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Ketosteroid C27-monooxygenase derived from mycobacterium neoaurum and application of ketosteroid C27-monooxygenase

A technology of mycobacteria and monooxygenase, applied in the field of sterone C27-monooxygenase, to achieve the effect of increasing ADD production and increasing yield

Active Publication Date: 2017-01-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Enzymes in other pathways have not been identified and used

Method used

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  • Ketosteroid C27-monooxygenase derived from mycobacterium neoaurum and application of ketosteroid C27-monooxygenase
  • Ketosteroid C27-monooxygenase derived from mycobacterium neoaurum and application of ketosteroid C27-monooxygenase
  • Ketosteroid C27-monooxygenase derived from mycobacterium neoaurum and application of ketosteroid C27-monooxygenase

Examples

Experimental program
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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 successfully constructed by enzyme digestion, and the successfully constructed recombinant plasmids were electrotransformed into the new Mycobacterium aureus JC-12 (Mycobacterium...

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 invention discloses Ketosteroid C27-monooxygenase derived from mycobacterium neoaurum and application of the ketosteroid C27-monooxygenase, and belongs to the technical field of gene engineering and enzyme engineering. By means of gene knockout and expression strengthening methods, three isoenzymes of a key enzyme SMO in the sterol side chain degradation process are screened out from the mycobacterium neoaurum; the isoenzymes are intensively expressed in mycobacterium neoaurum of high-yielding androstenedione (ADD) separately, the ADD yield is obviously increased, and the effect of SMO2 is most obvious. By overexpressing SMO2, the final yield of ADD is increased to 7.3 g / L from 5.2 g / L. The Ketosteroid C27-monooxygenase derived from the mycobacterium neoaurum and the application of the ketosteroid C27-monooxygenase provide beneficial guidance for industrialization of increasing the ADD yield through a microbiological fermentation method.

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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IPC IPC(8): C12N1/21C12N9/02C12N15/53C12P33/16C12P33/02C12P33/00C12R1/32
CPCC12N9/0073C12P33/00C12P33/02C12P33/16C12Y114/13072C12N15/67
Inventor 饶志明邵明龙张显杨套伟徐美娟
Owner JIANGNAN UNIV
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