Construction of co-enzyme regeneration system and application of co-enzyme regeneration system in high-efficiency catalysis of 5 alpha-AD

A coenzyme regeneration system, -AD technology, applied in the field of genetic engineering, can solve problems such as unsatisfactory catalytic efficiency

Active Publication Date: 2020-01-03
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the molecular modification of 5α-reductase is mainly to study the enzymatic activity of the substrate testosterone, and its catalytic efficiency is not ideal; 5α-reductase is the key enzyme that catalyzes the production of 5α-AD from AD. The transformation of 5α-reductase gene has not been reported

Method used

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  • Construction of co-enzyme regeneration system and application of co-enzyme regeneration system in high-efficiency catalysis of 5 alpha-AD
  • Construction of co-enzyme regeneration system and application of co-enzyme regeneration system in high-efficiency catalysis of 5 alpha-AD

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: Construction of mutant enzyme gene and recombinant expression vector

[0059]Using the synthetic recombinant plasmid pUC57-5α with the Treponema smegmatis 5α-reductase gene as a template, amplify the 5α-reductase gene whose nucleotide sequence is shown in SEQ ID NO.1, and amplify the amplified product It was connected with the Escherichia coli-mycobacterium shuttle plasmid pMV261, transformed into E.coli DH5α, and subjected to resistance screening, plasmid PCR, double enzyme digestion verification and DNA sequencing. The recombinant plasmid with correct sequencing and stable inheritance was selected and named pMV261-5α.

[0060] Using the pMV261-5α recombinant plasmid as a template, using F1 primer (sequence shown in SEQ ID NO.5) and R1primer (sequence shown in SEQ ID NO.6) as primers, site-directed mutagenesis was carried out by overlapping extension PCR to obtain The recombinant plasmid of the 5α-reductase mutant gene, named pMV261-5α Y187F .

[0061]...

Embodiment 2

[0063] Example 2: Recombinant bacterial strain MNR M3△ksdd / 261-5α Y187F build

[0064] The recombinant plasmid pMV261-5α obtained in Example 1 Y187F Electroporation to MNR M3△ksdd competent cells, screening strains that can be stably inherited is the 5α-reductase mutant mycobacterium engineering bacteria; the specific method is as follows:

[0065] 1) Take 10 μL of the plasmid that was sequenced correctly in the previous stage and add it to the melted MNR M3Δksdd competent cells, gently blow and mix with a gun, and treat in ice bath for about 15 minutes;

[0066] 2) Completely transfer the pre-cooled plasmid and competent cell mixed solution into a 1mm electric transfer cup (clean the electric transfer cup with absolute ethanol, place it on a sterile operating table, dry it, and place it on ice for pre-cooling for 5 minutes);

[0067] 3) Place the electroporation cup containing the mixture of plasmids and competent cells on a high-voltage pulse electroporation instrument, an...

Embodiment 3

[0071] Embodiment 3: Construction of recombinant bacterial strain MNR M3△ksdd / 261-5α

[0072] Use the 5α-reductase sequence shown in SEQ ID NO.1 as a template, and use the sequences shown in SEQ ID NO.9 and SEQ ID NO.10 as upstream and downstream primers to construct a recombinant strain MNR M3△ksdd / 261-5α, operate Process is the same as embodiment 1 embodiment 2.

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Abstract

The invention provides a co-enzyme regeneration system and particularly relates to a genetically engineered bacterium of 5 alpha-reductase constructed by tandem of glucose-6-phosphate-dehydrogenase (G6PDH) and 5alpha<Y187F>-reductase recombinant and tandem of NAD kinase and 5alpha<Y187F>-reductase recombinant and application of the 5 alpha<Y187F>-reductase in high-efficiency catalysis of 5 alpha-AD production. The 5 alpha<Y187F>-reductase is electrotransformed into a mycobacterium respectively with mycobacterium-derived G6PDH and NAD kinase recombinant plasmids to subject to heterologous expression, detection on production efficiency of the 5 alpha<Y187F>reductase discovers that the co-expressed genetically engineered bacterium MNR M3 / 261-5 alpha<Y187F>-G6PDH2 on production of the 5 alpha-AD is increased from previous 67.8 percent to 89.5 percent, the co-expressed strain MNR M3 / 261-5 alpha<Y187F>-NAD2 on production of the 5 alpha-AD is increased from previous 67.8 percent to 92.6 percent, the limitation problem of low catalytic activity of the 5 alpha-reductase is effectively solved, and a new thought is provided to molecular improvement of the 5 alpha-reductase.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the construction of a coenzyme regeneration system and its application in high-efficiency catalytic 5α-AD production. Background technique [0002] 5α-AD, as a key intermediate in the synthesis of dozens of steroid hormone drugs such as minandrolone and mesterolone, has important market value and research prospects. At present, androst-4ene-3,17-dione (AD) is mainly produced in industry through a series of chemical reactions, but there are also problems such as time-consuming and serious environmental pollution. The biotransformation method has the characteristics of green, environmental protection and high efficiency, and is becoming an alternative technology for many chemical synthesis processes. [0003] Steroidal 5α-reductase belongs to the reduced coenzyme II (NADPH)-dependent enzyme, which can catalyze the reduction of a series of steroid substrates at the 4,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/53C12N15/54C12N9/04C12N9/12C12N9/02C12P33/00C12R1/32
CPCC12N15/74C12P33/00C12N9/0006C12N9/1205C12N9/0004C12Y101/01049C12Y207/01023
Inventor 申雁冰王敏任小贤欧阳微夏梦雷骆健美
Owner TIANJIN UNIV OF SCI & TECH
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