Mycobacterium neoaurum-derived steroid C27-monooxygenase and application thereof

Patent metadata

Field	Value
Publication number	US-10851352-B2
Application number	US-201616063668-A
Country	US
Kind code	B2
Filing date	Aug 29, 2016
Priority date	Aug 10, 2016
Publication date	Dec 1, 2020
Grant date	Dec 1, 2020

Abstract

The present invention discloses a Mycobacterium neoaurum -derived steroid C27-monooxygenase and an application thereof, which belong to the technical fields of genetic engineering and enzyme engineering. By the method of gene knockout and intensive expression, the present invention screens out three isoenzymes of a key enzyme SMO in the process of degrading sterol side chains from Mycobacterium neoaurum . The three isoenzymes are intensively expressed respectively in the Mycobacterium neoaurum for the high yield of androsta-1,4-diene-3,17-dione (ADD), the yield of ADD is increased remarkably, wherein the effect of SMO2 is most remarkable. By overexpressing SMO2, the final ADD yield is increased from 5.2 g·L −1 to 7.3 g·L −1 . The present invention provides a helpful guidance for the industrialization of the microbial fermentation method for increasing the ADD yield.

First claim

What is claimed is:

1. Recombinant Mycobacterium neoaurum
wherein the recombinant Mycobacterium neoaurum overexpresses a steroid C27-monooxygenase gene;
wherein the steroid C27-monooxygenase gene is a Mycobacterium neoaurum C27-monooxygenase gene selected from the group consisting of: Mycobacterium neoaurum Smo1 gene, Mycobacterium neoaurum Smo2 gene, and Mycobacterium neoaurum Smo3 gene
wherein the amino acid sequences of the steroid C27-monooxygenase genes Smo1, Smo2, and Smo3 are SEQ ID NOS: 4, 5, and 6, respectively, and
wherein the recombinant Mycobacterium neoaurum produces an increased yield of androsta-1,4-diene-3,17-dione (ADD) when fermented in the presence of cholesterol at 30° C. in fermentation medium that supports growth of Mycobacterium neoaurum compared to the yield of ADD produced by the Mycobacterium neoaurum lacking overexpression of the steroid C27-monooxygenase gene.

2. The recombinant Mycobacterium neoaurum according to claim 1 , wherein the recombinant Mycobacterium neoaurum further comprises a recombinant plasmid comprising the steroid C27-monooxygenase gene.

3. A method for producing ADD which comprises;
providing the recombinant Mycobacterium neoaurum of claim 1 as a production strain; and
incubating the recombinant Mycobacterium neoaurum with phytosterol or cholesterol or both under conditions that support overexpression of the steroid C27-monooxygenase gene in the recombinant Mycobacterium neoaurum.

Assignees

• Univ Jiangnan

Inventors

• Rao Zhiming
• Shao Minglong
• Zhang Xian
• Yang Taowei
• Xu Meijuan

Classifications

• C12Y114/13072

  Methylsterol monooxygenase (1.14.13.72) · CPC title

• C12N9/0073 Primary

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

• C12P33/16

  Acting at 17 position · CPC title

• C12P33/00

  Preparation of steroids · CPC title

• C12P33/02

  Dehydrogenating; Dehydroxylating · CPC title