Recombinant penicillium chrysogenum gene engineering strain and construction method and application thereof

A technology of genetically engineered bacteria and Penicillium chrysogenum, applied in genetic engineering, microbial-based methods, applications, etc., can solve the problems of high kinetic energy consumption, high cost, and long cultivation time, and achieve good adsorption and combination and increase potency , the effect of shortening 24h

Active Publication Date: 2020-02-18
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Purpose of the invention: the technical problem to be solved by this invention is to provide a recombinant Penicillium chrysogenum genetically engineered bacterium for the production of penicillin seeds by fermentation of Penicillium chrysogenum in the prior art.

Method used

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  • Recombinant penicillium chrysogenum gene engineering strain and construction method and application thereof
  • Recombinant penicillium chrysogenum gene engineering strain and construction method and application thereof
  • Recombinant penicillium chrysogenum gene engineering strain and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Construction of Penicillium chrysogenum Pc-somA gene knockout bacteria.

[0058] (1) Extraction of the original Penicillium chrysogenum genome

[0059] Use the kit (FastPure plant DNA IsolationMini kit) that Vazyme company extracts plant genome, specific method is as follows:

[0060] The activation of bacteria is as follows: Gently scrape the Penicillium chrysogenum engineering bacteria activated on the IPM plate with the buffer solution of scraping spores to make spore liquid (hemocytometer plate counts 10 8 / mL).

[0061] 1. Inoculate 400uL of scraped Penicillium chrysogenum spore liquid into 100mL LYPD medium, and incubate at 25°C, 220r / min for 10.5h;

[0062] 2. Use a filter cloth to filter and collect mycelium balls, centrifuge at 5000r / min for 5min, wash twice with PBS, grind the collected mycelium balls with liquid nitrogen for at least 3 times, weigh 0.1g of ground powder and add to 1.5mL in a centrifuge tube. Immediately add 400uL of Buffer A1 a...

Embodiment 2

[0091] Embodiment 2: crystal violet dyeing experiment

[0092] Inoculate the original Penicillium chrysogenum and the genetically engineered bacteria on the IPM plate respectively, and when the spores are overgrown, add 2 mL of spore scraping buffer to the plate, scrape off the spores with a coating stick, then wash with 1 mL of spore scraping liquid and transfer to the sterilized plate. Bacteria in a 5mL centrifuge tube, dilute to 2mL with spore scraping buffer to obtain spore liquid. Quantify with a hemocytometer and dilute to 10 6 cells / mL, then continue to dilute to 10 6 、10 5 、10 4 、10 3 、10 2 .

[0093] Add 1 mL of synthetic medium in advance to the 24-well plate, and then inoculate 4 uL of different concentrations of spore liquid into the medium. Cultivate statically at 35°C for 72 hours to make Penicillium chrysogenum form a film on the bottom of the well plate; then pour out the medium, wash it twice with PBS, add 0.1% crystal violet to stain for 15 minutes; th...

Embodiment 3

[0097] Embodiment 3: Immobilized fermentation experiment of genetically engineered bacteria.

[0098] 1. Preparation of plastic medium as immobilization medium

[0099] (1) Wash the plastic medium-polymethyl methacrylate in deionized water for about 4 minutes, then extract the cleaned plastic medium in acetone for 24 hours to remove surface oil and impurities, and bake in an oven at 45°C for 30 minutes.

[0100] (2) Place the treated plastic medium in (1) on a SDCD16-3-20 type corona treatment equipment for corona treatment with a current intensity of 8A for 15 minutes, and store it in a refrigerator at 4°C for standby.

[0101] 2. Configuration of fermentation medium

[0102] Components of immobilized fermentation medium: soybean cake powder 4.65%, calcium carbonate 1%, lactose 3%, potassium dihydrogen phosphate 0.5%, ammonium sulfate 0.4%, sodium sulfate 0.15%, salad oil 0.03%, phenylacetic acid 2.5%, The solvent is water, pH=5.8.

[0103] 3. The fermentation steps are as...

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Abstract

The invention discloses a recombinant penicillium chrysogenum gene engineering strain and a construction method and an application thereof. The sequence of the Pc-somA gene in the penicillium chrysogenump gene engineering strain is replaced by the Bleo resistance gene. The penicillium chrysogenum is ATCC48271; the construction method is as follows: (1) extracting genomic DNA of penicillium chrysogenum; (2) constructing a gene knockout fragment; (3) preparing protoplasts of penicillium chrysogenum; and(4) introducing the gene knockout fragment into the prepared protoplast for homologous recombination to obtain recombinant penicillium chrysogenum gene engineering strain. Beneficial effects are as follows: the gene engineering strain provided by the invention can increase the biofilm amount OD570 of penicillium chrysogenum by nearly 2 times in the process of immobilized fermentation for penicillin production, the hypha can be better adsorbed and combined with a plastic medium, the titer of penicillin is increased to 1963.25 U/mL, the whole fermentation period is shortened by 24 hours, and the production cost is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant Penicillium chrysogenum genetically engineered bacterium and its construction method and application. Background technique [0002] So far, penicillin is still one of the most clinically used antibiotics in my country because of its good therapeutic effect and antibacterial ability. From the discovery of penicillin by Fleming in 1942 to the purification and mass production of penicillin, many years of efforts have been made. Its successful development has greatly enhanced people's resistance to bacteria and driven the development of antibiotic families. Penicillin antibiotics are a general term for β-lactam derivatives, which can be divided into penicillin G, penicillin V, enzyme-resistant penicillin, ampicillin, etc.; penicillin can also be divided into natural penicillin and semi-synthetic penicillin. Natural penicillin is mainly based on the fermentation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12N15/31C12P37/00C12R1/82
CPCC07K14/385C12N15/80C12P37/00
Inventor 应汉杰王芳娟陈勇刘丽张德力俞莹丁赛欧阳平凯
Owner NANJING UNIV OF TECH
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