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Method for improving regeneration of mycobacterium coenzyme and for simultaneously promoting production of androstenedione

A technology for androstenedione and androstenedione production, which is applied in the field of biocatalysis, can solve problems such as prolonging the production cycle, and achieve the effect of improving regeneration and increasing production

Inactive Publication Date: 2018-11-30
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

Previous studies promoted AD(D) production efficiency by regulating the coenzyme regeneration system in microorganisms, and promoted intracellular NAD by overexpressing NADH oxidase + The results showed that the production of AD(D) was increased, but the cell growth was significantly inhibited, prolonging the production cycle of AD(D) (Su LQ, Shen YB, Zhang WK, Gao T, Shang ZH , Wang M(2017) Cofactor engineering to regulate NAD + / NADH ratio with its application to phytosterols biotransformation. Microb Cell Fact 16(1):182-192)

Method used

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  • Method for improving regeneration of mycobacterium coenzyme and for simultaneously promoting production of androstenedione
  • Method for improving regeneration of mycobacterium coenzyme and for simultaneously promoting production of androstenedione
  • Method for improving regeneration of mycobacterium coenzyme and for simultaneously promoting production of androstenedione

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Experimental program
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Effect test

Embodiment 1

[0028] Construction of embodiment 1 recombinant strain MNR M3C3

[0029] 1. Construct the pMV306-cyp125-3 plasmid, the process including:

[0030] (1) Synthesize the target gene, according to the nucleotide sequence (SEQ ID NO.1) of the cyp125-3 monooxygenase gene cyp125-3 of the mycobacterium M3 sterol C27, add restriction sites (BamH I and Hind III) to The two ends of the cyp125-3 sequence, and the designed sequence was handed over to Jinweizhi Company for gene synthesis;

[0031] (2) PCR amplification of the target gene fragment cyp125-3 gene (the verification picture is figure 1 ), target fragment cyp125-3 and plasmid pMV261 were digested with BamHI and Hind III respectively, purified, and ligated to obtain recombinant plasmid pMV261-cyp125-3.

[0032] (3) Digest the recombinant plasmid pMV261-cyp125-3 and the expression plasmid pMV306 with XbaI and Hind III, recover the promoter and target gene fragments in pMV261-cyp125-3 from the gel, and connect them with the digeste...

Embodiment 2

[0040] NAD (H) content and NAD in the recombinant bacterium MNR M3C3 of embodiment 2 + / NADH ratio comparison.

[0041] 1. Bacteria activation culture:

[0042] Transfer the recombinant bacteria MNR M3C3 and the original bacteria MNR M3 to fresh slant medium, culture at 29°C for 2-4 days, wash the strains on the slant medium with 20mL 0.5% Tween 80 sterile aqueous solution to obtain the eluate, Take 1mL of the eluate and add it to 30mL of the seed medium, and culture it on a shaker at 29°C and 200r / min for 36h to obtain the seed solution;

[0043] Slant medium composition: K 2 HPO 4 0.5g / L, MgSO 4 ·7H 2 O 0.5g / L, ferric ammonium citrate 0.05g / L, citric acid 2g / L, ammonium nitrate 2g / L, glycerin 20g / L, glucose 5g / L, CaCO 3 10g / L, agar 20g / L, the rest is water, pH7.2;

[0044] Seed medium composition: K 2 HPO 4 0.5g / L, MgSO 4 ·7H 2 O 0.5g / L, ferric ammonium citrate 0.05g / L, citric acid 2g / L, ammonium nitrate 2g / L, glycerin 20g / L, glucose 5g / L, CaCO 3 10g / L, the r...

Embodiment 3

[0064] Embodiment 3 Recombinant bacterial strain growth comparison

[0065] 1. Bacteria activation culture:

[0066] Transfer the recombinant bacteria MNR M3C3 and the original bacteria MNR M3 to fresh slant medium respectively, culture at 29°C for 3 days, wash the bacteria on the slant medium with 20mL 0.5% Tween 80 sterile aqueous solution, and mix them evenly to elute solution, absorb 1mL of the eluent and add it to 30mL of the seed medium, and culture it on a shaking table at 29°C and 200r / min for 36h to obtain the seed culture solution;

[0067] Slant medium composition: K 2 HPO 4 0.5g / L, MgSO 4 ·7H 2 O 0.5g / L, ferric ammonium citrate 0.05g / L, citric acid 2g / L, ammonium nitrate 2g / L, glycerin 20g / L, glucose 5g / L, CaCO 3 10g / L, agar 20g / L, the rest is water, pH7.2;

[0068] Seed medium composition: K 2 HPO 4 0.5g / L, MgSO 4 ·7H 2 O 0.5g / L, ferric ammonium citrate 0.05g / L, citric acid 2g / L, ammonium nitrate 2g / L, glycerin 20g / L, glucose 5g / L, CaCO 3 10g / L, the...

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Abstract

The invention belongs to the technical field of bio-catalysis, and in particular relates to a method for improving regeneration of mycobacterium coenzyme and for simultaneously promoting production ofandrostenedione. According to the method, sterol C27 site monooxygenase Cyp125 in a phytosterol metabolic pathway is introduced into steroid transformation microorganism, so as to improve androstenedione conversion efficiency of recombinant microorganism in a fermentation system; and therefore, a novel method for solving a problem of slow metabolism of mycelial phytosterol is offered. Through over-expression of the Cyp125, specific enzyme activity of the mycobacterium sterol C27 site monooxygenase is improved by 22.2%; regeneration of intracellular NAD+ is improved, and an intracellular NAD+ / NADH ratio is improved by 131%; and meanwhile, it is unexpectedly discovered that a bacterium biomass amount can be improved by 18.7% through the over-expression of the enzyme, and finally, effects ofshortening a conversion cycle by 24h and improving an AD(D) yield by 10.0% can be achieved.

Description

Technical field: [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a method for improving the regeneration of mycobacterium coenzymes and simultaneously promoting the production of androstenedione. Background technique: [0002] Androstenedione, androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD), are cortisone-producing, mineralocorticoids Important intermediates of other steroid hormone drugs such as steroid drugs, conventional oral contraceptives and anabolic hormones, can be obtained by microbial degradation of phytosterol side chains. The metabolic pathway of phytosterols to produce AD(D) is complex, not only affected by their own metabolic pathways but also regulated by intracellular coenzymes. At present, the strategies to improve AD(D) production based on the analysis of phytosterol metabolic pathway mainly include: 1. The transformation of key enzymes in the phytosterol metabolic pathway; 2. The regul...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/53C12P33/02C12R1/32
CPCC12N9/0073C12N15/74C12P33/02
Inventor 申雁冰王敏苏立秋夏梦雷骆健美商志华许双平
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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