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Cofactor regeneration mycobacteria and its application in fermentation of oil-water two-liquid phase

A new mycobacterium aureus, two-liquid phase technology, applied in the field of biocatalysis, can solve the problems of low substrate solubility, slow metabolism of phytosterols, slow metabolism of strains, etc., and achieve the effect of increasing production

Pending Publication Date: 2018-03-09
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the slow metabolism of the strain and the low solubility of the substrate in the phytosterol biotransformation process, and introduce the cofactor regeneration system into the steroid transformation microorganism to improve the performance of the recombinant microorganism in the oil-water two-liquid phase fermentation system. The conversion efficiency of androstenedione provides a new method to solve the slow metabolism of phytosterols in bacteria, and provides the possibility for the wide application of oil-water two-liquid phase fermentation system in the industrial production of androstenedione

Method used

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  • Cofactor regeneration mycobacteria and its application in fermentation of oil-water two-liquid phase
  • Cofactor regeneration mycobacteria and its application in fermentation of oil-water two-liquid phase
  • Cofactor regeneration mycobacteria and its application in fermentation of oil-water two-liquid phase

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Experimental program
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Effect test

Embodiment 1

[0025] The construction of embodiment 1 recombinant bacterial strain MNR M3N2

[0026] 1. Construction of pMV261-nox-2 plasmid, the process includes:

[0027] (1) Synthesize the target gene, according to the nucleotide sequence (Gene ID: 13019046, SEQ ID NO.1) of the NZ9000 NADH oxidase gene nox-2 of Lactococcus lactis subsp. (BamH I and Sal I) were added to both ends of the nox-2 sequence, and the designed sequence was handed over to Jinweizhi Company for gene synthesis;

[0028] (2) PCR amplification of the target gene fragment nox-2 gene (the verification picture is figure 1 ), the target fragment nox-2 and the expression plasmid pMV261 were digested with BamHI and SalI respectively, purified and ligated to obtain the recombinant plasmid pMV261-nox-2.

[0029] 2. Construction of strain MNR M3N2:

[0030] (1) Preparation of Mycobacterium MNR M3 Competent Cells: Primary Seed Culture to OD 600 1.0, according to 10% inoculum amount transferred to the seed medium for seconda...

Embodiment 2

[0034] NAD (H) content and NAD in the recombinant bacterium MNR M3N2 of embodiment 2 + / NADH ratio comparison.

[0035] 1. Bacteria activation culture:

[0036] Transfer the recombinant bacteria MNR M3N2 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;

[0037] 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 310g / L, agar 20g / L, the rest is water, pH7.2;

[0038] 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 res...

Embodiment 3

[0056] Example 3 The MNR M3N2 strain catalyzes the production of androstenedione from phytosterols in an aqueous phase system

[0057] 1. Bacteria activation culture:

[0058] Transfer the recombinant bacteria MNR M3N2 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;

[0059] 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;

[0060] 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 nitr...

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Abstract

The invention belongs to the technical field of biological catalyst, and particularly relates to a mycobacteria genetically engineered bacterium with cofactor regeneration function and its applicationin fermentation of two liquid phases. Through a NADH oxidizing system in the cell, the genetically engineered bacterium for transforming plant sterol biology is structured and transformed for the NADH oxidizing system in the cell, thus the problem of low transforming efficiency of androstenedione in the oil water two-liquid-phase fermenting system is solved; the genetically engineered bacterium also provides possibility for the wide application of the oil water two-liquid-phase fermenting system in industrial production of the androstenedione. The genetically engineered bacterium MNR M3N2 structured by the invention can improve the AD (D) output by 1.4-2.9 times during the preparation process of androstenedione by degrading the phytosterol through the oil water two-liquid-phase fermentingsystem fermenting method.

Description

Technical field: [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a mycobacterium genetically engineered bacterium with cofactor regeneration function and its application in a two-liquid phase fermentation system. 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. Phytosterols are hydrophobic compounds with low solubility and are easy to gather on the surface of the water phase, while steroid invertase belongs to intracellular enzymes, and the substrate can only be in contact with the enzyme to carry out the transformation reaction after diffusing into the cell, which leads to the conversion reaction of phytosterols an...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P33/02C12R1/32
CPCC12N9/0036C12N15/74C12P33/02C12Y106/03001
Inventor 王敏苏立秋申雁冰商志华高田张文凯骆健美
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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