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Poor oxygen resistance high density fermentation natamycin gene engineering strain constructed by using vgb gene, and application thereof

A genetically engineered strain and high-density fermentation technology, applied in the field of genetic engineering, can solve problems such as low local dissolved oxygen concentration, slowed bacterial growth, and high oxygen consumption, so as to improve oxygen supply shortage, reduce production costs, and improve production efficiency. The effect of oxygen uptake capacity

Inactive Publication Date: 2014-02-05
INST OF BIOPHARM OF SHANDONG PROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Natamycin-producing bacteria such as Streptomyces chrysospora are aerobic bacteria. As the fermentation time prolongs, the density of bacteria increases rapidly, and the oxygen consumption is extremely high. The physical parameters of the fermentation tank cannot meet the oxygen supply, especially It is the intertwining of high-density Streptomyces chrysosporium mycelia, which makes the local dissolved oxygen concentration lower. As a result, the growth of the bacteria slows down, and the expression of key enzymes for natamycin synthesis decreases, which directly leads to a decrease in the production of natamycin

Method used

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  • Poor oxygen resistance high density fermentation natamycin gene engineering strain constructed by using vgb gene, and application thereof
  • Poor oxygen resistance high density fermentation natamycin gene engineering strain constructed by using vgb gene, and application thereof

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Effect test

Embodiment 1

[0024] Construction of recombinant plasmid pSET152-vgb.

[0025] (1) According to the sequence of vgb gene and its promoter included in GenBank (NO.M30794.1), design upstream and downstream primers respectively:

[0026] Upstream primer vgbL: 5'-GGC GAATTC AGTTTTAAAGAGGCAAT-3' (underlined as EcoRI restriction site); downstream primer vgbR: 5'-CT TCTAGA TTATTCAACCGCTTGAGCGTAC-3' (underlined as XbaI restriction site).

[0027] p BBR -MCS5-vgb was used as a template, and the vgb gene and its promoter sequence were obtained by PCR amplification.

[0028] (2) The PCR product and the vector pSET152 were digested with EcoRI / XbaI respectively, after gel cutting and recovery, they were mixed at a molar ratio of 3:1 to 10:1, added with T4 ligase, and ligated overnight at 16°C. Connecting solution with CaCl 2 The E. coli competent cells E.coli ET12567 prepared by the method were mixed evenly, heat-shocked at 42°C for 90s, and cultured at 37°C for 1 hour, and then coated with LB so...

Embodiment 2

[0030] The construction of recombinant vgb gene Streptomyces chrysanthosporium adopts the mode of Escherichia coli-Streptomyces indirect conjugative transfer, and the specific steps are as follows:

[0031] (1) Pick a single colony of Escherichia coli E. coli ET12567 containing the recombinant plasmid pSET152-vgb in LB medium (containing 25 μg / mL of kanamycin, 25 μg / mL of chloramphenicol and 50 μg / mL of Amp Mycin), cultured overnight at 37°C with shaking.

[0032] (2) Transfer the overnight activated recombinant E.coli ET12567 into fresh LB medium (containing kanamycin, chloramphenicol and apramycin) at 2% inoculum size, and culture at 37°C until OD 600 =0.4~0.6, centrifuge, wash the bacteria twice with equal volume of LB medium, resuspend in 0.1 times volume of LB medium;

[0033] (3) While processing recombinant E.coli ET12567, take about 10 8 Put the spores of Streptomyces chrysosporium in 500 μl 2×YT culture concentrate, heat shock at 50°C for 10 minutes and then cool do...

Embodiment 3

[0041] Application of S.gilvosporeus sw-807 in natamycin fermentation.

[0042] Wash the slanted spores of S. gilvosporeus S. gilvosporeus and recombinant bacteria S. gilvospores sw-807 with sterile water to make a spore suspension (about 10 8 each / mL); inoculate into 5mL seed culture medium according to 2%~5% inoculum amount, shake and cultivate at 28~30°C for 48h; 16 to 20 hours; then transfer 2% to 5% of the inoculum to a 250mL Erlenmeyer flask containing 30mL and 50mL of fermentation medium, and ferment for 3 to 5 days at 28 to 30°C with a rotational speed of 200 to 300r / min.

[0043] After the fermentation, the activity of VHb and the yield of natamycin were measured respectively.

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Abstract

The invention relates to a poor oxygen resistance high density fermentation natamycin gene engineering strain (Streptomyces gilvosporeus sw-807, CGMCC No.6890) constructed by using vgb gene, and an application thereof. According to the present invention, vitreoscilla hemoglobin gene vgb is adopted to construct recombinant plasmid, a conjugal transfer manner is adopted to integrate the vgb gene into Streptomyces gilvosporeus genome to obtain the recombinant gene engineering strain capable of being directly used for natamycin fermentation production, and VHb expression in the recombinant strain can be provided for effectively solving oxygen supply and demand contradiction in natamycin fermentation production so as to substantially increase natamycin yield, reduce production cost and bring great economic benefits.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to constructing a recombinant Streptomyces chrysospora engineering strain by utilizing the hemoglobin (vgb) gene of Vitella hyaline, through the expression of the Vgb gene, improving the recombinant chrysosporium chains in the process of high-density fermentation of natamycin Oxygen utilization by molds to increase natamycin production. Background technique [0002] Natamycin is a natural, broad-spectrum, highly effective polyene macrolide antifungal agent. Compared with other antibacterial ingredients, natamycin has extremely low toxicity to mammalian cells and can be widely used in diseases caused by fungi. For example, natamycin is used in suspensions, emulsions, ointments and sheath tablets. In the fight against fungal infections of the skin and mucous membranes. In addition, natamycin has low solubility and can be used to treat the surface of food to e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/76C12N15/70C12N1/21C12P19/62
Inventor 刘飞王绍花朱希强凌沛学
Owner INST OF BIOPHARM OF SHANDONG PROVINCE
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