Attorney Docket Ne. GEVO-O4143US (310142-21728)
`
`PATENT
`
`US. PATENT AND TRADEMARE OFFICE
`PROVISIONAL APPLICATION COMER SHEET
`
`This is a requestfor filing a PROVISIONAL
`APPLICATION under 37 CER. §1.S3Cc}.
`
`.
`Docket Number
`
`GEVO-
`aa03U8
`
`ne
`this
`issside
`£47
`- sien
`“
`‘Fype a plus sign (4) inside this tox—+
`
`INVENTORGVAPPLICANYG)
`
`Last Nawe Prrsreae|fess MAME AMdiopie Berrian RESIDENCE qIry ANR ETHER STATE ORFOREIGNCOUNTRY¥
`
`
`
`
`
`Fuglewood, Colorade
`
`_
`
`BUELTER
`
`|
`
`Denver, Colorado
`
`CePORTER-SCHEINMAN |stephanieff Conifer, Colorada
`
`
`
`SMITH
`
`Christopher Po Englewood, Colorado
`
`FITLE OF INVENTION
`
`CYPTOSGLICALLY ACTIVE DIBYDROXY’ACHES DEHYDRATASES
`
`CORRESPONDENCE ADDRESS
`
`COOLEY GOPWARD KRONISH LLP
`INDIVIDUAL AND FIRM NAME!
`CUSTOMER NUMBER:
`58249
`
`ADDRESS _
`
`ATYR: PATENT GROEP,
`
`777 6" Street NW, Suite 1 £00
`Tek: (2023 842-7800 Fax: (202) 842-7899
`
`Crry Washington
`
`Zi Cope
`
`20001
`
`COUNTRY U.S.A.
`
`ENCLOSED APPLICATION PARTStspccx an ena spay}
`
`t [X}
`[x]
`
`Specification - No. af Pages 332
`Drawing(s}- No.of Sheets
`1
`
`{] Application Data Sheet
`f} CD{s), number
`EN] Citeer fanecrty): Sequence Listing (21 pages)
`
`Application Size Fee: Ifthe specification and drawings exceed 1X} sheets of paper, the application sizefee duc is $270 (3125 for small entity) |
`for each additional $0 sheets or fraction thereof. See 34 USC. 41(a1G) and 37 CFR Lats}
`
`METHOD OF PAYMENT (siect oor}
`
`IX} The Commissioner is hereby authorized to charge the filing fee of $248.00 required§PROVISIONALFILING FRE AMOUNT (3)
`
`by this paper, and to credit any overpayment, io Deposit Account No. 50-1283,
`f] 8220.00 Large Entity
`{Xj Sid.0 Small Entiry
`The Invention was made by an agency of the United States Government er uudera contract with an agency of the United States Gevernment,
`PX}
`Yes, the names of the US. Government agency and the Government contract number are: Phe National Science Foundation, Grant
`Number HP-0823122 and the United States Environmental Protection Agency, Grant Number EP-D-09-023,.
`
`Respeetfally submi
`
`
`
`SIGNATURE aBeltafye bower‘a
`
`Dated:
`
`f[larlaoee
`
`TYPED er PRINTED NAME:
`103206 + HDC
`
`Pau] Wickman
`
`REGISTRATION NO.
`
`65.242
`
`BUTAMAX 1010
`
`

`

`CYTOSOLICALLY ACTIVE DIHYDROXYACID DEHYDRATASES
`
`Atty. Docket No. GEVO-041/03US
`
`SPECIFICATION
`
`TO WHOM IT MAY CONCERN:
`
`Be it known that we, with names, residences, and citizenships fisted below, have
`invented the Inventions described in the following specification entitled:
`
`CYTOSOLICALLY ACTIVE DIHYDROXYACID DEHYDRATASES
`
`dun Urano
`
`Residence:
`Citizenship:
`
`Aurora, Colorado
`USA
`
`Catherine Asieson Dundon
`Englewood, Colorado
`USA
`
`Residence:
`Citizenship:
`
`Peter Meinhold
`
`Residence:
`Citizenship:
`
`Denver, Colorado
`Germany
`
`Renny Feldman
`Residence:
`Citizenship:
`
`Aristos Aristidou
`
`Denver, Colorado
`USA
`
`Residence:
`Citizenship:
`
`Highlands Ranch, Colorado
`Cyprus
`
`Andrew Hawkins
`
`Residence:
`Citizenship:
`
`Parker, Colorado
`USA
`
`Thomas Buelter
`Residence:
`Citizenship:
`
`Denver, Colorada
`Germany
`
`Matthew Peters
`
`Residence:
`Citizenship:
`
`Highlands Ranch, Colorado
`USA
`
`Doug Lies
`Rasidence:
`
`LOS182 viBC
`
`Parker, Colorado
`
`

`

`Atty. Docket No. GEVO-047/03US
`
`Citizenship: USA
`
`Stephanie Porter-Scheinman
`Residence: Conifer, Colorado
`Citizenship: USA
`
`Christopher Smith
`Residence: Englewood, Colorado
`Citizenship: USA
`
`Melissa Dey
`Residence: Aurora, Colorado
`Citizenship: USA
`
`208182 vAfc
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`

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`CYTOSOLICALLY ACTIVE DIHYDROXYACID DEHYDRATASES
`
`TECHNICAL FIELD
`
`Alty. Docket No. GEVO-041/03US
`
`Recombinant microorganisms and methods of producing such organisms
`[0001]
`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
`[6002]
`century. As early as 1900, Rudolf Diesel demonstrated at the World Exhibition in
`Paris, France, an engine running on peanut oil.
`Soon thereafter, Henry Ford
`demonstrated his Model T running on ethanol derived from corn. Petroleum-dcerived
`fuels displaced biofuels in the 1930s and 1940s due fo increased supply, and
`efficiency at a lower cost.
`{0003} Market fluctuations in the 1970s coupled fo the decrease in US oil
`production led to an increase in crude oil 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.
`{O004]
`One is the threat of ‘peak oil, 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.
`{0005}
`Ethanol is the most abundant fermentatively producedfuel today but has
`several drawbacks when compared fo gasoline. Butanal, in comparison, has several
`advantages over ethanol as a fuel:
`it can be made fram the same feedstocks as
`ethanolbut, 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 does not absorb water and can thus be stored and distributed 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 ethanol-gasoline
`blends, which is important in reducing evaporative hydrocarbon emissions.
`[0006]
`Isobutanol has the same advantages as butanol with the additional
`advantage of having a higher octane number due to its branched carbon chain.
`isobutanol is also useful as a commodity chemical and is also a precursor to MTBE.
`
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`Atty. Docket No. GEVO-041/03US
`
`isobutanal can be produced in microorganisms expressing a heterologous metabolic
`pathway, but these microorganisms are not af commercial relevance due to their
`inherent low performance characteristics, which include low productivity,
`low titer,
`low yield, 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
`isobutanol
`productivity, titer, and/or yield.
`
`SUMMARY OF THE INVENTION
`dihydroxyacid
`cytosolically active
`provides
`[6008]
`The present
`invention
`dehydratase (DHAD) enzymes and recombinant microorganisms comprising said
`cytosolicaily 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.
`[6010]
`In various embodiments described herein, the DHAD enzymes may be
`derived from a prokaryotic organism.
`in one embodiment, the prokaryotic organism
`is a bacterial organism.
`In another embodiment,
`the bacterial arganism is
`Lactococcus factis.
`In a specific embodiment, the DHAD enzyme from L.
`lactis
`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. coli comprises the amino acid sequence of SEQ ID NO: 129.
`[0017]=In alternative embodiments described herein, the DHAD enzyme may be
`derived from a eukaryotic organism.
`In one embodiment. the eukaryotic organismis
`a fungal organism.
`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 Enamoeba and Giardia.
`[0012]
`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 comprised 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
`embodiment, said modified or mutated DHAD enzyme has at least about 10 amino
`
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`Atty. Docket No. GEVO-041/03US
`
`in another embodiment, said modified or mutated
`acid deletions at the N-terminus.
`DHAD enzyme has at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 22, 23,
`24, 25, 30 or amino acid deletions at the N-terminus.
`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
`deletions at the N-terminus.
`[0014]
`In further embodiments, the invention provides DHAD snzymes comprising
`the amino acid sequence P(/L)XXXGX(VL)XIL (SEQ ID NO: 19), wherein X is any
`amino acid, and wherein said DHAD enzymes exhibit the ability to convert 2,3-
`dihydroxyisovaierate to ketoisovalerate in the cytosol.
`[0015]
`in additional embodiments,
`the invention provides DHAD enzymes
`comprising the amino acid sequence CPGXGXC (SEQ ID NO: 123), wherein X is
`any amino acid, and wherein said DHAD enzymes exhibit the ability to convert 2,3-
`dihydroxyisovalerate to ketoisovalerate in the cytosol.
`{0016}
`In another embodiment, the invention provides DHAD enzymes comprising
`the amino acid sequence CPGXG(A/S)C (SEQ ID NO: 124), whereinX is any amino
`acid, and wherein said DHAD enzymes exhibit
`the ability to convert 2,3-
`dihydroxyisovalerate to ketoisovalerate in the cytosol.
`[0017]
`In yet another embodiment,
`the invention provides DHAD enzymes
`comprising the amino acid sequence CXXXPGXGXC (SEQ ID NO: 125), 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 enzymesof the present invention exhibit
`a 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 cytosol.
`In one embodiment, the DHAD enzyme is derived
`from a bacterial organism.
`In a specific embodiment, the DHAD enzyme is derived
`from L. factis and comprises the amino acid sequence of SEQ ID NO: 9.
`In 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
`exhibits Increased cytosolic activity as compared to the DHAD enzyme comprised by
`the amino acid sequence of SEQ ID NO: 714.
`
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`Atty. Docket No. GEVO-041/03US
`
`recombinant
`provides
`invention
`the
`embodiment,
`another
`[mn
`{0021}
`microorganisms comprising a DHAD enzyme comprising the amino acid sequence
`P(UVLPOXGX(VLIXIL (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,
`microorganisms 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.
`In one embodiment, the peptide
`tag is non-cleavable.
`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 selected from the group consisting of ubiquitin, ubiquitin-like (UBL) proteins, myc,
`HA-tag, green fluorescent protein (GFP), and the maltose binding protein (MBP).
`[0023]
`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 embodiment, the protein exhibiting cytosolic activity is DHAD.
`In one embodiment, the chaperone may bea native protein.
`in another embodiment,
`the chaperone protein may be an exogenous protein.
`In some embodiments, the
`chaperone protein may be selected from the group consisting of: endoplasmic
`reticulum oxidoreductin 1 (Ero1, accession no. NP_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 Trx1, accession no.
`NP_013144.4; and Trx2, accession no. NP_0O1 1725.1), thioredoxin reductase (Trrt,
`accession no. NP_010640.1); glutaredoxins (which includes Grxt, accession no.
`NP_008895.1;
` Grx2,
`accession
`no. NP_010801.1; Gre,
`accession
`no.
`NP_010383.1; Grx4, accession no. NP_01101.1; GrxS, accession no. NP_015266.1;
`Grx6, accession no. NP_010274.1: Grx?, accession no. NP_009570.1; Grx8,
`accession
`no. NP_013468.1):
`glutathione
`reductase Glr7
`{accession
`no.
`NP_015234.1); and Jac? (accession no. NP_011497.1), including variants of Jaci
`that have been sulfably altered to reduce ar prevent
`its normal mitochondrial
`localization; Hsp10, Hsp60, GroEL, and GroES and homolags or variants thereof.
`[0024]
`In seme 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 to Escherichia coli, L.
`lactis, Helicobacter pyiori, and Entamoeba
`histolytica,
`in specific embodiments,
`the bacterially derived iron-sulfur cluster
`assembly protein encoding genes are selected from the group consisting of cyaY,
`iseS, iscU, iscA, hscB, hiscA, fdx, isuX, sufA, sufB, sufC, sufD, sufS, sufE, apbC, and
`homologs or variants thereof.
`[0025]
`in another embodiment, the iron-sulfur cluster assemblyprotein encoding
`genes may be derived from eukaryotic organisms, including, but not limited to yeasts
`Page 6 of 144
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`Atty. Docket No. GEVO-041/03US
`
`In one embodiment, the jron-sulfur cluster protein encoding genes are
`and plants.
`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_0O1 1424.1), 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 transiocated
`to chloroplast or plant genes found in the chloroplast genome itself.
`[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 genesto the mitochondria or other
`subcellular compartment is prevented.
`In certain embodiments, it may be preferable
`to overexpress one or more genes encoding an iron-sulfur cluster assembly protein.
`[0027]
`in certain embodiments described herein, it may be desirable to reduce or
`eliminate the activity and/or proteins levels of one or more iron-sulfur cluster
`containing cytosolic proteins.
`In a specific embodiment,
`the iron-sulfur cluster
`containing cytosolic protein is 3-isoprpyimalate dehydratase (Leuip}.
`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 a partial deletion in the LEU? gene resulting
`in the reduction of Leutp 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 requiatory region associated with the
`LEU? 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 fo increase
`
`the levels of iron within the yeast cytosol and mitochondria, such that this iron is
`more available for the production of iron-sulfur cluster-containing proteins in the
`cytosol. Thus, in certain embodiments, the recombinant microorganism may further
`been engineered to overexpress one or more genes selected from the group
`consisting of AFT?, AFT2, GRX3, and GRX4, or homologs thereof.
`In alternative
`embodiments, the microorganism may be engineered to delete and/or attenuate one
`or more genes selected from the group consisting of GRX3 and GRX4, or homologs
`thereof,
`it may be desirable to reduce
`[In various embodiments described herein,
`{0029]
`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
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`Atty. Docket No. GEVO-047/03US
`
`from catalases, superoxide diamutases, metallothioneins, and methionine sulphoxide
`reductases.
`In a specific embodiment, said catalase may be encoded by one of
`more of the genes selected from the group consisting of the E. coli genes kaiG 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. cofi 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 CUP4-2 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 SmiA gene or
`homologs 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 MXR7 and MXR2, or homologsthereof.
`[0030]
`In some embodiments,
`it may be desirable to increase the level of
`available glutathione in the cylosal, 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 fo be expressed to increase the level of
`available glutathione in the cytosol can be selected from 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
`GLRI or homologs thereof.
`In another specific embodiment, said giutathione
`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 cytosol 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 from the group the S. cerevisiae genes GSH? and GSH2, or homologs
`thereof.
`it may be desirable to overexpress one or more
`in some embodiments,
`[0031]
`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 TRXT and TRX2 genes or homologs thereof.
`In another specific
`embodiment, said thioredoxin reductase is encoded by S. cerevisiae TRR? gene or
`homologs thereof.
`In additional embodiments, the recombinant microorganism may
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`Atty. Docket No. GEVO-041/03US
`
`In one
`further be engineered to overexpress the mitochondrial thioredoxin system.
`embodiment, the mitochandrial thioredoxin system is comprised of the mitochondrial
`thioredoxin and mitochondrial thioredoxin reductase.
`In a specific embodiment, said
`mitochondrial thioredoxin is encoded by the S. cerevisiae TRX3 gene or homologs
`thereof.
`In another specific embodiment, said mitochondrial thioredoxin reductase is
`encoded by the S. cerevisiae TRR2 gene or homologs thereof.
`[0632]
`in various embodiments described herein, it may be desirable to engineer
`the recombinant microorganism to overexpress one or more mitochondrial export
`proteins.
`in a specific embodiment, said mitochondrial export protein may be
`selected from the group consisting of the S. cerevisiae ATM1, the S. cerevisiae
`ERV?, and the S. cerevisiae BAT1, or homologs thereof.
`[0033],
`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/ADP carrier protein is encoded bythe S. cerevisiae
`AAGT, AAC2, and/or AAC3 genesor homologs thereof.
`In another embodiment, the
`inner mitochondrial membrane electrical potential, AW,, is increased via a mutation in
`the mitochondrial ATP synthase cornplex that increases ATP hydrolysis activity. Ina
`specific embodiment, said mutation is an ATP1-111 suppressor mutation or a
`corresponding mutation in a homologous protein.
`it may further be desirable to
`[0034]
`in various embodiments described herein,
`engineer the recombinant microorganism fo express one or more enzymes in the
`cytosol that reduce the concentration of reactive nitrogen species (RNS) and/ornitric
`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.
`In a specific embodiment, said nitric oxide reductase is
`encoded by one of more of the genes selected from 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-nitrosothio! reductase is encoded
`by the S. cerevisiae gene SFA7 or homologs thereof.
`In one embodiment, said
`giutathions-S-nitrosothio)
`reductase gene SFA is overexpressed.
`In another
`specific embodiment, said one or more enzymes is encoded by a gene selected from
`the group consisting of the E. coff gene yifE, the Staphylococcus aureus gene scdA,
`and Neisseria gonorrhoeae gene dnrN, or homologs thereof.
`[0035]
`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.
`In one embodiment, the
`recombinant microorganism has been engineered to overexpress one or more of the
`genes selected from the S. cerevisiae genes MET1, MET2, MET3, MET5, METS,
`METIO, MET14, MET16, MET17, HOM2, HOM3, HOM6, CYS3, CYS4, SUL1, and
`SUL2, or homologs thereof. The recombinant microorganism may additionally or
`optionally also overexpress one or more of the genes selected from the S. cerevisiae
`
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`Atty. Docket No. GEVO-041/03US
`
`genes YCT?, MUPT, GAPT, AGPI, GNP1, BAP1, BAP2, TAT1, and TAT2. or
`homologs thereof,
`[0036]=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
`embadiment, the recombinant microorganism may exhibit at feast about 10 percent,
`ai 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 100 percent, at least about 200
`percent, or at least about 500 percent greater dihydroxyacid dehydratase (DHAD)
`activity in the cytosol as compared to the parental microorganism.
`[0037]
`In certain embodiments described herein,
`it may be desirable to further
`overexpress an additional enzyme that converts 2,3-dihydroxyisovalerate to
`ketoisovalerate in the cytosol.
`In a specific embodiment,
`the enzyme may be
`selected
`from the
`group
`consisting
`of
`3-isopropyimalate
`(Leutp)
`and
`imidazoleglycerol-phosphate dehydrogenase (His3p).
`recombinant
`the
`herein,
`[0038]
`In
`various
`embodiments
`described
`microorganisms may be further engineered to express an isobutanol producing
`metabolic pathway comprising at least one exogenous gene that catalyzes a stepin
`the conversion of pyruvate to isobutanol.
`in one embodiment, the recombinant
`microorganism may be engineered to express an isobutanol producing metabolic
`pathwaycomprising at least two exogenous genes.
`In another embodiment, the
`recombinant microorganism may be engineered to express an isobutanol producing
`metabolic pathway comprising at Jeast
`three exogenous genes.
`in another
`embodiment, the recombinant microorganism may be engineered to express an
`isobutanol producing metabolic pathway comprising at feast four exogenous genes.
`In another embodiment,
`the recombinant microorganism may be engineered to
`express an isobutanol producing metabolic pathway comprising five exogenous
`genes.
`the isobutanol pathway
`in various embodiments described herein,
`{0039}
`enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS),
`ketol-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2-keto-acid
`decarboxylase (KIVD), and isobutyraidehyde dehydrogenase (IDH).
`In a preferred
`embodiment, said dihydroxyacid dehydratase (DHAD)
`is a cytosolically active
`(DHAD) enzyme.
`recombinant
`the
`herein,
`described
`embodiments
`{0040}
`in
`various
`microorganisms may be engineered to express native genes that catalyze a step in
`the canversion of pyruvate to isobutanol.
`in one embodiment, the recombinant
`microorganism is engineered to increase the activity of a native metabolic pathway
`gene for conversion of pyruvate to isobutanol.
`[In another embodiment,
`the
`recombinant microorganism is further engineered to include at least one enzyme
`encoded by a heterologous gene and at least one enzyme encoded by a native
`gene.
`In yet another embodiment,
`the recombinant microorganism comprises a
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`Atty. Docket No. GEVO-044/038US
`
`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
`catalyzing the conversion of pyruvate to isobutanolis/are localized in the cytosol.
`[n
`a preferred embodiment, the enzyme Is dihydroxyacid dehydratase (DHAD).
`[0042],
`[In some embodiments,
`the present
`invention provides
`recombinant
`microorganisms that have been engineered to express a heterologous metabolic
`pathway for conversion of pyruvate to isobutanol.
`In another embodiment,
`the
`recombinant microorganism further comprises a pathway for the fermentation of
`isobutanol from a pentose sugar.
`In one embodiment, the pentose sugar is xylose.
`in one embodiment,
`the recombinant microorganism is engineered to express 2
`functional xylose isomerase (XI).
`In another embodiment,
`the recombinant
`microorganism further comprises a detetion or disruption of a native gene encoding
`for an enzyme that catalyzes the conversion of xylose to xylitol.
`In one embodiment,
`the native gene is xylose reductase (XR).
`In another embodiment, the native gene is
`xylitol dehydrogenase (XDH).
`In yet another embodiment, both native genes are
`deleted or disrupted.
`In yet another embodiment, the recombinant microorganism is
`engineered to express a xylulose kinase enzyme.
`recombinant
`[0043]
`in another aspect,
`the present
`invention provides a
`microorganism engineered to include reduced pyruvate decarboxylase (PDC) activity
`as compared to a parental microorganism.
`In one embodiment, PDC activity is
`eliminated. PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is
`reduced to ethanol by alcohol dehydrogenases via the oxidation of NADH to NAD+.
`In one embodiment, the recombinant microorganism includes a mutation in at feast
`one PDC gene resulting in a reduction of PDC activity of a polypeptide encoded by
`said gene.
`in another embodiment,
`the recombinant microorganism includes a
`partial deletion of a PDC gene resulting in a reduction of PDC activity of a
`polypeptide encoded by said gene.
`in another embodiment,
`the recombinant
`microorganism comprises a complete deletion of a PDC gene resulting in a reduction
`of PDC activity of a polypeptide encoded by said gens.
`In yet another embodiment,
`the recomb