Aspergillus niger genetically engineered bacteria as well as construction method and application thereof

A technology of genetically engineered bacteria, Aspergillus niger, applied in the field of genetic engineering, can solve the problems of long fermentation period and low yield of citric acid, achieve the effects of increasing yield and sugar conversion rate, shortening fermentation period, and increasing the effect of oxygen and mass transfer

Active Publication Date: 2020-05-01
NANJING HIGH TECH UNIV BIOLOGICAL TECH RES INST CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Purpose of the invention: In order to solve the problems of low yield and long fermentation period when Aspergillus niger is fermented to produce citric acid in the existing industry, the present invention provides a strain of Aspergill...

Method used

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  • Aspergillus niger genetically engineered bacteria as well as construction method and application thereof
  • Aspergillus niger genetically engineered bacteria as well as construction method and application thereof
  • Aspergillus niger genetically engineered bacteria as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of Aspergillus niger glucan synthesis regulatory gene VosA knockout bacteria

[0038] (1) Extraction of the original Aspergillus niger genome

[0039] The plant genome extraction kit (takara minibest plant genomicDNAextraction kit) was used by takara company, and the specific method was as follows:

[0040]1. Take 1 mL of the scraped Aspergillus niger spore liquid and inoculate it in 50 mL DP medium, and cultivate it at 35 °C and 200 r / min for 24 hours; the formula of the DP medium is as follows: 10 g / L dextrin, 5 g / 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. ;

[0041] 2. Centrifuge at 8000r / min for 5min to collect mycelial balls, wash them twice with normal saline, grind the collected mycelial balls with liquid nitrogen for 3 times, weigh 100mg of the ground powder and add it to 500μL of Buffer HS II in advance Mix well in...

Embodiment 2

[0072] Embodiment 2 crystal violet dyeing experiment

[0073] Inoculate the original Aspergillus niger (Aspergillus Niger ATCC 12846) and the genetically engineered bacteria 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 rod, and transfer to a sterile 5mL In a centrifuge tube, dilute to 2 mL 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 5 / mL, 10 4 / mL.

[0074] 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 36 hours to allow Aspergillus niger to form a film at 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 w...

Embodiment 3

[0080] Embodiment 3 Genetic Engineering Bacteria Immobilization Fermentation Experiment

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

[0082] Soak the porous fiber material (pore size 1 mm) in absolute ethanol for 1 h, then rinse with water until there is no obvious smell of ethanol, and dry it in an oven at 65°C to constant weight. cut into 0.5cm 3 Carriers of the same size left and right.

[0083] 2. Preparation of Fermentation Medium

[0084] Weigh 250g / L corn flour and place it in a 75°C water bath. When the corn flour liquid reaches 65°C, add 1mL of liquefying enzyme (60000U / L) to liquefy for 40min, then heat the water bath to 95°C, and wait for the corn flour liquid to reach 65°C. When the temperature reaches 85°C, add 1mL of liquefying enzyme and liquefy for 60 minutes until the iodine solution does not turn blue. Filter the corn flour liquid to obtain the corn flour clear liquid, and add unfiltered corn flour liquid as the feedstock [unfi...

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Abstract

The invention discloses aspergillus niger genetically engineered bacteria. The aspergillus niger genetically engineered bacteria are aspergillus niger in which glucan synthesis regulating gene VosA isknocked out. The invention also discloses a construction method of the above genetically engineered bacteria, and further discloses an application of the genetically engineered bacteria in the production of citric acid through immobilized fermentation. The aspergillus niger genetically engineered bacteria in which the glucan synthesis regulating gene VosA is knocked out are constructed, so that the hydrophobicity of the aspergillus niger during the production of the citric acid through immobilized fermentation is reduced, the yield of a biological film is reduced, a vector agglomeration phenomenon is reduced, the citric acid yield and sugar conversion efficiency are improved, and the aspergillus niger genetically engineered bacteria are suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetic engineering bacterium of Aspergillus niger whose glucan synthesis regulating gene VosA is knocked out, and a construction method and application thereof. Background technique [0002] As one of the organic acids with the largest demand in the world, citric acid is widely used in food, medicine, daily chemical and other industries. Among them, 75% of citric acid is used in the food industry, mainly as sour agent, antioxidant, and pH regulator in food additives. At the same time, because citric acid has a mild and refreshing sour taste, it is widely used in beverages, pastries, wine, Manufacture of food such as dairy products. Another 15% is used in chemical industry and textile industry, which can be used as buffer, complexing agent, masking agent, metal builder, mordant and so on. There is also 10% citric acid used as anticoagulant, antacid, fl...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12P7/48C12N11/00C12R1/685
CPCC12N1/14C12N11/00C12N15/80C12P7/48
Inventor 余斌刘庆国陈勇应汉杰刘丽赵南柳东
Owner NANJING HIGH TECH UNIV BIOLOGICAL TECH RES INST CO LTD
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