21-hydroxylation of steroids
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.
1. A process for the hydroxylation of the carbon atom 21 of a steroid, comprising the steps of:
(a) providing a prokaryotic 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 prokaryotic cell.
2. The process of claim 1 , further comprising a step (c) of extracting the 21-hydroxylated steroid from the supernatant of the prokaryotic cell.
3. The process of claim 1 , further comprising adding one or more cell permeabilizing agents to the prokaryotic 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, 17OH-progesterone, medroxyprogesterone, and 5-α-dihydro-progesterone.
6. The process of claim 1 , wherein the prokaryotic cell is a resting prokaryotic cell.
7. 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.
8. The process of claim 1 , wherein the one or more chaperones are recombinantly expressed chaperones.
9. The process of claim 1 , wherein the expression of at least one tryptophanase gene is reduced or abolished in the prokaryotic cell.
10. The process of claim 1 , wherein the prokaryotic cell further expresses a heterologous gene encoding for an enzyme catalyzing a step in the heme biosynthesis pathway.
11. 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 prokaryotic cell genome.
12. A prokaryotic 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.
13. A kit comprising the prokaryotic cell of claim 12 .
14. The process of claim 1 , wherein the prokaryotic cell is an E. coli cell.
15. The process of claim 10 , 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 7 , wherein the ferredoxin reductase is an NADPH-dependent ferredoxin reductase and wherein the heterologous electron transfer system further comprises a ferredoxin.
17. A kit for the hydroxylation of the carbon atom 21 of a steroid, comprising a prokaryotic cell expressing:
(a) a heterologous CYP21A2 protein or a functional variant thereof;
(b) at least one heterologous electron transfer system capable of transferring electrons to CYP21A2; and
(c) one or more chaperones facilitating folding of CYP21A2;
wherein the prokaryotic cell is an E. coli cell; and
wherein the prokaryotic cell further expresses a heterologous gene encoding for an enzyme catalyzing a step in the heme biosynthesis pathway.
18. The process of claim 7 , wherein the ferredoxin reductase is an NADPH-dependent ferredoxin reductase.