Biosynthesis of mogrosides

Patent metadata

Field	Value
Publication number	US-12234464-B2
Application number	US-201917292266-A
Country	US
Kind code	B2
Filing date	Nov 9, 2019
Priority date	Nov 9, 2018
Publication date	Feb 25, 2025
Grant date	Feb 25, 2025

Abstract

Provided herein are enzymes (e.g., cucurbitadienol synthases (CDS), UDP-glycosyltransferases (UGT), C11 hydroxylases, epoxide hydrolases (EPH), squalene epoxidases, and/or cytochrome P450 reductases), host cells expressing the enzymes, and methods of producing mogrol precursors, mogrol, and/or mogrosides using such host cells.

First claim

What is claimed is:

1. A host cell that comprises 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 amino acid sequence of the CDS encoded by the heterologous polynucleotide is at least 90% identical to SEQ ID NO: 43, and wherein the host cell produces 11-hydroxycucurbitadienol, 24-25 epoxy-cucurbitadienol or cucurbitadienol, and wherein the host cell is not a plant cell.

2. The host cell of claim 1 , wherein the CDS comprises a leucine at the amino acid residue corresponding to the amino acid residue at position 123 of SEQ ID NO: 73.

3. The host cell of claim 1 , wherein the host cell further comprises one or more heterologous polynucleotides encoding a UDP-glycosyltransferase (UGT), a C11 hydroxylase, a cytochrome P450 reductase, an epoxide hydrolase (EPH), a lanosterol synthase, and/or a squalene epoxidase.

4. A method of producing 11-hydroxycucurbitadienol, 24-25 epoxy-cucurbitadienol or cucurbitadienol, comprising contacting the host cell of claim 1 with 2-3-oxidosqualene or 2,3; 22,23-diepoxysqualene, thereby producing the 11-hydroxycucurbitadienol, 24-25 epoxy-cucurbitadienol or cucurbitadienol.

5. The method of claim 4 , wherein the method further comprises isolating the 11-hydroxycucurbitadienol, 24-25 epoxy-cucurbitadienol or cucurbitadienol.

6. The host cell of claim 1 , wherein the host cell is a yeast cell or a bacterial cell.

7. The host cell of claim 6 , wherein the host cell is a Saccharomyces cell or a Yarrowia cell.

8. The host cell of claim 6 , wherein the host cell is an E. coli cell.

9. The host cell of claim 1 , wherein the host cell produces at least 10%, 20%, or 30% more 11-hydroxycucurbitadienol, 24-25 epoxy-cucurbitadienol or cucurbitadienol relative to a control, wherein the control is a host cell that expresses S. grosvenorii CDS, encoded by a polynucleotide corresponding to SEQ ID NO: 33.

10. The host cell of claim 1 , wherein the CDS comprises the motif DQGWL (SEQ ID NO: 335).

11. The host cell of claim 10 , wherein the motif DQGWL (SEQ ID NO: 335) is located at residues in the CDS corresponding to residues 479-483 in SEQ ID NO: 73.

12. The host cell of claim 1 , wherein the CDS comprises the motif GHWANDLGGP (SEQ ID NO: 336).

13. The host cell of claim 12 , wherein the motif GHWANDLGGP (SEQ ID NO: 336) is located at residues in the CDS corresponding to residues 117-126 in SEQ ID NO: 73.

14. The host cell of claim 1 , wherein the CDS comprises the motif CWGVCYTYAGW (SEQ ID NO: 337).

15. The host cell of claim 14 , wherein the motif CWGVCYTYAGW (SEQ ID NO: 337) is located at residues in the CDS corresponding to residues 612-622 in SEQ ID NO: 73.

16. The host cell of claim 1 , wherein the amino acid sequence of the CDS comprises SEQ ID NO: 43.

17. The host cell of claim 7 , wherein the host cell is a Saccharomyces cerevisiae cell.

18. The host cell of claim 7 , wherein the host cell is a Yarrowia lipolytica cell.

19. The host cell of claim 1 , wherein the host cell is further modified to downregulate ERG7.

Assignees

• Ginkgo Bioworks Inc

Inventors

• Boucher Jeffrey Ian
• Flores Nicholas
• Jensen Jaide
• Saran Dayal
• Wang Jue

Classifications

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12P33/00

  Preparation of steroids · CPC title

• C12P19/18

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title

• C12P33/12

  Acting on D ring {(carbons 13 and 14 belong to the C ring; degradation of lateral chains C12P33/005)} · CPC title

• A23L27/36

  Terpene glycosides · CPC title