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17[beta]-hydroxysteroid dehydrogenase mutant of mycobacteria and heterologous expression of mutant

A technology of hydroxysteroids and mycobacteria, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems of low substrate concentration and conversion rate, and cannot meet the requirements of industrial production, so as to improve production efficiency, enzyme activity, and enzyme activity Effect

Active Publication Date: 2021-04-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many microorganisms can use androst-1,4-diene-3,17-dione (ADD) as a substrate to produce BD through 17β-hydroxysteroid dehydrogenase reduction reaction, but their low substrate concentration and conversion rate Can not meet the requirements of industrial production

Method used

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  • 17[beta]-hydroxysteroid dehydrogenase mutant of mycobacteria and heterologous expression of mutant
  • 17[beta]-hydroxysteroid dehydrogenase mutant of mycobacteria and heterologous expression of mutant
  • 17[beta]-hydroxysteroid dehydrogenase mutant of mycobacteria and heterologous expression of mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Mining of the 17β-HSD gene in Mycobacterium LY-1

[0037] Final sequencing analysis report based on the full gene annotation results of Mycobacterium LY-1. A 17β-HSD gene was obtained, the base sequence of which is shown in SEQ ID NO: 1, and the corresponding amino acid sequence deduced from the complete target gene is SEQ ID NO: 2, named 17β-HSDy1. The 17β-HSDy1 sequence was compared by Blastp, and it was confirmed that there was a highly consistent DNA sequence encoding 17β-HSD enzyme.

Embodiment 2

[0038] Example 2: Extraction of Mycobacterium LY-1 Genomic DNA

[0039] (1) Mycobacterium LY-1 strain in liquid seed medium (15.0g·L -1 Yeast powder, 0.6g·L -1 (NH 4 ) 2 HPO 4 , 5.4g·L -1 NaNO 3 , 2.0g·L -1 glycerol) for 2-3 days at a culture temperature of 30° C. and a rotation speed of 120 rpm / min. Pipette 1 mL of the well-growing bacterial solution into a 1.5 mL EP tube, centrifuge at 12,000 rpm for 1 min, and discard the medium completely.

[0040] (2) Add 150 μL pH 8.0 TE buffer to the centrifuge tube to fully suspend the bacteria, and add 8 μL 3mg·mL -1 lysozyme, use the tip to crush the bacteria to fully lyse;

[0041](3) Add 300 μL of Digestion Solution and 4 μL of RNase A to the centrifuge tube and mix well, then place in a metal bath at 55°C for 10 minutes; then add 4 μL of Proteinase K, and place in a metal bath at 55°C for 30 minutes.

[0042] (4) Add 300 μL Ext solution and 300 μL PB solution in sequence and shake fully, then centrifuge at 12000 rpm for ...

Embodiment 3

[0046] Example 3: Cloning of the 17β-HSDy1 gene of mycobacterium LY-1 and construction of the plasmid pMA5-17β-HSDy1

[0047] The primers were designed according to the 17β-HSDy1 gene sequence obtained from the analysis of the whole genome of Mycobacterium LY-1.

[0048] Primer P1: aaagtgaaatcaggggggatccTCATCGGGGTGCCATCCT

[0049] Primer P2: atttcgacctctagaacgcgtGTGCACCACCACCACCACCAAGTTAGAGAACTCCATCGCA

[0050] Using the extracted mycobacterium LY-1 genome as a template, the gene encoding 17β-HSD was amplified by PCR with the above primers. The PCR reaction was carried out in a 25 μL system, and the reaction conditions were as follows: pre-denaturation at 95°C for 3 min, followed by cycling; denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 1 min, a total of 34 cycles; final extension at 72°C for 10 min. The PCR products were recovered after agarose gel electrophoresis.

[0051] A one-step cloning kit was used to construct the recombinant Baci...

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Abstract

The invention discloses a 17[beta]-hydroxysteroid dehydrogenase mutant of mycobacteria and heterologous expression of the mutant, and belongs to the field of enzyme gene engineering and enzyme engineering. The invention provides actinomycete 17[beta]-hydroxysteroid dehydrogenase 17[beta]-HSDy1 from mycobacterium sp. LY-1 and a gene sequence of the 17[beta]-HSDy1. By taking pMA5 as an expression plasmid and bacillus subtilis WB600 as an expression host, heterogenous expression of the 17[beta]-HSDy1 of the mycobacterium sp. LY-1 is realized. A key T239 site of the dehydrogenase obtained throughsoftware simulation is mutated into tyrosine, and the enzyme activity is improved by 19.2% and reaches 9571.77 U.mg<1>. An enzyme activity-growth curve of recombinant bacillus subtilis WB600-pMA5-17[beta]HSDy1-T239Y obtained by mutation is measured, and the specific enzyme activity of the recombinant bacillus subtilis WB600-pMA5-17[beta]HSDy1-T239Y reaches the maximum after fermentation is performed for 28 hours. The heterologously expressed strain can be used for production of androstane-1,4-ene-3,17-dione converted boldenone, and has potential industrial application.

Description

technical field [0001] The invention belongs to the field of genetic engineering and fermentation engineering, and specifically relates to a 17β-hydroxysteroid dehydrogenase mutant derived from mycobacterium and its heterologous expression. Background technique [0002] Steroid drugs are the second largest class of drugs after antibiotics, and are widely used in the medical field. They are often used in the treatment of allergic diseases, rheumatoid arthritis, contraception and surgical anesthesia in clinical practice. Common steroid drug intermediates are divided into C19 and C22, among which Boldenone (BD), androstene-4-ene-3,17-dione (AD), androstene-1,4- Steroid drug intermediates based on ene-3,17-dione (ADD), 9α-hydroxyandrostene-4-ene-3,17-dione (9α-OH-AD) and testosterone (T) are in The market demand in the field of medicine is increasing year by year. [0003] Boldenone (BD) is an androgenic anabolic steroid and testosterone (TS) derivative that promotes protein s...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/75C12N1/21C12P33/06C12R1/125
CPCC12N9/0006C12N15/75C12P33/06C12Y101/01051
Inventor 许正宏李会刘伟
Owner JIANGNAN UNIV
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