Method for producing 9alpha-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion

Patent metadata

Field	Value
Publication number	US-11001871-B2
Application number	US-201916256001-A
Country	US
Kind code	B2
Filing date	Jan 24, 2019
Priority date	Dec 15, 2017
Publication date	May 11, 2021
Grant date	May 11, 2021

Abstract

The present disclosure discloses a method for producing 9α-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion, and belongs to the fields of gene engineering and enzyme engineering. According to the present disclosure, oxidation subunit KshA, reduction subunit KshB and unknown active subunit KshC of 3-ketosteroid-9α-hydroxylase sourcing from Mycobacterium sp. Strain VKM Ac-1817D are successfully expressed in E. coli BL21, and KshC is identified as an oxidation subunit, the enzyme activity of which is far higher than that of KshA. BL21/pET-28a(+)-fdh constructed in the laboratory is used for expressing formate dehydrogenase (FDH), and by using crude enzyme liquid of KSH (KshB+KshC) and FDH engineering bacteria as a biocatalyst and a steroidal compound (AD) as a substrate, optimum reaction temperature is determined as 30° C. and optimum pH is determined as 7.0. In optimum conditions, AD is converted to produce a product 9-OH-AD, and within 20 hours, the output of 9-OH-AD is 4.7 g/L, and the molar conversion rate reaches 96.7%. According to the present disclosure, in production of 9-OH-AD, coupling of a 3-ketosteroid-9α-hydroxylase hydroxylation system and a coenzyme recycling system is realized, and the method has the advantages of being high in efficiency, low in cost, green, environmentally friendly and the like.

First claim

What is claimed is:

1. A composition comprising an Escherichia coli bacterium comprising genes encoding 3-ketosteroid-9α-hydroxylase reduction subunit KshB and 3-ketosteroid-9α-hydroxylase oxidation subunit KshC.

2. The composition of claim 1 , further comprising coenzyme NAD + , NADH, formate dehydrogenase, or a combination thereof.

3. The composition of claim 1 , wherein an amino acid sequence of the 3-ketosteroid-9α-hydroxylase reduction subunit KshB is SEQ ID NO:4.

4. The composition of claim 1 , wherein an amino acid sequence of the 3-ketosteroid-9α-hydroxylase oxidation subunit KshC is SEQ ID NO:5.

5. The composition of claim 1 , wherein the composition further comprises NADH and formate dehydrogenase.

6. The composition of claim 5 , wherein the additive proportion of the 3-ketosteroid-9α-hydroxylase oxidation subunit KshC to the 3-ketosteroid-9α-hydroxylase reduction subunit KshB to the formate dehydrogenase is 1-10:1-10:1-10 according to enzyme activity.

7. The composition of claim 5 , wherein NADH is present in an amount of 0.01 to −1 mol/L.

8. A method of preparing a composition, wherein the composition comprises 3-ketosteroid-9α-hydroxylase reduction subunit KshB and 3-ketosteroid-9α-hydroxylase oxidation subunit KshC, and wherein the method comprises:
(a) amplifying a nucleotide sequence set forth in SEQ ID NO:2 to obtain a gene encoding 3-ketosteroid-9α-hydroxylase reduction subunit KshB, and cloning the gene encoding 3-ketosteroid-9α-hydroxylase reduction subunit KshB into an Escherichia Coli expression vector pET-28a to obtain a recombinant plasmid pET-28a- kshB;
(b) amplifying a nucleotide sequence set forth in SEQ ID NO:3 to obtain a gene encoding 3-ketosteroid-9α-hydroxylase oxidation subunit KshC, and cloning the gene encoding 3-ketosteroid-9α-hydroxylase oxidation subunit KshC to an Escherichia Coli expression vector pET- Duet1(+) to obtain a recombinant plasmid pET-Duet1(+)-kshC;
(c) respectively transforming the recombinant plasmids obtained in step (a) and step (b) into Escherichia Coli to obtain a recombinant Escherichia Coli comprising BL21/pET-28a(+)-kshB and BL21/pET-Duet 1 (+)-kshC; and
(d) culturing the recombinant Escherichia Coli obtained in step (3) to obtain the composition.

9. A method for increasing the efficiency of converting androstane-4-alkene-3,17-diketone to produce 9α-hydroxy androstane-4-alkene-3,17-diketone, which comprises adding 3 ketosteroid -9α-hydroxylase reduction subunit KshB and 3-ketosteroid-9α-hydroxylase oxidation subunit KshC as a catalyst to a biotransformation reaction.

10. The method of claim 8 , which further comprises:
(e) rupturing the recombinant Escherichia Coli cells to obtain a crude enzyme mixture;
(f) adding an amount of NADH and androstane-4-alkene-3,17-diketone (AD) to the crude enzyme mixture;
(g) incubating the crude enzyme mixture under conditions that support formation of 9α-hydroxy androstane-4-alkene-3,17-diketone from AD; and
(h) adding the 9α-hydroxy androstane-4-alkene-3,17-diketone to a medicine.

11. The composition of claim 1 , wherein the genes encoding 3-ketosteroid-9α-hydroxylase reduction subunit KshB and 3-ketosteroid-9α-hydroxylase oxidation subunit KshC are Mycobacterium genes.

Assignees

• Univ Jiangnan

Inventors

• Rao Zhiming
• Sha Zongyan
• Zhang Xian
• Yang Taowei
• Xu Meijuan

Classifications

• C12N9/0008 Primary

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12P33/06 Primary

  Hydroxylating · CPC title

• C12Y114/13142

  3-Ketosteroid 9alpha-monooxygenase (1.14.13.142) · CPC title

• C12N9/0077

  with a reduced iron-sulfur protein as one donor (1.14.15) · CPC title

• C12N9/0073

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13 · CPC title