Host cells and methods for producing isopentenol from mevalonate

{
  "publication_number": "US10273506B2",
  "country": "US",
  "kind": "B2",
  "title": "Host cells and methods for producing isopentenol from mevalonate",
  "abstract": "The present invention provides for a genetically modified host cell capable of producing isopentenol and/or 3-methyl-3-butenol, comprising (a) an increased expression of phosphomevalonate decarboxylase (PMD) (b) an increased expression of a phosphatase capable of converting isopentenol into 3-methyl-3-butenol, (c) optionally the genetically modified host cell does not express, or has a decreased expression of one or more of NudB, phosphomevalonate kinase (PMK), and/or PMD, and (d) optionally one or more further enzymes capable of converting isopentenol and/or 3-methyl-3-butenol into a third compound, such as isoprene.",
  "claims": [
    "1. A genetically modified host cell capable of producing isopentenol and/or 3-methyl-3-butenol, comprising (a) an increased expression of phosphomevalonate decarboxylase (PMD), wherein the PMD has an amino acid sequence having at least 90% identity with SEQ ID NO:1, and (i) amino acid residue at position 74 is histidine, (ii) amino acid residue at position 145 is phenylalanine, or (iii) amino acid residue at position 74 is histidine and amino acid residue at position 145 is phenylalanine, (b) an increased expression of a phosphatase capable of converting isopentenol into 3-methyl-3-butenol, (c) optionally the genetically modified host cell does not express, or has a decreased expression of one or more of dihydroneopterin triphosphate diphosphate (NudB), phosphomevalonate kinase (PMK), and/or PMD, and (d) optionally one or more further enzymes capable of converting isopentenol and/or 3-methyl-3-butenol into a third compound; wherein the host cell is a bacterial or fungal cell.",
    "2. The genetically modified host cell of claim 1, wherein the decreased expression is a disruption of the promoter or knock out of the gene encoding NudB, PMK, and/or PMD.",
    "3. The genetically modified host cell of claim 1, wherein the third compound is isoprene.",
    "4. The genetically modified host cell of claim 1, further comprising an increased expression of one or more of acetyl-CoA acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), and/or mevalonate kinase (MK).",
    "5. The genetically modified host cell of claim 1, wherein the PMD is encoded on a nucleotide sequence which is in nucleic acids which is transformed into the genetically modified host cell, or host cell prior to genetic modification.",
    "6. The genetically modified host cell of claim 3, wherein one or more of the PMD, phosphatase, AtoB, HMGS, HMGR, and MK, are encoded on one or more nucleotide sequences which are in one or more nucleic acids which are transformed into the genetically modified host cell, or host cell prior to genetic modification.",
    "7. A method for producing isopentenol and/or 3-methyl-3-butenol and/or the third compound, comprising: (a) providing a genetically modified host cell of claim 1, 2, or 3, (b) culturing the genetically modified host cell under a condition wherein phosphomevalonate decarboxylase (PMD) and/or phosphatase are expressed, and (c) optionally recovering the isopentenol and/or 3-methyl-3-butenol and/or the third compound.",
    "8. The method of claim 7, wherein the (b) culturing step further comprises expressing acetyl-CoA acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), and/or mevalonate kinase (MK).",
    "9. The method of claim 7, wherein the (b) culturing step is under an anaerobic or microaerobic condition.",
    "10. The method of claim 8, wherein the (b) culturing step is under an anaerobic or microaerobic condition.",
    "11. The genetically modified host cell of claim 1, wherein the genetically modified host cell is a species of the genus Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Paracoccus, or Clostridia.",
    "12. The genetically modified host cell of claim 11, wherein the genetically modified host cell is a species of the genus Escherichia.",
    "13. The genetically modified host cell of claim 1, wherein the genetically modified host cell is a yeast cell.",
    "14. The genetically modified host cell of claim 13, wherein the yeast cell is a species of the Saccharomyces genus.",
    "15. The genetically modified host cell of claim 1, wherein the PMD comprises the following amino acid residues: (a) E at position 71, S at position 108, N at position 110, A at position 119, S at position 120, S at position 121, A at position 122, S at position 155, R at position 158, S at position 208, and D at position 302 corresponding to SEQ ID NO:1; or (b) E at position 73, S at position 94, N at position 96, A at position 105, S at position 106, S at position 107, A at position 108, S at position 141, R at position 144, S at position 192, and D at position 283 corresponding to SEQ ID NO:2.",
    "16. The genetically modified host cell of claim 1, wherein the PMD has an amino acid sequence having at least 95% identity with SEQ ID NO:1.",
    "17. The genetically modified host cell of claim 16, wherein the PMD has an amino acid sequence having at least 99% identity with SEQ ID NO:1."
  ],
  "description_excerpt": "The application claims priority as a continuation application to PCT International Patent Application No. PCT/US16/18984, filed Feb. 22, 2016, which claims priority to U.S. Provisional Patent Application Ser. No. 62/119,071, filed Feb. 20, 2015; both of which are incorporated herein by reference.\n\nThe invention was made with government support under Contract Nos. DE-AC02-05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in the invention.\n\nThe present invention is in the field of producing isopentenol.\n\nThe mevalonate pathway has been extensively used to produce a range of valuable chemicals via isopentenyl pyrophosphates (IPP) or dimethylallyl pyrophosphates (DMAPP) as essential intermediates for terpene synthesis. In addition to terpene-based chemicals, Chou and colleagues engineered the mevalonate pathway in Escherichia coli to produce isopentenols via hydrolysis of IPP to 3-methyl-3-butenol (Chou and Keasling, Appl. Environ. Microbiol. 2012). In this pathway, 3 ATPs are required to produce one molecule of IPP from one molecule of mevalonate. Among these 3 ATPs, two ATPs are consumed for two-step phosphorylations to produce mevalonate pyrophosphate, but the pyrophosphate group is subsequently hydrolyzed to produce isopentenol from IPP. As a result, the overall pathway is not only energetically demanding but also inefficient because of unnecessary phosphorylation-dephosphorylation steps.",
  "cpc": [
    "C07F 9/091",
    "B60K 2360/173",
    "B60K 35/28",
    "B65G 69/006",
    "C07F 9/098",
    "C08F 136/08",
    "C12N 9/0006",
    "C12N 9/1025",
    "C12N 9/1205",
    "C12N 9/16",
    "C12N 9/88",
    "C12P 5/007",
    "C12P 7/04",
    "C12Y 101/01088",
    "C12Y 203/0301",
    "C12Y 207/01036",
    "C12Y 401/01033",
    "G01S 2013/9314",
    "G01S 2013/9317",
    "G01S 2013/9323",
    "G01S 2013/9324"
  ],
  "ipc": [
    "C07F 9/09",
    "C08F 136/08",
    "C12N 1/20",
    "C12N 9/04",
    "C12N 9/10",
    "C12N 9/12",
    "C12N 9/16",
    "C12N 9/88",
    "C12P 5/00",
    "C12P 7/04"
  ],
  "assignees": [
    "UNIV CALIFORNIA"
  ],
  "inventors": [
    "LEE TAEK SOON",
    "KANG ARAM"
  ],
  "filing_date": "2017-08-21",
  "publication_date": "2019-04-30",
  "grant_date": "2019-04-30",
  "priority_date": "2015-02-20",
  "application_number": "US-201715682325-A",
  "family_id": "56692485",
  "citations": [
    "US2013089906A1",
    "WO2014100726A2"
  ]
}