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Recombinant aspergillus niger genetically engineered bacterium, construction method and application

A technology of genetically engineered bacteria and Aspergillus niger, applied in the field of genetic engineering, can solve the problems of rising food prices and rising production costs of citric acid.

Active Publication Date: 2021-05-11
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, my country's current grain reserves cannot meet national needs, and grain prices have generally risen, and the production cost of citric acid has also increased. Therefore, it is necessary to improve the citric acid fermentation method and reduce the fermentation cost.

Method used

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  • Recombinant aspergillus niger genetically engineered bacterium, construction method and application
  • Recombinant aspergillus niger genetically engineered bacterium, construction method and application
  • Recombinant aspergillus niger genetically engineered bacterium, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Construction of Aspergillus niger iron acquisition regulatory gene AcuM knockout bacteria

[0075] (1) Extraction of the original Aspergillus niger genome

[0076] The plant genome extraction kit (takara minibest plant genomicDNAextraction kit) was used by takara company, and the specific method was as follows:

[0077] 1. Inoculate 1 mL of the scraped Aspergillus Niger ATCC12846 spore liquid into 50 mL of 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 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.

[0078] 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...

Embodiment 2

[0113] Embodiment 2: crystal violet dyeing experiment

[0114] Inoculate the original Aspergillus Niger ATCC12846 (original bacterium) and the recombinant Aspergillus niger genetically engineered bacteria (ΔAcuM strain) on the PDA plate respectively, wait for the spores to grow, add 3mL of spore scraping buffer to the plate, and scrape the spores with a coating stick Next, transfer to a sterilized 5mL centrifuge tube, and dilute to 2mL with spore scraping buffer to obtain spore liquid. Quantify with a hemocytometer and dilute to 10 5 cells / mL, then continue to dilute to 10 4 / mL, 10 3 / mL.

[0115] 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 ...

Embodiment 3

[0120] Embodiment 3: Immobilized fermentation experiment of genetically engineered bacteria

[0121] 1. Preparation of porous fibrous material immobilization medium

[0122] Soak the polyurethane material in 1M NaOH aqueous solution for 1h, wash it with pure water, soak it in 1M HCl aqueous solution for 1h, then rinse it with pure water until the pH is neutral, and then put it in an oven at 65°C and dry it to constant weight. cut into 0.5cm 3 Carriers of the same size left and right.

[0123] 2. Preparation of Immobilized Fermentation Medium

[0124] (i) Weigh 250g / L corn flour and place it in a water bath at 75°C. When the corn flour liquid reaches 65°C, add 1mL of liquefied enzyme (60000U / L) and keep it warm for 40min to obtain corn flour liquid;

[0125] (ii) Heat the water bath to 95°C, and when the corn flour solution reaches 85°C, add 1mL of liquefaction enzyme, and enzymatically hydrolyze for 60min, until the iodine solution does not turn blue, filter to obtain corn ...

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Abstract

The invention discloses a recombinant aspergillus niger genetically engineered bacterium, a construction method and application. In the recombinant aspergillus niger genetically engineered bacterium, an iron acquisition regulatory gene is inactivated. The genetically engineered bacterium enables the amount of a biological membrane to be reduced in a process of producing citric acid by immobilized fermentation of aspergillus niger, so that a phenomenon that the pore diameter of a carrier is blocked is effectively alleviated, and oxygen and mass transfer effects between the carrier and the outside are improved, and furthermore, the advantages of the immobilized fermentation are expressed, the yield of the citric acid and the conversion rate of saccharic acid are improved, the fermentation period is shortened, and the problems in the prior art that the yield is low and the fermentation period is long when the citric acid is produced by the aspergillus niger through fermentation are effectively solved; and the recombinant aspergillus niger genetically engineered bacterium can be continuously fermented for 12 batches and the fermentation strength is not obviously reduced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant Aspergillus niger genetically engineered bacterium, its construction method and application. Background technique [0002] Because citric acid is widely used, it plays an important role in medicine, food, agriculture and other industries, and a large amount of citric acid is consumed every year. In the organic acid market, citric acid has a market share as high as 70%. In the food industry, citric acid is widely used in the production of various beverages, candies, biscuits and other foods, and can also be used as a flavoring agent or antioxidant. Citrate also plays an important role in the food industry; in the pharmaceutical industry, citrate It can help the rapid dissolution of active ingredients in drugs, or be used as an anticoagulant; in the chemical industry, citric acid is widely used as a complexing agent, masking agent, builder, et...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/87C12N15/31C12P7/48C12R1/685
CPCC07K14/38C12N15/87C12P7/48
Inventor 应汉杰张涛陈勇孙文俊刘丽蒋颖赵伟朱家庆柳东余斌
Owner NANJING UNIV OF TECH
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