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Chitin synthase transcriptional regulation gene MedA inactivated aspergillus niger genetically engineered bacterium and application thereof

A technology of genetically engineered bacteria and chitin synthase, applied in the field of genetic engineering, can solve the problems of bacterial residue accumulation, short catalytic aging, easy aging, etc., to increase the effect of oxygen and mass transfer, shorten the fermentation period, and solve the problem of yield. low effect

Active Publication Date: 2021-12-21
NANJING UNIV OF TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, liquid submerged fermentation has natural defects such as poor tolerance, easy inactivation, easy aging, and short catalytic time limit, which will gradually cause the cell proliferation and differentiation ability of Aspergillus niger to decline, and the autolysis phenomenon is serious, so it cannot be used continuously for a long time. This leads to the accumulation of a large amount of bacterial residues in the fermentation process, resulting in the waste of nutrients and the low conversion rate of substrates.

Method used

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  • Chitin synthase transcriptional regulation gene MedA inactivated aspergillus niger genetically engineered bacterium and application thereof
  • Chitin synthase transcriptional regulation gene MedA inactivated aspergillus niger genetically engineered bacterium and application thereof
  • Chitin synthase transcriptional regulation gene MedA inactivated aspergillus niger genetically engineered bacterium and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of Aspergillus niger Iron Acquisition Regulatory Gene MedA Knockout Bacteria

[0042] (1) Extraction of the original Aspergillus niger genome

[0043] The plant genome extraction kit (takara minibest plant genomicDNAextraction kit) was used by takara company, and the specific method was as follows:

[0044] 1. Take 1 mL of scraped Aspergillus niger ATCC12846 spore liquid and inoculate it in 50 mL DP medium, and cultivate it at 35°C and 200 r / min for 24 hours; the formulation of the DP medium is as follows: 10g / L dextrin, 5g / L peptone , 2.5g / L Potassium Dihydrogen Phosphate, 1g / L Sodium Nitrate, 0.5g / L Magnesium Sulfate, 10g / L Glycine, dilute to 100mL with water and then divide into 50mL.

[0045] 2. Centrifuge the product obtained in step 1 at 8000r / min for 5min to collect mycelial balls, wash twice with normal saline, grind the collected mycelial balls with liquid nitrogen for 3 times, weigh 100mg of ground powder and add to the previously added...

Embodiment 2

[0073] Embodiment 2 crystal violet dyeing experiment

[0074] Inoculate the original Aspergillus niger (ATCC12846) and the genetically engineered bacteria (ΔMedA strain) on the PDA plate respectively, wait until the spores are overgrown, add 3mL of spore scraping buffer to the plate, scrape off the spores with a coating stick, and transfer them to a sterile In a 5mL centrifuge tube, dilute to 2mL with spore scraping buffer to obtain spore liquid. Quantify with a hemocytometer and dilute to 10 4 cells / mL, then continue to dilute to 10 3 / mL, 10 2 / mL.

[0075]Add 1 mL of synthetic medium in advance to the 24-well plate, and then inoculate 2 μL of different concentrations of spore liquid into the medium. Cultivate statically at 35°C for 72 hours to allow Aspergillus niger to form a film on the bottom of the orifice plate. Afterwards, the culture medium was discarded, washed twice with PBS, and stained with 0.1% crystal violet for 15 minutes. Afterwards, the crystal violet ...

Embodiment 3

[0080] Embodiment 3 Genetic Engineering Bacteria Immobilization Fermentation Experiment

[0081] 1. Preparation of Porous Fibrous Material Immobilization Medium

[0082] Soak the polyurethane material (molecular weight 3000-3500) in 1M NaOH for 1h, wash it with pure water, soak it in 1M HCl for 1h, then wash it with pure water until the pH is neutral, and then put it in an oven at 65°C and dry it until constant weight. cut into 0.5cm 3 Carriers of the same size left and right.

[0083] 2. Preparation of Fermentation Medium

[0084] Weigh 250g of corn flour, add 1L of water and mix to prepare an aqueous corn flour solution, place it in a 75°C water bath, add 1mL of liquefying enzyme (60000U / L) to liquefy for 40 minutes, and then put it in a water bath Heat to 95°C, and when the cornflour aqueous solution reaches 85°C, add 1mL of liquefaction enzyme and liquefy for 60min until the iodine solution does not turn blue to obtain cornflour liquefaction; filter the cornflour liqui...

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Abstract

The invention discloses a chitin synthase transcriptional regulation gene MedA inactivated aspergillus niger genetically engineered bacterium and application thereof. According to the invention, an aspergillus niger genetically engineered bacterium with chitin synthase transcriptional regulation gene MedA deletion is constructed through a gene knockout means. According to the genetically engineered bacterium, the biofilm quantity of aspergillus niger in the process of producing citric acid through immobilized fermentation is reduced, the phenomenon that the pore diameter of a carrier is blocked is effectively relieved, the oxygen and mass transfer effect between the carrier and the outside is improved, so that the advantages of immobilized fermentation are exerted, the yield of citric acid is increased, the fermentation period is shortened, and the problems of low yield and long fermentation period in citric acid production through aspergillus niger fermentation in existing industrial engineering are effectively solved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an Aspergillus niger genetically engineered bacterium with inactivated chitin synthase transcription regulation gene MedA, a construction method and application thereof. Background technique [0002] As the most important acid in biochemical products, citric acid is widely used in food, medicine, daily chemical and other industries. It is one of the organic acids with the largest demand and the most widely used in the world. my country is the main producer of citric acid, and its annual output accounts for 53% of the world's total output. At present, the domestic strains producing citric acid are mainly filamentous fungus Aspergillus niger, and the substrates are mainly cassava flour, corn flour and other rough grain products. In recent years, due to the price of food in our country, labor costs have generally risen, followed by a sharp increase in the pr...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12N15/31C12N11/093C12P7/48C12R1/685
CPCC07K14/38C12N15/80C12N11/093C12P7/48Y02E50/10
Inventor 余斌应汉杰陈勇刘庆国赵南邹亚男周勇卢宗梅熊结青杨儒文陈天鹏孙文俊张涛姚建忠
Owner NANJING UNIV OF TECH
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