21-hydroxylation of steroids

Patent metadata

Field	Value
Publication number	US-11965199-B2
Application number	US-202217589667-A
Country	US
Kind code	B2
Filing date	Jan 31, 2022
Priority date	Oct 30, 2014
Publication date	Apr 23, 2024
Grant date	Apr 23, 2024

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, wherein the at least one electron transfer system is selected from the group consisting of:
the combination of a flavodoxin reductase (Fpr) and an adrenodoxin;
the combination of a Fpr and the ferredoxin domain of an electron transfer domain (etp fd );
the combination of an adrenodoxin reductase homolog (Arh) and an adrenodoxin; and
the combination of an Arh and etp fd; and
(iii) one or more chaperones facilitating folding of CYP21A2; and

(b) adding the steroid to the cell.

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 steroid is a 3-keto steroid.

5. The process of claim 4 , wherein the 3-keto steroid is selected from the group consisting of medrane, deltamedrane, progesterone, 170 H-progesterone, medroxyprogesterone, and 5-α-dihydro-progesterone.

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

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

8. The process of claim 7 , wherein the cell is an E. coli cell.

9. The process of claim 7 , wherein the cell is a yeast cell.

10. The process of claim 1 , wherein:
(a) the Fpr is an E. coli Fpr;
(b) the adrenotoxin is human or bovine Adx4-108;
(c) the Arh is S. pombe adrenodoxin reductase homolog (Arh1); and/or (d) etp fd is the ferredoxin domain of the S. pombe electron transfer domain (etp1 fd ).

11. The process of claim 10 , wherein the ferredoxin reductase is an NADPH-dependent ferredoxin reductase.

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

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

14. 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.

15. The process of claim 14 , wherein the heterologous gene encoding for an enzyme catalyzing a step in the heme biosynthesis pathway is a hemA gene.

16. The process of claim 1 , wherein the functional variant of a CYP21A2 protein has at least 20% of the ability of the unaltered CYP21A2 protein to 21-hydroxylate a steroid.

17. 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.

18. 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, wherein the at least one electron transfer system is selected from the group consisting of:
the combination of a flavodoxin reductase (Fpr) and an adrenotoxin;
the combination of a Fpr and the ferredoxin domain of an electron transfer domain (etp fd );
the combination of an adrenotoxin reductase homolog (Arh) and an adrenotoxin; and
the combination of an Arh and etp fd ; and

(iii) one or more chaperones facilitating folding of CYP21A2.

19. A multicistronic expression vector comprising:
(i) a nucleic acid encoding for a CYP21A2 protein or a functional variant thereof;
(ii) one or more nucleic acids encoding for at least one heterologous electron transfer system capable of transferring electrons to CYP21A2, wherein the at least one electron transfer system is selected from the group consisting of:
the combination of a flavodoxin reductase (Fpr) and an adrenotoxin;
the combination of a Fpr and the ferredoxin domain of an electron transfer domain (etp fd );
the combination of an adrenotoxin reductase homolog (Arh) and an adrenotoxin; and
the combination of an Arh and etp fd ; and optionally

(iii) one or more nucleic acids encoding for chaperones facilitating folding of CYP21A2.

20. A kit comprising:
(a) the cell of claim 18 ;
(b) a multicistronic expression vector of claim 19 ; or
(c) (i) an expression vector comprising a nucleic acid encoding for a CYP21A2 protein or a functional variant thereof;
(ii) one or more expression vectors comprising one or more nucleic acids encoding for at least one heterologous electron transfer system capable of transferring electrons to CYP21A2, wherein the at least one electron transfer system is selected from the group consisting of:
the combination of a flavodoxin reductase (Fpr) and an adrenotoxin;
the combination of a Fpr and the ferredoxin domain of an electron transfer domain (etp fd );
the combination of an adrenotoxin reductase homolog (Arh) and an adrenotoxin; and

the combination of an Arh and etp fd ; and optionally
(iii) one or more expression vectors comprising one or more nucleic acids encoding for chaperones facilitating folding of CYP21A2.

Assignees

• Euroapi
• 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

• C12N9/0087

  Steroid 21-monooxygenase (1.14.99.10) · CPC title

• C12N9/0095

  acting on iron-sulfur proteins as donor (1.18) · CPC title

• C12Y118/01006

  Adrenodoxin-NADP+ reductase (1.18.1.6) · CPC title

• C12Y114/9901

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