Biosynthetic production of gamma- and delta-lactones using cytochrome P450 enzymes having subterminal hydroxylase activity

Patent metadata

Field	Value
Publication number	US-12416028-B2
Application number	US-202117480177-A
Country	US
Kind code	B2
Filing date	Sep 21, 2021
Priority date	Mar 21, 2019
Publication date	Sep 16, 2025
Grant date	Sep 16, 2025

Abstract

Provided herein are methods for making lactones comprising incubating a cellular system expressing a heterologous cytochrome P450 (CYP450) protein with a medium comprising straight chain fatty acids to produce subterminal hydroxy fatty acids, and incubating a yeast cell culture with the subterminal hydroxy fatty acids to produce lactones.

First claim

What is claimed is:

1. A method of making lactones, the method comprising:
(a) incubating a cellular system expressing a heterologous cytochrome P450 (CYP450) protein with a medium comprising a straight chain fatty acid to produce a subterminal hydroxy fatty acid, wherein the straight chain fatty acid and the subterminal hydroxy fatty acid have 5 to 40 carbon atoms, and wherein the heterologous cytochrome P450 protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of a cytochrome P450 protein from Umbelopsis isabellina , and either:
(b1) incubating the subterminal hydroxy fatty acid with a yeast cell culture to produce lactones, wherein the lactones comprise at least one gamma-lactone, at least one delta-lactone, or combinations thereof, or
(b2) incubating the subterminal hydroxy fatty acids in an acidic environment to produce lactones, wherein the lactones comprise at least one gamma-lactone, at least one delta-lactone, or combinations thereof.

2. The method of claim 1 , wherein the heterologous cytochrome P450 protein comprises the amino acid sequence of SEQ ID NO: 6.

3. The method of claim 1 , wherein the cellular system expresses an exogenous isocitrate dehydrogenase (Icd) protein besides expressing an heterogenous cytochrome 450 protein.

4. The method of claim 3 , wherein the Icd protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of an Icd protein from E. coli.

5. The method of claim 3 , wherein the Icd protein comprises the amino acid sequence of SEQ ID NO: 4.

6. The method of claim 1 , wherein the straight chain fatty acid and the hydroxy fatty acid have 7 carbons to 20 carbons.

7. The method of claim 1 , wherein the straight chain fatty acid is lauric acid or tridecylic acid.

8. The method of claim 1 , wherein the subterminal hydroxy fatty acid is a hydroxylated lauric acid, or a hydroxylated tridecylic acid.

9. The method of claim 1 , wherein the cellular system comprises bacterial cells, yeast cells, or a combination thereof.

10. The method of claim 1 , wherein the lactones comprise five carbons to eight carbons.

11. The method of claim 1 , wherein the lactones comprise one or more of δ-hexalactone, γ-hexalactone, and δ-octalactone.

12. The method of claim 1 , wherein the lactones comprise one or more of γ-valerolactone, δ-heptalactone, and γ-heptalactone.

13. The method of claim 1 , wherein the straight chain fatty acid is lauric acid, the subterminal hydroxyl fatty acid comprises one or more of ω-1 hydroxy lauric acid, ω-2 hydroxy lauric acid, and ω-3 hydroxy lauric acid, and the lactones comprise one or more of δ-hexalactone, γ-hexalactone, and δ-octalactone.

14. The method of claim 1 , wherein the straight chain fatty acid is tridecylic acid, the subterminal hydroxy fatty acid comprises one or more of ω-1 hydroxy tridecylic acid, ω-2 hydroxy tridecylic acid, and ω-3 hydroxy tridecylic acid, and the lactones comprise one or more of γ-valerolactone, δ-heptalactone, and γ-heptalactone.

15. The method of claim 1 , wherein the subterminal hydroxy fatty acid is isolated from the cellular system prior to step (b1) or (b2).

16. The method of claim 1 , further comprising isolating the lactones from the yeast cell culture.

17. A method of making lactones, the method comprising:
(a) incubating a cellular system expressing a heterologous cytochrome P450 protein with a medium comprising a straight chain fatty acid to produce a subterminal hydroxy fatty acid, wherein the heterologous cytochrome P450 protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of a cytochrome P450 protein from Umbelopsis isabellina ; and
wherein the straight chain fatty acid and the subterminal hydroxy fatty acid have 5 to 40 carbon atoms;
(b) incubating the subterminal hydroxy fatty acid from step (a) with a cell culture to produce subterminal hydroxy fatty acid with the length shorter than the subterminal hydroxy fatty acid from step (a); and
(c) incubating the subterminal hydroxy fatty acids obtained at the end of step (b) in an acidic environment to produce lactones, wherein the lactones comprise at least one gamma-lactone, at least one delta-lactone, or combinations thereof.

Assignees

• Conagen Inc

Inventors

• Chen Hui
• Yu Xiaodan Oliver

Classifications

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12P7/62

  Carboxylic acid esters · CPC title

• C07D309/30

  Oxygen atoms, e.g. delta-lactones · CPC title

• C12P17/04

  containing a five-membered hetero ring, e.g. griseofulvin {, vitamin C} · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title