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Genetically engineered bacterium for producing 5alpha-androstanedione and application thereof

A technology of genetically engineered bacteria and genes, applied in genetic engineering, application, plant genetic improvement and other directions, to achieve the effects of low raw material prices, high production efficiency, and clean and environmentally friendly preparation methods

Active Publication Date: 2020-08-04
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at many problems in the production of 5α-AD by the existing chemical method, the present invention explores a new way of producing 5α-AD by the green biological method

Method used

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  • Genetically engineered bacterium for producing 5alpha-androstanedione and application thereof
  • Genetically engineered bacterium for producing 5alpha-androstanedione and application thereof
  • Genetically engineered bacterium for producing 5alpha-androstanedione and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The acquisition of the target gene 5α-reductase gene of embodiment 1

[0035] According to literature research, 5α-reductase from mice has been successfully expressed in Saccharomyces cerevisiae cells and exhibited corresponding activity; combined with NCBI sequence search and comparison analysis, it was found that 5α-reductase from Treponema denticola was closely related to small The 5α-reductase in the mouse has a higher homology and is of bacterial origin. Therefore, the original 5α-reductase gene (ie, SEQ ID NO.1) in Treponema denticola was selected as the original gene of this invention.

[0036] The original 5α-reductase gene sequence from Treponema denticola was sent to Jinweizhi Company to synthesize a 5α-reductase gene that conforms to the codon preference of mycobacteria.

[0037] Using the synthesized plasmid PUC57-5α containing the 5α-reductase gene sequence (this plasmid is a common conventional plasmid used in gene synthesis) as a template, and the restrict...

Embodiment 2

[0040] The construction of embodiment 2 genetically engineered bacteria

[0041] 1. Construct the pMV261-5α plasmid, the process including:

[0042] The 5α-reductase gene of the target fragment obtained in Example 1 was double-digested with BamH I and HindIII respectively with the shuttle plasmid pMV261 in a certain ratio and purified, then ligated overnight at 16°C, transformed into Escherichia coli DH5α competent cells, and used Kana Screen the genetically engineered bacteria on a mycin plate, pick the transformants for PCR and double enzyme digestion verification, that is, after the recombinant plasmid is digested with BamHI and Hind III, gene fragments of about 4.5 kb and 0.8 kb in size are released, and the double enzymes The correct plasmid was verified and sent to Jinweizhi Company for sequencing, and the correctly sequenced plasmid was the recombinant plasmid pMV261-5α.

[0043] 2. Construction of genetic engineering strain MNR M3△ksdD / 261-5α:

[0044] (1) Preparatio...

Embodiment 3

[0048] Example 3 The method and product identification of genetically engineered bacteria MNR M3△ksdD / 261-5α transforming PS to produce 5α-AD

[0049] The strains were divided into two groups, and the performances of the following strains were measured respectively. Grouped as follows:

[0050] Experimental group: genetically engineered bacteria prepared in Example 2 of the present invention: MNR M3△ksdD / 261-5α

[0051] Control group: Mycobacterium aureus control strain MNR M3△ksdD / 261

[0052] The construction method of the control bacterium MNR M3△ksdD / 261: the plasmid pMV261 plasmid is directly introduced into the host bacterium MNRM3△ksdD, the specific import method is the same as step 2 in Example 2, the only difference is that the plasmid is different (i.e. (1) Competent Cell preparation (2) Electrotransformation: (3) Recombinant screening and verification), the following Mycobacterium aureus control strain MNR M3△ksdD / 261 is the same strain.

[0053] 1. Bacteria acti...

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Abstract

The invention belongs to the technical field of biocatalysis, and particularly relates to construction and application of genetically engineered bacteria for one-step conversion from cheap substrate phytosterol (PS) as a raw material to 5alpha-androstanedione (5alpha-AD). The 5alpha-reductase gene from treponema pallidum is heterologously expressed into mycobacteria mainly producing androstenedione (AD), and one-step biotransformation from phytosterol to 5alpha-AD is realized, so that green and efficient production of 5 alpha-AD is realized.

Description

Technical field: [0001] The invention belongs to the technical field of biocatalysis, and specifically relates to a genetically engineered bacterium capable of directly transforming cheap substrate phytosterol (PS) into 5α-androstanedione (5α-AD) in one step and an application thereof. Background technique: [0002] As a class of hormonal drugs that play an important role in medicine, more than 250 kinds of steroid drugs have been identified so far. Their market demand is second only to antibiotics. The global annual output exceeds 1 million tons, which has a very broad market prospect. . Steroid hormone drugs are widely used in anti-tumor, anti-inflammation, anti-allergy, etc., and they are also essential drugs in the treatment of endocrine diseases such as rheumatoid arthritis, bronchitis and Addison. [0003] As an important intermediate of steroid drugs, 5α-AD is widely used in the synthesis of steroid hormone drugs such as mesterolone, androsandrolone, and primobolone,...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/74C12N15/66C12P33/02C12R1/32
CPCC12N9/001C12N15/74C12N15/66C12P33/02C12Y103/99005
Inventor 王敏申雁冰赵云秋骆健美夏梦雷马赛
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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