Production of non-yeast sterols by yeast

Patent metadata

Field	Value
Publication number	US-9624521-B2
Application number	US-201013510636-A
Country	US
Kind code	B2
Filing date	Nov 24, 2010
Priority date	Dec 3, 2009
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

This invention relates to the production of 7-dehydrocholesterol, 25-hydroxy-7-dehydrocholesterol, and 25-hydroxy ergosterol in yeast such as Saccharomyces cerevisiae . It also relates to various enzymes catalyzing the reduction of the double bond at position 24 of lanosterol, dimethyl zymosterol, methyl zymosterol, zymosterol, cholesta-7, 24-dienol, or cholesta-5,7,24-trienol; or the hydroxylation at position 25 of ergosterol, 7-dehydrocholesterol, cholesta-8-enol, and cholesta-7-enol. It also relates to various nucleic acids encoding cholesterol C25-hydroxylases and sterol Δ24-reductases and their use to produce and hydroxylate 7-dehydrocholesterol or ergosterol. It also relates to the yeast strains so produced, and methods of making these sterols that include the steps of cultivaton a transformed yeast cell, and harvesting the resulting sterol(s).

First claim

What is claimed is:

1. Yeast comprising a nucleic acid encoding cholesterol C25-hydroxylase which has been codon-optimized for expression in yeast and encodes a cholesterol C25 hydroxylase with an amino acid sequence that is at least 90% identical with an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 29;
wherein the yeast possesses no active Erg5p and Erg6p enzymes, and the yeast also expresses a sterol Δ24-reductase.

2. The yeast of claim 1 , wherein the yeast expresses a truncated version of HMG1.

3. A method of producing 25-hydroxy-7-dehydrocholesterol in a genetically modified yeast comprising:
a) transforming the yeast with a nucleic acid encoding cholesterol C25-hydroxylase; wherein said nucleic acid encoding cholesterol C25 hydroxylase has been codon-optimized for expression in yeast and encodes a cholesterol C25 hydroxylase with an amino acid sequence that is at least 90% identical with an amino acid sequence selected from the croup consisting of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 29; and
b) contacting 7-dehydrocholesterol with said cholesterol C25-hydroxylase expressed in yeast;
wherein the yeast possesses no active Erg5p and Erg6p enzymes, and the yeast also expresses a sterol Δ24-reductase.

4. The method according to claim 3 , wherein the yeast expresses a truncated version of HMG1.

5. The method according to claim 3 further comprising prior to step a):
contacting cholesta-5,7,24-trienol with a yeast comprising a nucleic acid sequence that encodes a functional sterol Δ24-reductase enzyme, whereby the yeast converts lanosterol, dimethyl zymosterol, methyl zymosterol, zymosterol, cholesta-7, 24-dienol, or cholesta-5,7,24-trienol to 3β-hydroxy-8-lanosta-8-ene, 4,4-dimethyl-cholesta-8-enol, 4α-methyl-cholesta-8-enol, cholesta-8-enol, lathosterol or 7-dehydrocholesterol, respectively.

6. The method according to claim 3 , wherein the expression in the codon-optimized C25-hydroxylase in the genetically modified yeast is at least as in the range of the expression of the C25-hydroxylase according to SEQ ID NO:5.

7. The method according to claim 3 , wherein the codon-optimized C25-hydroxylase is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 8, 19, 20, 22, 23, 25, 26, 27, 28, 30, and 31.

Assignees

• Hohmann Hans-Peter
• Lehmann Martin
• Merkamm Muriel
• Pompon Denis
• Dsm Ip Assets Bv

Inventors

• Hohmann Hans-Peter
• Lehmann Martin
• Merkamm Muriel
• Pompon Denis

Classifications

• C12N9/1007

  Methyltransferases (general) (2.1.1.) · CPC title

• C12N9/0067

  acting on hydrogen as donor (1.12) · CPC title

• C12N9/001

  acting on the CH-CH group of donors (1.3) · CPC title

• C12N9/0083

  Miscellaneous (1.14.99) · CPC title

• C12P33/06

  Hydroxylating · CPC title