21-hydroxylation of steroids

Patent metadata

Field	Value
Publication number	US-10385376-B2
Application number	US-201515523107-A
Country	US
Kind code	B2
Filing date	Oct 29, 2015
Priority date	Oct 30, 2014
Publication date	Aug 20, 2019
Grant date	Aug 20, 2019

Abstract

Generally, the present invention relates to the field of steroid hydroxylation. More specifically, the present invention relates to a method for the 21-hydroxylation of steroids in cells. It also relates to cells expressing a steroid 21-hydroxylating enzyme or steroid 21-hydroxylase, expression vectors comprising a nucleic acid encoding for a steroid 21-hydroxylase and a kit for carrying out the method for the 21-hydroxylation of steroids in cells.

First claim

The invention claimed is:

1. A process for the hydroxylation of the carbon atom 21 of a steroid, comprising the steps of:
(a) providing a cell expressing:
(i) a heterologous CYP21A2 protein or a functional variant thereof;
(ii) at least one heterologous electron transfer system capable of transferring electrons to CYP21A2; and
(iii) one or more chaperones facilitating folding of CYP21A2; and

(b) adding the steroid to the cell,
wherein the steroid is medrane or deltamedrane.

2. The process of claim 1 , further comprising a step (c) of extracting the 21-hydroxylated steroid from the supernatant of the cell.

3. The process of claim 1 , further comprising adding one or more cell permeabilizing agents to the cell after step (b).

4. The process of claim 1 , wherein the cell is a resting cell.

5. The process of claim 1 , wherein the cell is a prokaryotic cell or a eukaryotic cell.

6. The process of claim 1 , wherein the at least one heterologous electron transfer system comprises:
(a) a CYP21A2 reductase, and/or
(b) a ferredoxin reductase.

7. The process of claim 1 , wherein the one or more chaperones are recombinantly expressed chaperones.

8. The process of claim 1 , wherein the expression of at least one tryptophanase gene is reduced or abolished in the cell.

9. The process of claim 1 , wherein the cell further expresses a heterologous gene encoding for an enzyme catalyzing a step in the heme biosynthesis pathway.

10. The process of claim 1 , wherein the genes encoding for (i), (ii), and optionally (iii) are comprised in one or more expression cassettes which are integrated into the cell genome.

11. The process of claim 5 , wherein the cell is an E. coli cell.

12. The process of claim 5 , wherein the cell is a yeast cell.

13. The process of claim 9 , wherein the heterologous gene encoding for an enzyme catalyzing a step in the heme biosynthesis pathway is a hemA gene.

14. The process of claim 6 , wherein the at least one heterologous electron transfer system comprises an NADPH-dependent ferredoxin reductase and a ferredoxin.

Assignees

• Sanofi Sa

Inventors

• Lattemann Claus
• Stillger Thomas
• Janocha Bernd
• Rasser Hans-Falk
• Rissom Sebastian
• Anderko Simone
• Bernhardt Rita
• Hannemann Frank

Classifications

• C12P33/06 Primary

  Hydroxylating · CPC title

• C12Y118/01006

  Adrenodoxin-NADP+ reductase (1.18.1.6) · CPC title

• C12Y118/01002

  Ferredoxin-NADP+ reductase (1.18.1.2) · CPC title

• C12Y114/9901

  Steroid 21-monooxygenase (1.14.99.10), i.e. steroid cytochrome P450 21-hydroxylase · CPC title

• C12Y114/14

  with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14) · CPC title