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Construction method of tetracycline anoxia-tolerance strains

A construction method, tetracycline technology, applied in the field of construction of tetracycline-resistant low-oxygen strains, can solve the problems that restrict the improvement of antibiotic fermentation titer, the reduction of solubility and mass transfer efficiency, and the consumption of manpower and material resources

Inactive Publication Date: 2015-06-24
NINGXIA QIYUAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tetracycline fermenter (Streptomyces aureus) belongs to actinomycetes. During the fermentation process, the dense structure of mycelium and the viscosity of the fermentation medium often limit the dissolved oxygen; while the solubility of oxygen in the fermentation broth is low, And with the increase of biomass and antifoaming agent in the fermentation broth, its solubility and mass transfer efficiency will gradually decrease. Oxygen supply will usually cause restrictions on the industrial fermentation of tetracyclines, which seriously restricts the improvement of antibiotic fermentation titers and seriously affects the production of tetracyclines. mass production
[0004] ① Improve the hypoxia problem in the process of bacterial fermentation by increasing the ventilation of the fermentation tank and reducing the liquid volume of the tank, but this method is costly and low-yield
[0005] ② Use physical and chemical methods to conduct microbial mutation breeding to select low-oxygen-tolerant strains. This method consumes a lot of manpower and material resources, and the cycle is long and the effect is slow.

Method used

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  • Construction method of tetracycline anoxia-tolerance strains
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  • Construction method of tetracycline anoxia-tolerance strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1. Preparation of competent cells of Escherichia coli ET12567

[0050] a) Inoculate 2uL of ET12567 glycerol frozen-preserved bacterial solution into LB medium, and culture overnight at 37°C and 180r / min on a shaking table. Transplanted in LB medium with 1% inoculum, cultured on a shaker at 37°C until the OD600 in the mid-log phase was 0.458.

[0051] b) 0-4°C, 4000r / min centrifuge to collect the bacteria, suspend the bacteria pellet in 2mL of pre-cooled 0.1 mol / L CaCl 2 solution, 1-5 ℃ ice bath for 20-30 min. Centrifuge at 0-4°C, 4000r / min for 10min, discard the supernatant, and collect the bacteria. Suspend the bacteria in 100uL pre-cooled 0.1 mol / L CaCl 2 solution, stored in a 4°C refrigerator for later use.

[0052] 2. Transform Escherichia coli competent cells

[0053] a) Take 100ul ET12567 competent cells into EP tubes, add 10uL expressable plasmid pSET152 of the Vitiligo hyaline hemoglobin gene, and bathe in ice (1-5°C) for 30min. Heat shock in a constant te...

Embodiment 2

[0065] 1. Preparation of competent cells of Escherichia coli ET12567

[0066] a) Inoculate 3uL of ET12567 glycerol frozen-preserved bacterial solution into LB medium, and culture overnight at 33°C and 200r / min on a shaking table. Transplanted in LB medium with 3% inoculum, cultured on a shaker at 35°C until the OD600 in the mid-log phase was 0.516.

[0067] b) Collect the bacteria by centrifugation at 3°C ​​and 4000r / min, suspend the bacteria pellet in 2 mL of pre-cooled 0.1 mol / L CaCl 2 solution, ice-bathed for 30 min. Centrifuge at 3°C ​​and 4000r / min for 5min, discard the supernatant, and collect the bacteria. Suspend the bacteria in 100uL pre-cooled 0.1 mol / L CaCl 2 solution, stored in a 4°C refrigerator for later use.

[0068] 2. Transform Escherichia coli competent cells

[0069] a) Take 60ul of ET12567 competent cells into an EP tube, add 8uL of expressable plasmid pSET152 of the Vitiligo hyaline hemoglobin gene, and bathe in ice at 1-5°C for 30-40min. Heat shock ...

Embodiment 3

[0082] 1. Preparation of competent cells of Escherichia coli ET12567

[0083] a) Inoculate 3uL of ET12567 glycerol frozen-preserved bacterial solution into LB medium, and culture overnight at 36°C and 200r / min on a shaking table. Transfer to LB medium with 3% inoculum amount, and culture on a shaker at 36°C until the OD600 in the mid-log phase is 0.433.

[0084] b) Collect the bacteria by centrifugation at 0-2°C and 4500r / min, suspend the bacteria pellet in 2 mL of pre-cooled 0.1 mol / L CaCl 2 solution in an ice bath for 30 min. Centrifuge at 0-2°C, 4500r / min for 7min, discard the supernatant, and collect the bacteria. Suspend the bacteria in 100uL pre-cooled 0.1 mol / L CaCl 2 solution, stored in a refrigerator at 4°C for later use.

[0085] 2. Transform Escherichia coli competent cells

[0086] a) Take 80ul of ET12567 competent cells into an EP tube, add 5uL of expressible plasmid pSET152 of Vibrella hyaline hemoglobin gene, and ice-bath for 20-30min. Heat shock in a cons...

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Abstract

The invention relates to a construction method of tetracycline anoxia-tolerance strains. The construction method comprises the following steps: constructing a plasmid pSET152 capable of expressing a vitreoscilla hemoglobin gene; transforming the constructed plasmid pSET152 into escherichia coli PUZ8002; and carrying out conjugational transfer on the transformed escherichia coli PUZ8002 and streptomyces aureus, so that the tetracycline anoxia-tolerance strain is obtained. The tetracycline anoxia-tolerance strain constructed by using the method disclosed by the invention can improve the protein expression level of recombinant tetracycline under low oxygen conditions, thereby finally achieving the purpose of energy saving and cost reducing.

Description

technical field [0001] The invention belongs to the technical field of molecular biological fermentation medicine, and in particular relates to a method for constructing a tetracycline-resistant low-oxygen strain. Background technique [0002] In industry, high-density cell fermentation is usually carried out, and the high-density cell fermentation process of tetracycline needs to consume a lot of oxygen. Tetracycline fermenter (Streptomyces aureus) belongs to actinomycetes. During the fermentation process, the dense structure of mycelium and the viscosity of the fermentation medium often limit the dissolved oxygen; while the solubility of oxygen in the fermentation broth is low, And with the increase of biomass and antifoaming agent in the fermentation broth, its solubility and mass transfer efficiency will gradually decrease. Oxygen supply will usually cause restrictions on the industrial fermentation of tetracyclines, which seriously restricts the improvement of antibioti...

Claims

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Application Information

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IPC IPC(8): C12N15/76C12N1/21C12R1/49
Inventor 张燕玉马学霞李学兵孙瑞君崔莉
Owner NINGXIA QIYUAN PHARMA
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