US008273565B2
`
`(12) United States Patent
`Dundon et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,273,565 B2
`*Sep. 25, 2012
`
`(54)
`
`METHODS OF INCREASING DIHYDROXY
`ACID DEHYDRATASE ACTIVITY TO
`
`IMPROVE PRODUCTION OF FUELS,
`CHEMICALS, AND AMINO ACIDS
`
`(75)
`
`Inventors: Catherine Asleson Dundon,
`Englewood, CO (US); Aristos
`Aristidou, Highlands Ranch, CO (US);
`Andrew Hawkins, Parker, CO (US);
`Doug Lies, Parker, CO (US); Lynne
`Albert, Golden, CO (US)
`
`(73)
`
`Assignee: Gevo, Inc., Englewood, CO (US)
`
`(*)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. l54(b) by 0 days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21)
`
`Appl. No.: 13/246,693
`
`(22)
`
`Filed:
`
`Sep. 27, 2011
`
`Prior Publication Data
`
`US 2012/0028322 A1
`
`Feb. 2, 2012
`
`Related U.S. Application Data
`
`Division of application No. 13/228,342, filed on Sep.
`8, 2011, now Pat. No. 8,071,358, and a division of
`application No. 12/953,884, filed on Nov. 24, 2010,
`now Pat. No. 8,017,376.
`
`Provisional application No. 61/263,952, filed on Nov.
`24, 2009, provisional application No. 61/350,209,
`filed on Jun. 1, 2010.
`
`Int‘ Cl‘
`(200601)
`CIZN 1/00
`(2006:01)435/254 2 536/23 1
`ICJ'0S7HCf1/02
`.
`.
`.
`.................................. ..
`.
`;
`.
`Field of Classification Search ...................... .. None
`
`(65)
`
`(62)
`
`(60)
`
`(51)
`
`(52)
`(58)
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
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`8,017,376 B2
`8,071,358 B1
`8,232,089
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`
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`
`(Continued)
`
`Primary Examiner — Alexander Kim
`(74) Attorney, Agent, or Firm — Cooley LLP
`
`(57)
`
`ABSTRACT
`
`The present invention is directed to recombinant microorgan-
`isms comprising one or more dihydroxyacid dehydratase
`(DHAD)-requiring biosynthetic pathways and methods of
`using said recombinant microorganisms to produce beneficial
`metabolites derived from said DHAD-requiring bio synthetic
`pathways. In various aspects of the invention, the recombi-
`nant microorganisms may be engineered to overexpress one
`or more polynucleotides encoding one or moreAft proteins or
`homologs thereof. In some embodiments, the recombinant
`microorganisms may comprise a cytosolically localized
`DHAD enzyme. In additional embodiments, the recombinant
`microorganisms may comprise a mitochondrially localized
`DHAD enzyme. In various embodiments described herein,
`the recombinant microorganisms may be microorganisms of
`the Saccharomyces clade, Crabtree-negative yeast microor-
`ganisms, Crabtree-positive yeast microorganisms, post-
`WGD (whole genome duplication) yeast microorganisms,
`pre-WGD (whole genome duplication) yeast microorgan-
`isms, and nomfermeming yeast microorganisms.
`
`See application file for complete search history.
`
`19 Claims, 7 Drawing Sheets
`
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`BUTAMAX 100 1
`
`

`
`US 8,273,565 B2
`Page 2
`
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`* cited by examiner
`
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`Sep. 25, 2012
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`US 8,273,565 B2
`
`1
`METHODS OF INCREASING DIHYDROXY
`ACID DEHYDRATASE ACTIVITY TO
`
`IMPROVE PRODUCTION OF FUELS,
`CHEMICALS, AND AMINO ACIDS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`2
`
`higher than those found in non-engineered microorganisms in
`order to sustain commercially viable productivities, yields,
`and titers. The present application addresses this need by
`engineering recombinant microorganisms to improve their
`DHAD activity.
`
`SUMMARY OF THE INVENTION
`
`This application is a divisional ofU.S. application Ser. No.
`13/228,342, filed Sep. 8, 2011, which is a divisional applica-
`tion of U.S. application Ser. No. 12/953,884, filed Nov. 24,
`2010, now U.S. Pat. No. 8,017,376, which claims the benefit
`of U.S. Provisional Application Ser. No. 61/263,952, filed
`Nov. 24, 2009, and U.S. Provisional Application Ser. No.
`61/350,209, filed Jun. 1, 2010, each of which are herein
`incorporated by reference in their entireties for all purposes.
`
`ACKNOWLEDGMENT OF GOVERNMENTAL
`SUPPORT
`
`This invention was made with government support under
`Contract No. IIP-0823122, awarded by the National Science
`Foundation, and under Contract No. EP-D-09-023, awarded
`by the Environmental Protection Agency. The government
`has certain rights in the invention.
`
`TECHNICAL FIELD
`
`Recombinant microorganisms and methods of producing
`such organisms are provided. Also provided are methods of
`producing beneficial metabolites including fuels, chemicals,
`and amino acids by contacting a suitable substrate with
`recombinant microorganisms and enzymatic preparations
`therefrom.
`
`DESCRIPTION OF THE TEXT FILE SUBMITTED
`ELECTRONICALLY
`
`The contents of the text file submitted electronically here-
`with are incorporated herein by reference in their entirety: A
`computer readable format copy ofthe Sequence Listing (file-
`name:
`GEVO_041_13US_SeqList_ST25.txt,
`date
`recorded: Sep. 27, 2011, file size: 658 kilobytes).
`
`BACKGROUND
`
`Dihydroxyacid dehydratase (DHAD) is an enzyme that
`catalyzes the conversion of2,3-dihydroxyisovalerate to 0t-ke-
`toisovalerate and of 2,3-dihydroxy-3-methylvalerate to
`2-keto-3-methylvalerate. This enzyme plays an important
`role in a variety of biosynthetic pathways, including path-
`ways producing valine, isoleucine, leucine and pantothenic
`acid (vitamin B5). DHAD also catalyzes the conversion of
`2,3-dihydroxyisovalerate to ot-ketoisovalerate as part of
`isobutanol biosynthetic pathways disclosed in commonly
`owned and co-pending US Patent Publication Nos. 2009/
`0226991 and 2010/0143997. In addition, biosynthetic path-
`ways for the production of 3-methyl- 1 -butanol and 2-methyl-
`1-butanol use DHAD to convert 2,3-dihydroxyisovalerate to
`ot-ketoisovalerate and 2,3-dihydroxy-3 -methylvalerate to
`2-keto-3-methylvalerate, respectively (Atsumi et al., 2008,
`Nature 451(7174): 86-9).
`DHAD is an essential enzyme in all of these biosynthetic
`pathways, hence, it is desirable that recombinant microorgan-
`isms engineered to produce the above-mentioned compounds
`exhibit optimal DHAD activity. The optimal level of DHAD
`activity will typically have to be at levels that are significantly
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`The present inventors have discovered that overexpres sion
`of the transcriptional activator genes AFT1 and/or AFT2 or
`homologs thereof in a recombinant yeast microorganism
`improves DHAD activity. Thus,
`the invention relates to
`recombinant yeast cells engineered to provide increased het-
`erologous or native expression of AFT1 and/or AFT2 or
`homologs thereof. In general, cells that overexpress AFT1
`and/orAFT2 or homologs thereof exhibit an enhanced ability
`to produce beneficial metabolites such as isobutanol, 3-me-
`thyl-1-butanol, 2-methyl-1-butanol, valine, isoleucine, leu-
`cine, and pantothenic acid.
`One aspect of the invention is directed to a recombinant
`microorganism comprising a DHAD-requiring biosynthetic
`pathway, wherein said microorganism is engineered to over-
`express one or more polynucleotides encoding one or more
`Aft proteins or homologs thereof. In one embodiment, the Aft
`protein is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ
`ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
`SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID
`NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
`SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
`NO: 34, SEQ ID NO: 36, SEQ ID NO: 209, SEQ ID NO: 211,
`SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ
`ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, and SEQ ID
`NO: 225. In another embodiment, one or more of the poly-
`nucleotides encoding said one or more Aft proteins or
`homologs thereof is a native polynucleotide. In yet another
`embodiment, one or more of the polynucleotides encoding
`said one or more Aft proteins or homologs thereof is a heter-
`ologous polynucleotide.
`In a specific embodiment according to this aspect, the
`invention is directed to a recombinant microorganism com-
`prising a DHAD-requiring biosynthetic pathway, wherein
`said microorganism has been engineered to overexpress a
`polynucleotide encoding Aftl (SEQ ID NO: 2) and/or Aft2
`(SEQ ID NO: 4) or a homolog thereof. In one embodiment,
`the polynucleotide encoding the Aft protein or homolog
`thereof is native to the recombinant microorganism.
`In
`another embodiment, the polynucleotide encoding the Aft
`protein or homolog thereof is heterologous to the recombi-
`nant microorganism.
`Another aspect of the invention is directed to a recombi-
`nant microorganism comprising a DHAD-requiring biosyn-
`thetic pathway, wherein the activity of one or more Aft pro-
`teins or homologs thereof is increased. In one embodiment,
`the Aft protein is selected from SEQ ID NO: 2, SEQ ID NO:
`4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
`NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,
`SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID
`NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32,
`SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 209, SEQ ID
`NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
`217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223,
`and SEQ ID NO: 225. In one embodiment, the polynucleotide
`encoding the Aft protein or homolog thereof is native to the
`recombinant microorganism. In another embodiment, the
`polynucleotide encoding the Aft protein or homolog thereof
`is heterologous to the recombinant microorganism.
`
`

`
`US 8,273,565 B2
`
`3
`Another aspect of the invention is directed to a recombi-
`nant microorganism comprising a DHAD-requiring biosyn-
`thetic pathway, wherein said microorganism has been engi-
`neered to overexpress one or more polynucleotides encoding
`one or more proteins or homologs thereof regulated by anAft
`protein or homolog thereof. In one embodiment, the proteins
`regulated by an Aft protein or homolog thereof are selected
`from FET3, FET4, FET5, FTR1, FTH1, SMF3, MRS4,
`CCC2, COT1, ATX1, FRE1, FRE2, FRE3, FRE4, FRE5,
`FRE6, FIT1, FIT2, FIT3, ARN1, ARN2, ARN3, ARN4,
`ISU1,
`ISU2, TIS11, HMX1, AKR1, PCL5, YOR387C,
`YHL035C, YMR034C, ICY2, PRY1, YDL124W, BNA2,
`ECM4, LAP4, YOL083W, YGR146C, BIO5, YDR271C,
`OYE3, CTH1, CTH2, MRS3, MRS4, HSP26,YAP2, VMR1,
`ECL1, OSW1, NFT1, ARA2, TAF1/TAF130/TAF145,
`YOR225W, YKR104W, YBR012C, and YMR041C or
`homologs thereof. In a specific embodiment, the protein regu-
`lated by an Aft protein or homolog thereof is ENB1. In
`another specific embodiment, the protein regulated by an Aft
`protein or homologs thereof is FET3. In yet another specific
`embodiment,
`the protein regulated by an Aft protein or
`homolog thereof is SMF3. In one embodiment, all genes
`demonstrated to increase DHAD activity and/or the produc-
`tion of a metabolite from a DHAD-requiring biosynthetic
`pathway are overexpressed. Where none of the AFT regulon
`genes expressed alone are effective in increasing DHAD
`activity and/or the production of a metabolite from a DHAD-
`requiring biosynthetic pathway, then 1, 2, 3, 4, 5, or more of
`the genes in the AFT regulon may be overexpres sed together.
`In various embodiments described herein, the DHAD-re-
`quiring biosynthetic pathway may be selected from isobu-
`tanol, 3-methyl-1-butanol, 2-methyl-1-butanol, valine, iso-
`leucine,
`leucine, and/or pantothenic acid biosynthetic
`pathways. In various embodiments described herein,
`the
`DHAD enzyme which acts as part ofan isobutanol, 3-methyl-
`1-butanol, 2-methyl-1-butanol, valine,
`isoleucine,
`leucine,
`and/or pantothenic acid biosynthetic pathway may be local-
`ized to the cytosol. In alternative embodiments, the DHAD
`enzyme which acts as part of an isobutanol, 3-methyl-1-
`butanol, 2-methyl-1-butanol, valine, isoleucine, leucine, and/
`or pantothenic acid biosynthetic pathway may be localized to
`the mitochondria. In additional embodiments, a DHAD
`enzyme which acts as part of an isobutanol, 3-methyl-1-
`butanol, 2-methyl-1-butanol, valine, isoleucine, leucine, and/
`or pantothenic acid biosynthetic pathway is localized to the
`cytosol and the mitochondria.
`In one embodiment, the invention is directed to a recom-
`binant microorganism for producing isobutanol, wherein said
`recombinant microorganism comprises an isobutanol pro-
`ducing metabolic pathway and wherein said microorganism
`is engineered to overexpress one or more polynucleotides
`encoding one or more Aft proteins or homologs thereof. In
`one embodiment, the Aft protein is selected from SEQ ID
`NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ
`ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,
`SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID
`NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,
`SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID
`NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
`215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221,
`SEQ ID NO: 223, and SEQ ID NO: 225.
`In a specific embodiment, the invention is directed to a
`recombinant microorganism for producing isobutanol,
`wherein said recombinant microorganism comprises an
`isobutanol producing metabolic pathway and wherein said
`microorganism is engineered to overexpress a polynucleotide
`encoding Aft1 (SEQ ID NO: 2) or a homolog thereof. In
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`4
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`another specific embodiment, the invention is directed to a
`recombinant microorganism for producing isobutanol,
`wherein said recombinant microorganism comprises an
`isobutanol producing metabolic pathway and wherein said
`microorganism is engineered to overexpress a polynucleotide
`encoding Aft2 (SEQ ID NO: 4) or a homolog thereof. In yet
`another embodiment, the invention is directed to a recombi-
`nant microorganism for producing isobutanol, wherein said
`recombinant microorganism comprises an isobutanol pro-
`ducing metabolic pathway and wherein said microorganism
`is engineered to overexpress a polynucleotide encoding Aft1
`(SEQ ID NO: 2) or a homolog thereof and Aft2 (SEQ ID NO:
`4) or a homolog thereof.
`In each of the aforementioned aspects and embodiments,
`the Aft protein may be a constitutively active Aft protein or a
`homolog thereof. In one embodiment,
`the constitutively
`active Aft protein or homolog thereofcomprises a mutation at
`a position corresponding to the cysteine 291 residue of the
`native S. cerevisiae Aft1 (SEQ ID NO: 2). In a specific
`embodiment, the cysteine 291 residue is replaced with a phe-
`nylalanine residue. In another embodiment, the constitutively
`active Aft protein or homolog thereofcomprises a mutation at
`a position corresponding to the cysteine 187 residue of the
`native S. cerevisiae Aft2 (SEQ ID NO: 2). In a specific
`embodiment, the cysteine 187 residue is replaced with a phe-
`nylalanine residue.
`In another embodiment, the invention is directed to a
`recombinant microorganism for producing isobutanol,
`wherein said recombinant microorganism comprises an
`isobutanol producing metabolic pathway, wherein said
`microorganism has been engineered to overexpress one or
`more polynucleotides encoding one or more proteins or
`homologs thereof regulated by an Aft protein or homolog
`thereof. In one embodiment, the proteins regulated by Aft or
`a homolog thereof are selected from FET3, FET4, FET5,
`FTR1, FTH1, SMF3, MRS4, CCC2, COT1, ATX1, FRE1,
`FRE2, FRE3, FRE4, FRE5, FRE6, FIT1, FIT2, FIT3,ARN1,
`ARN2, ARN3, ARN4, ISU1, ISU2, TIS11, HMX1, AKR1,
`PCL5, YOR387C, YHL035C, YMR034C,
`ICY2, PRY1,
`YDL124W, BNA2, ECM4, LAP4, YOL083W, YGR146C,
`BIO5, YDR271C, OYE3, CTH1, CTH2, MRS3, MRS4,
`HSP26, YAP2, VMR1, ECL1, OSW1, NFT1, ARA2, TAF1/
`TAF130/TAF145, YOR225W, YKR104W, YBR012C, and
`YMR041C or homologs thereof. In a specific embodiment,
`the protein regulated by an Aft protein or homolog thereof is
`ENB 1. In another specific embodiment, the protein regulated
`by an Aft protein or homologs thereof is FET3. In yet another
`specific embodiment, the protein regulated by an Aft protein
`or homolog thereof is SMF3. In one embodiment, all genes
`demonstrated to increase DHAD activity and/or the produc-
`tion of a metabolite from a DHAD-requiring biosynthetic
`pathway are overexpressed. Where none of the AFT regulon
`genes expressed alone are effective in increasing DHAD
`activity and/or the production of a metabolite from a DHAD-
`requirin