engineered to Increase the inner mitochondrial membrane
`electrical potential, AWy.
`in one
`embodiment,
`this
`is
`accomplished via
`overexpression of an ATP/ADP carrier protein, wherein said overexpression
`increases ATP* import into the mitochondrial matrix in exchange for ADP*.
`In a
`specific embodiment, said ATP/ADPcarrier protein is encoded by the S. cerevisiae
`AAC, AAC2, and/or AAC3 genes or homologs thereof.
`in another embodiment, the
`inner mitochondrial membrane electrical potential, AY, is increased via a mutation in
`the mifochondrial ATP synthase complex that increases ATP hydrolysis activity.
`In a
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`specific embodiment, said mutation is an ATP1-111 suppressor mutation or a
`corresponding mutation in a homologous protein.
`it may further be desirable to
`[0037]
`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 (RNS) and/or nitric
`oxide (NO) in said cytosol.
`In one embodiment, said one or more enzymes are
`selected from the group consisting of nitric oxide reductases and glutathione-S-
`nitrosothio! reductase.
`Jn a specific embodiment, said nitric oxide reductase is
`encoded by one of more of the genes selected frorn the group consisting of the E.
`coli gene norV and the Fusarium oxysporum gene P-450dNIR, or homologs thereof.
`In another specific embodiment, said glutathione-S-nitrosothiol reductase is encoded
`by the S. cerevisiae gene SFAT or homologs thereof.
`In one embodiment, said
`glutathione-S-nitrosothiol
`reductase gene SFA7 is overexpressed.
`in another
`specific embodiment, said one or more enzymes is encoded by a gene selected fram
`the group consisting of the E. colf gene yifE, the Staphylococcus aureus gene scdA,
`and Neisseria gonorrhoeae gene dnrN, or homologs thereof.
`{[O638}]
`Also provided herein are recombinant microorganisms that demonstrate
`increased the levels of sulfur-containing compounds within yeast cells, including the
`amino acid cysteine, such that this sulfur is more available for the production of iron-
`sulfur cluster-containing proteins in the yeast cytosol or mitochondria.
`in one
`embodiment, the recombinant microorganism has been engineered to overexpress
`one or more of the genes selected from the S. cerevisiae genes METI, MET2,
`METS, METS, METS, MET1O, MET1I4, MET16, MET17, HOM2, HOM3, HOMG6,
`CYS3, CYS4 SUL1,
`and SUL2, or homologs
`thereof.
`The
`recombinant
`microorganism mayadditionally or optionally also overexpress one or more of the
`genes selected from the S. cerevisiae genes YCT?, MUPT, GAP1, AGP1, GNP.
`BAP1, BAP2, TAT, and TAT2, ar homologs thereof.
`[0038]
`in various embodiments described herein, the recombinant microorganism
`may exhibit at least about 5 percent greater dihydroxyacid dehydratase (DHAD)
`activity in the cytosol as compared to the parental microorganism.
`In another
`embodiment, the recombinant microorganism may exhibit at least about 10 percent,
`at least about 15 percent, about least about 20 percent, at