JPWO2020097588A5 - - Google Patents

Claims (15)

A host cell comprising a heterologous polynucleotide encoding a cucurbitadienol synthase (CDS) enzyme, wherein the heterologous polynucleotide sequence is at least 90% identical to SEQ ID NO: 3 and/or the CDS encoded by the heterologous polynucleotide is at least 90% identical to SEQ ID NO: 43, and wherein said host cell produces a cucurbitadienol compound. 2. The host cell of claim 1, wherein said CDS comprises a leucine at the amino acid residue corresponding to amino acid residue 123 of SEQ ID NO: 73 . 3. The host cell of claim 1 or 2 , wherein said CDS enzyme comprises a substrate groove and an active site cavity. 4. The host cell further comprises one or more heterologous polynucleotides encoding UDP-glycosyltransferase (UGT), C11 hydroxylase, cytochrome P450 reductase, epoxide hydrolase (EPH) , lanosterol synthase, and/or squalene epoxidase. The host cell according to any one of 1-3. A host cell according to any one of claims 1-4, wherein said host cell is a yeast cell, a plant cell or a bacterial cell. 6. A host cell according to claim 5, wherein said host cell is a Saccharomyces cell, optionally a Saccharomyces cerevisiae cell; a Yarrowia cell, optionally a Yarrowia lipolytica cell; or an E. coli cell. wherein said host cell produces at least 10%, 20%, or 30% or more of a cucurbitadienol compound relative to a control, wherein said subject is a swingle CDS encoded by a polynucleotide corresponding to SEQ ID NO:33; 7. The host cell of any one of claims 1-6, which is a host cell that expresses 8. Any one of claims 1-7, wherein said CDS comprises the amino acid motif DQGWL (SEQ ID NO:335), optionally said motif is present at residues in the CDS corresponding to residues 479-483 in SEQ ID NO:73. A host cell as described in . 9. Any one of claims 1-8, wherein the CDS comprises the amino acid motif GHWANDLGGP (SEQ ID NO:336), optionally wherein the motif is present at residues in the CDS corresponding to residues 117-126 in SEQ ID NO:73. A host cell according to paragraph. 10. Any one of claims 1-9, wherein the CDS comprises the amino acid motif CWGVCYTYAGW (SEQ ID NO:337), optionally wherein the motif is present at residues in the CDS corresponding to residues 612-622 in SEQ ID NO:73. A host cell according to paragraph. 11. The host cell of any one of claims 1-10, wherein the amino acid sequence of said CDS comprises SEQ ID NO:43. 12. The host cell of any one of claims 1-11, wherein said host cell is further modified to down-regulate lanosterol synthase (ERG7). 13. A cucurbita comprising contacting the host cell of any one of claims 1-12 with oxidosqualene and optionally isolating the cucurbitadienol compound, thereby producing a cucurbitadienol compound. A method for producing a dienol compound. 14. The method of claim 13 , wherein the oxidosqualene is 2-3-oxidosqualene or 2,3;22,23-diepoxysqualene. The method of claim 13 or 14 or any of claims 1-12, wherein the cucurbitadienol compound is 11-hydroxy cucurbitadienol, 24-25 epoxy-cucurbitadienol, or cucurbitadienol. or the host cell of claim 1.