Method for efficiently producing steroid medicine precursor by fermentation

A technology of precursors and steroids, applied in the field of biocatalysis, can solve problems such as low production efficiency, reduced production cost, and long fermentation cycle

Active Publication Date: 2019-05-03
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In view of the above problems, the present invention will efficiently ferment and produce steroid drug precursors by improving the vitality of steroid drug production strains, to solve the problems of long fermentation period and low production efficiency in the production of steroid drug precursors by microbial methods, and provide a new solution for the production of steroid drug precursors. Cost Reduction Provides New Approach

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  • Method for efficiently producing steroid medicine precursor by fermentation
  • Method for efficiently producing steroid medicine precursor by fermentation
  • Method for efficiently producing steroid medicine precursor by fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Acquisition of type II NADH dehydrogenase gene (ndh)

[0027] The bacterial strain (hereinafter referred to as MNR) with the preservation number CICC 21097 was inoculated in the basal medium containing phytosterols (glucose 10g / L, MgSO 4 0.5g / L, K 2 HPO 4 0.5g / L, (NH 4 ) 2 HPO 4 3.5g / L, citric acid 2g / L, ferric ammonium citrate 0.05g / L, phytosterol 5g / L) cultivated for 60 hours to obtain samples for transcriptome sequencing. The obtained gene transcript sequences were compared in NCBI's non-redundant protein sequence database and KEGG's protein database to obtain the protein with the highest sequence similarity to each gene transcript sequence. The protein information is the functional annotation information of the protein corresponding to each gene transcript sequence. The gene transcript sequence obtained by searching NADH dehydrogenase from the annotation information is the type Ⅱ NADH dehydrogenase gene. Primers ndh-F and ndh-R were designed usin...

Embodiment 2

[0031] Example 2 Constructing Genetic Engineering Expression Vectors for Type II NADH Dehydrogenase Gene Overexpression

[0032] Constructing a genetic engineering expression vector for the overexpression of the type II NADH dehydrogenase gene itself, the process includes: recovering the type II NADH dehydrogenase gene obtained in Example 1 through double digestion with BamHI and HindIII, and recovering it with the same The digested pMV261 plasmid was ligated overnight at 16°C under the action of T4 DNA ligase, and the ligated product was transformed into Escherichia coli DH5α by chemical transformation, and positive clones were obtained by kanamycin screening. The plasmids in the positive clones were extracted and verified by enzyme digestion ( figure 2 ) and sequencing, the successfully constructed genetic engineering expression vector pMV261-ndh for type II NADH dehydrogenase gene self-overexpression.

Embodiment 3

[0033] Example 3 Construction of type II NADH dehydrogenase gene self-overexpression strain NdhN

[0034] Preparation of mycobacterial MNR competent cells: Inoculate MNR strains into LB medium and culture at 30°C until OD 600 1.0, according to 10% inoculum amount transferred to the seed medium for secondary seed culture; 24h after adding 2% glycine to continue culturing for 24h. Collect the bacteria by centrifugation, wash the suspended bacteria with 10% pre-cooled glycerin of 1 times, 3 / 4 times, 1 / 2 times and 1 / 4 times the volume of fermentation broth respectively and centrifuge, finally add 1 / 25 times of 10% glycerol Suspend the bacteria and store in sub-packages;

[0035] Electroporation: Take 10 μL of the pMV261-ndh genetic engineering expression vector obtained in Example 2, add it to 100 μL of competent bacteria and let it stand for 30 minutes, then transfer to the electroporation cuvette for clicking. Electroporation conditions were 2kV / cm, 25μF, 720Ω for 3-6ms, place...

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Abstract

The invention belongs to the technical field of biocatalysis, and particularly relates to a method for improving steroid medicine production strain vitality to efficiently produce a steroid medicine precursor by fermentation. The method improves steroid medicine production strain vitality to efficiently produce a steroid medicine precursor by fermentation through the overexpression of type II NADHdehydrogenase gene, and solves the problems of long fermentation cycle and low production efficiency during the steroid medicine precursor production by a microbiological method. A new method for reducing production cost of steroid medicine precursor is provided.

Description

Technical field: [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a method for improving the activity of a steroid drug-producing strain and efficiently fermenting and producing a steroid drug precursor. Background technique: [0002] Steroid drugs, referred to as steroid drugs, are the main products sold in the pharmaceutical industry and are widely used clinically. It is known that phytosterols can be catabolized by many microorganisms into a series of steroid precursors. Among them, 4-androstene-3,17-dione (AD) and 1,4-androstene-3,17-dione (ADD) are the main components of synthetic steroid drugs such as progestins, contraceptives and estrogens. precursor. It has been reported that fast-growing Mycobacterium agrobacterium has a strong ability to degrade phytosterol side chains to accumulate AD and ADD. As an alternative to chemical synthesis, biotransformation has become the main production method for steroid precursors ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P33/00C12R1/32
Inventor 王敏张扬申雁冰周秀玲骆健美夏梦雷张振建
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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