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High-androstenedione-producing recombinant mycobacterium and construction method and application of high-androstenedione-producing recombinant mycobacterium

A technology of androstenedione and mycobacteria, which is applied in the field of biotechnology and biochemical industry, can solve the problems of poor transformation ability of strains, influence on production efficiency, and easy degradation of steroids, so as to reduce the generation of by-products and improve production efficiency , The effect of simple process method

Active Publication Date: 2020-07-28
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the main problems in the production of androstenedione by microbial transformation of phytosterols are: the transformation ability of the strains is poor, the steroids are easily degraded in the transformation project, and there are By-products, etc., seriously affect production efficiency

Method used

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  • High-androstenedione-producing recombinant mycobacterium and construction method and application of high-androstenedione-producing recombinant mycobacterium
  • High-androstenedione-producing recombinant mycobacterium and construction method and application of high-androstenedione-producing recombinant mycobacterium
  • High-androstenedione-producing recombinant mycobacterium and construction method and application of high-androstenedione-producing recombinant mycobacterium

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Embodiment 1

[0031] A method for constructing a recombinant mycobacterium with high production of androstenedione, comprising the steps of:

[0032] (1) Construction of three gene overexpression recombinant plasmid pMV261-ChoM2-Smo2-Hsd4A:

[0033] ① Synthesize the target gene, according to the cholesterol oxidase gene ChoM2 (SEQ ID NO.1), the sterone C27 monooxygenase gene Smo2 carrying the RBS fragment (the RBS fragment is connected to the 5' end of the gene Smo2, and the sequence of the RBS fragment is as follows: Shown in SEQ ID NO.2, the sequence of the gene Smo2 is shown in SEQ ID NO.3) and 17β-hydroxysterol dehydrogenase Hsd4A carrying the RBS fragment (the RBS fragment is connected to the 5' end of the gene Hsd4A, the sequence of the gene Hsd4A The nucleotide sequence shown in SEQ ID NO.4), the restriction site BamHI / EcoRI is added to the two ends of the gene ChoM2 sequence, the restriction site EcoRI is added to the two ends of the gene Smo2 carrying the RBS fragment, and the The...

Embodiment 2

[0047] A recombinant mycobacterium that produces high androstenedione, named MN CSH (abbreviation for recombinant strain) fermentation method for preparing androstenedione

[0048] With 1% (v / v) inoculum, the recombinant mycobacterium MN CSH The strains were inoculated into 5ml of seed medium, shaken at 30°C (200rpm) for 48h, and then transferred to 200mL fermentation transformation medium with 10% inoculum size, and transformed at 37°C at 200rpm for 168h.

[0049] The composition of the fermentation transformation medium is 0.5g / L K 2 HPO 4 , 0.5g / L MgSO 4 ·7H 2 O, 0.05g / L ferric ammonium citrate, 2.0g / L citric acid, 3.5g / L diammonium hydrogen phosphate, 20g / L phytosterol, 60g / L methylated-β-cyclodextrin, the rest is water, pH 8.0.

[0050] Take 1 mL of fermentation broth every 24 hours, extract with 2 times the volume of ethyl acetate, and detect the production of androstenedione by high performance liquid chromatography. Such as Figure 4 Shown, different fermentati...

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Abstract

The present invention discloses a high-androstenedione-producing recombinant mycobacterium and a construction method and an application of the high-androstenedione-producing recombinant mycobacterium.The construction method is as follows: cholesterol oxidase gene ChoM2, sterone C27 monooxygenase gene Smo2 carrying an RBS fragment and 17[beta]-hydroxysterol dehydrogenase gene Hsd4A carrying an RBSfragment are introduced into mycobacterium sp. CICC 21097 to obtain the high-androstenedione-producing recombinant mycobacterium MNCSH. A fermentation main product of the recombinant strain obtainedby the method is 4-androstenedione, the yield is 90% or more, the selectivity is 90% or more, formation of by-products is reduced and the yield of the androstenedione is increased. Compared with an original strain, the recombinant strain has better catalytic conversion performance and can improve production efficiency, and the processing method is simple and efficient.

Description

technical field [0001] The invention belongs to the fields of biotechnology and biochemical industry, and specifically relates to a recombinant mycobacterium with high androstenedione production, a construction method and application thereof. Background technique [0002] Steroid hormone drugs refer to hormone drugs with a steroid structure in their molecular structure, which is an important class of clinically important drugs, mainly including adrenocortical hormones and sex hormones. Steroid hormone drugs are widely used in the treatment of various diseases such as rheumatism, cardiovascular disease, cancer, skin disease and endocrine disorders due to their numerous pharmacological effects, such as anti-infection, anti-allergy, anti-virus and anti-shock Wait. After decades of research and development, steroid hormone drugs have formed a large class of steroid drugs with various types, wide clinical applications and strong demand, and have become the second largest drug af...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P33/02C12R1/32
CPCC12N9/0006C12N9/0073C12Y101/03006C12Y101/01239C12N15/74C12P33/02
Inventor 齐崴尤生萍张伟常红星钱建武苏荣欣
Owner TIANJIN UNIV
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