Optimization of C-5 sterol desaturation

Patent metadata

Field	Value
Publication number	US-12018309-B2
Application number	US-201917051610-A
Country	US
Kind code	B2
Filing date	May 21, 2019
Priority date	May 22, 2018
Publication date	Jun 25, 2024
Grant date	Jun 25, 2024

Abstract

The present invention is related to an improved method for production of 7-dehydrocholesterol (7-DHC), an important intermediate towards biotechnological production of vitamin D3 or derivatives/metabolites thereof. The invention features modified host strains expressing enzymes having improved C-5 sterol 5 desaturase activity and their use in a process for production of vitamin D3 or derivatives and/or metabolites thereof.

First claim

The invention claimed is:

1. A cholesterol-producing yeast cell expressing (i) a heterologous C5-sterol desaturase with at least 90% sequence identity to SEQ ID NO: 2, 4, 6 or 8 and (ii) a heterologous C8-isomerase with at least 90% sequence identity to SEQ ID NO:_14, wherein the yeast cell is capable of producing a sterol mix comprising 7-dehydrocholesterol (7-DHC), lanosterol, and lathosterol, wherein the ratio of 7-DHC to lanosterol and lathosterol combined is at least 5% higher than the ratio of 7-DHC to lanosterol and lathosterol combined in a sterol mix produced by a reference yeast cell that does not express the heterologous C5-sterol desaturase and C8-isomerase.

2. The cholesterol-producing yeast cell according to claim 1 , wherein at least 84% of the sterol mix produced is 7-DHC.

3. The cholesterol-producing yeast cell according to claim 1 , wherein 7-DHC is produced at a ratio to lanosterol of 18 or more.

4. The cholesterol-producing yeast cell according to claim 1 , wherein the heterologous C5-sterol desaturase is from S. cerevisiae, Y. lipolytica, K. lactis, S. pombe, P. pastoris, C. albicans, P. roqueforti, A. nidulans, C. neoformans, M. oryzae, M. acridum , or U. maydis.

5. The cholesterol-producing yeast cell according to claim 1 in which ERG5 and ERG6 are inactivated.

6. The cholesterol-producing yeast cell according to claim 1 , wherein the yeast cell further expresses a heterologous plant or vertebrate sterol 424-reductase.

7. A process for production of 7-DHC, comprising cultivating the yeast cell according to claim 1 under conditions suitable for sterol production.

8. The process according to claim 7 , wherein the 7-DHC is further converted into vitamin D3.

9. The process according to claim 7 , wherein the 7-DHC is further converted into 25-hydroxyvitamin D3.

10. A process for production of a sterol mix, said process comprising cultivating the yeast cell according to claim 4 under conditions suitable for sterol production.

11. A process for production of a sterol mix, comprising cultivating the yeast cell according to claim 6 under conditions suitable for sterol production, wherein
(a) ERG5 and ERG6 are inactivated in the yeast cell,
(b) the heterologous sterol Δ24-reductase is a vertebrate sterol Δ24-reductase, and
(c) the heterologous C5-sterol desaturase is from Pichia pastoris, Penicillium roqueforti, Schizosaccharomyces pombe , or Saccharomyces cerevisiae.

12. The cholesterol-producing yeast cell according to claim 1 ,
wherein
(a) the heterologous C5-sterol desaturase is from Pichia pastoris, Penicillium roqueforti, Schizosaccharomyces pombe , or Saccharomyces cerevisiae,
(b) ERG5 and ERG6 are inactivated in the yeast cell, and
(c) the yeast cell expresses a heterologous vertebrate sterol Δ24-reductase.

13. A process for production of a sterol mix comprising cultivating the yeast cell according to claim 12 under conditions suitable for sterol production.

Assignees

• Dsm Ip Assets Bv

Inventors

• Farrell Christopher Mark
• Laprade Lisa Ann
• Lehmann Otto Martin
• Trueheart Joshua
• Chevreux Bastien Jean Wolfgang

Classifications

• C12N9/0071

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

• C12R2001/645

  Fungi {; Processes using fungi} · CPC title

• C12N1/165

  Yeast isolates · CPC title

• C12P33/02 Primary

  Dehydrogenating; Dehydroxylating · CPC title