A new sterone c27-monooxygenase derived from mycobacterium aureus and its application

A technology of mycobacteria and monooxygenase is applied in the field of steroid C27-monooxygenase to achieve the effects of increasing the yield of ADD and increasing the yield

Active Publication Date: 2019-09-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzymes in other pathways have not been identified and used

Method used

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  • A new sterone c27-monooxygenase derived from mycobacterium aureus and its application
  • A new sterone c27-monooxygenase derived from mycobacterium aureus and its application
  • A new sterone c27-monooxygenase derived from mycobacterium aureus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of SMO knockout strains and corresponding complementation strains

[0037] By querying the whole genome information of Mycobacterium aureus, three SMO isozymes were screened. The new Mycobacterium aureus with SMO enzyme activity in the laboratory was used as the starting strain, and its chromosome was used as a template to obtain the genes of these three enzymes by means of PCR. Through the design of gene knockout primers, the knockout gene was obtained by means of PCR, and the mycobacterial knockout plasmid p2NIL was connected to construct the knockout plasmid. After the successful construction was verified by PCR, it was transformed into Mycobacterium aureus. Using cholest-4-en-3-one as the substrate, the degradation of the substrate by the knockout strain was detected. On the basis of the knockout strain, the knockout gene complementation strain was constructed, the complete SMO gene was amplified by PCR means by designing primers, and the in...

Embodiment 2

[0051] Embodiment 2: Construction of SMO enhanced expression recombinant strain

[0052] Plasmid pMV261 was used for gene overexpression in M. neoaureus. Double-digest pMD18-T-Smo1 with Sac I and Hind III, double-digest pMD18-T-Smo2 and pMD18-T-Smo3 with BamH I and EcoR I respectively, and simultaneously digest plasmid pMV261 with corresponding restriction sites, Gel recovery and purification of the corresponding gene fragments and plasmid pMV261 fragments, T 4 DNA ligase was used to ligate the two fragments overnight. After overnight, the ligated material was heat-shocked to transform E.coli JM109 competent cells, and positive transformants were screened using a kanamycin resistance plate. The transformant plasmids were extracted, and the recombinant plasmids p261-Smo1, p261-Smo2 and p261-Smo3 were verified to be successfully constructed by enzyme digestion, and the successfully constructed recombinant plasmids were electrotransformed into Mycobacterium neoaurum JC-12 (Mycob...

Embodiment 3

[0053] Example 3: SMO enhances the expression of recombinant strains to improve the production of ADD

[0054] recombinant strain JC-12 S1, JC-12 S2 and JC-12 S3 After being activated on the seed medium, it was inserted into the fermentation medium according to the inoculum size of 5%, with 20g / L cholesterol as the substrate, and fermented at 30°C and 160rpm for 168h to carry out the fermentation conversion experiment. Among them, seed medium: glucose 10g / L, peptone 10g / l, beef extract 6g / L, NaCl 10g / L, pH 7.5; fermentation medium: cholesterol 20g / L, glucose 20g / L, peptone 10g / L, beef Paste 6g / L, K 2 HPO 4 3g / L, MgSO 4 ·7H 2 O0.5g / L, MnCl 2 4H 2 O 5×10 -4 g / L, hydroxypropyl-β-cyclodextrin 60g / L, pH 7.5.

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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.

Description

technical field [0001] The invention relates to a sterone C27-monooxygenase derived from Mycobacterium aureus and its application, and belongs to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Steroid hormones are widely used clinically because they play a very important role in regulating the body due to their various physiological functions. Steroid hormone drugs are widely used for anti-tumor, anti-inflammation, anti-bacterial, anti-viral, anti-hormone and anti-allergic etc. In addition, various sex hormone drugs are the main drugs for the treatment of sexual organ degeneration and gynecological diseases, and are the main components of oral contraceptives. At the same time, steroid hormone drugs can also be used as active ingredients of anti-obesity drugs, used to prevent coronary heart disease, and as inhibitors of HIV integrase, to prevent HIV infection and to treat AIDS. Androst-4-ene-3,17-dione (AD) and androst-1,4-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N9/02C12N15/53C12P33/16C12P33/02C12P33/00C12R1/32
CPCC12N9/0073C12P33/00C12P33/02C12P33/16C12Y114/13072
Inventor 饶志明邵明龙张显杨套伟徐美娟
Owner JIANGNAN UNIV
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