Host cells and methods for producing isopentenol from mevalonate

{
  "publication_number": "US10814724B2",
  "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 3-methyl-3-butenol, comprising: (a) expression of acetyl-CoA acetyltransferase (AtoB), (b) expression of hydroxymethylglutaryl-CoA synthase (HMGS), (c) expression of hydroxymethylglutaryl-CoA reductase (HMGR), (d) expression of phosphomevalonate decarboxylase (PMD), wherein the PMD has an amino acid sequence having at least 90% identity with SEQ ID NO:1, and (e) the genetically modified host cell does not express phosphomevalonate kinase (PMK); wherein the host cell is a bacterial or fungal cell.",
    "2. The genetically modified host cell of claim 1, comprising wherein the PMD comprises (i) a histidine at position 74, (ii) a phenylalanine at position 145, or (iii) a histidine at position 74 and a phenylalanine at position 145, corresponding to the numbering of SEQ ID NO:1.",
    "3. The genetically modified host cell of claim 2, 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.",
    "4. 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.",
    "5. The genetically modified host cell of claim 4, wherein the PMD has an amino acid sequence having at least 99% identity with SEQ ID NO:1.",
    "6. The genetically modified host cell of claim 1, comprising one or more further enzymes capable of converting 3-methyl-3-butenol into a third compound.",
    "7. The genetically modified host cell of claim 6, wherein the third compound is isoprene.",
    "8. The genetically modified host cell of claim 1, wherein one or more of the PMD, AtoB, HMGS, and HMGR, 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 prior to genetic modification.",
    "9. 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.",
    "10. The genetically modified host cell of claim 9, wherein the genetically modified host cell is a species of the genus Escherichia.",
    "11. The genetically modified host cell of claim 1, wherein the genetically modified host cell is a yeast cell.",
    "12. The genetically modified host cell of claim 11, wherein the yeast cell is a species of the Saccharomyces genus.",
    "13. A method for producing 3-methyl-3-butenol, comprising: (a) providing a genetically modified host cell comprising: (i) expression of acetyl-CoA acetyltransferase (AtoB), (ii) expression of hydroxymethylglutaryl-CoA synthase (HMGS), (iii) expression of hydroxymethylglutaryl-CoA reductase (HMGR), (iv) expression of phosphomevalonate decarboxylase (PMD), wherein the PMD has an amino acid sequence having at least 90% identity with SEQ ID NO:1, and (v) the genetically modified host cell does not express phosphomevalonate kinase (PMK), wherein the host cell is a bacterial or fungal cell; and (b) culturing the genetically modified host cell under a condition wherein phosphomevalonate decarboxylase (PMD), acetyl-CoA acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), and hydroxymethylglutaryl-CoA reductase (HMGR) are expressed.",
    "14. The method of claim 13, wherein the PMD comprises (i) a histidine at position 74, (ii) a phenylalanine at position 145, or (iii) a histidine at position 74 and a phenylalanine at position 145, corresponding to the numbering of SEQ ID NO:1.",
    "15. The method of claim 13, comprising (c) recovering the 3-methyl-3-butenol.",
    "16. The method of claim 13, wherein the genetically modified host cell comprises one or more further enzymes capable of converting 3-methyl-3-butenol into a third compound.",
    "17. The method of claim 16, comprising (c) recovering the third compound.",
    "18. The method of claim 16, wherein the third compound is isoprene.",
    "19. The method of claim 18, comprising (c) recovering the isoprene.",
    "20. The method of claim 13, wherein the (b) culturing step is under an anaerobic or microaerobic condition."
  ],
  "description_excerpt": "The application claims priority as a continuation application to U.S. patent application Ser. No. 15/682,325, filed Aug. 21, 2017, now U.S. Pat. No. 10,273,506, issued Apr. 30, 2019, which 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; all 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 2370/173",
    "B60K 35/00",
    "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": [
    "B60K 35/00",
    "B65G 69/00",
    "C07F 9/09",
    "C08F 136/08",
    "C12N 9/04",
    "C12N 9/10",
    "C12N 9/12",
    "C12N 9/16",
    "C12N 9/88",
    "C12P 5/00",
    "C12P 7/04",
    "G01S 13/931"
  ],
  "assignees": [
    "University of California San Diego UCSD"
  ],
  "inventors": [
    "Taek Soon Lee",
    "Aram Kang"
  ],
  "filing_date": "2019-04-18",
  "publication_date": "2020-10-27",
  "grant_date": "2020-10-27",
  "priority_date": "2015-02-20",
  "application_number": "US-201916388556-A",
  "family_id": "56692485",
  "cited_by_count": 1,
  "citations": [
    "US20130089906A1",
    "WO2014100726A2",
    "US20160002672A1",
    "US10273506B2"
  ]
}