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Method and strain for reducing by-product fumaric acid in L-malic acid fermentation process and application

A fermentation process, malic acid technology, applied in the field of bioengineering, to achieve the effect of reducing by-product fumaric acid and reducing costs

Active Publication Date: 2021-12-28
南京昊禾生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the problems existing in the prior art, and to provide a method, strain and application for reducing the by-product fumaric acid in the L-malic acid fermentation process

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  • Method and strain for reducing by-product fumaric acid in L-malic acid fermentation process and application
  • Method and strain for reducing by-product fumaric acid in L-malic acid fermentation process and application
  • Method and strain for reducing by-product fumaric acid in L-malic acid fermentation process and application

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Embodiment 1

[0053] Embodiment 1: Aspergillus niger fum Construction of knockout strains

[0054] This embodiment includes the following steps:

[0055] (1) fum Construction of gene knockout vector

[0056] for amplification fum The upstream sequence fragment of the gene, using the Aspergillus niger genome as a template to design amplification primers fum -F-F and Fum -F-R, recovered by PCR amplification fum The upstream sequence fragment of the gene, via Eco R I and Sac After I double enzyme digestion and gel recovery, it was ligated with the vector pLH594 obtained by the same restriction endonuclease treatment with the help of the One-Step Clone Kit kit, and the ligated product was transformed into E. coli In JM109 competent cells, spread evenly in LB solid medium containing 100 µg / mL kanamycin resistance, culture upside down at 37°C overnight, pick a single clone and verify it by colony PCR and plasmid double enzyme digestion ( figure 2 ), get a successful connecti...

Embodiment 2

[0074] Example 2: Application of engineering strains to produce L-malic acid by fermentation

[0075] Aspergillus niger constructed by the present invention fum The method for producing malic acid by fermentation of gene knockout engineering strain M1 in shake flasks, the specific steps are as follows:

[0076] First, the obtained engineering strain M1 was inoculated on PDA medium and placed in an incubator at 28°C for upside-down culture for 5 days until sufficient conidia were produced;

[0077] The preparation method of the PDA medium: accurately weigh 200 g of peeled potatoes, cut into about 1 cm 3Add distilled water and stir continuously and boil for 30 min, filter and collect the filtrate with double-layer gauze, add 20 g glucose and stir until completely dissolved, distill water to 1 L and divide into jars, autoclave at 121 °C 20 min. Add 1.5% agar to the solid medium.

[0078] Then, the conidia of strain M1 were collected and inoculated in malic acid fermentation ...

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Abstract

The invention discloses an Aspergillus niger engineering strain for reducing a by-product fumaric acid in an L-malic acid fermentation process. A fumaric acid hydratase gene fum is knocked out of theAspergillus niger engineering strain. In order to overcome the defects in the prior art that in the existing process of producing malic acid through Aspergillus niger fermentation, the by-product fumaric acid is accumulated along with the generation of malic acid, and the cost of a subsequent malic acid purification process is increased, the invention provides the Apergillus niger engineering strain with the fum gene knocked out, and a method for greatly reducing the by-product fumaric acid in the Aspergillus niger fermentation process. The by-product fumaric acid accumulated in the process of producing the malic acid by fermenting the aspergillus niger is obviously reduced, the cost in the downstream separation and purification process of the malic acid is reduced, and an excellent strain is provided for industrial fermentation production of the malic acid.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method, bacterial strain and application for reducing the by-product fumaric acid in the fermentation process of L-malic acid. Background technique [0002] As an important organic acid, L-malic acid is ubiquitous in plants, animals and microorganisms. It is an important intermediate metabolite of the tricarboxylic acid cycle in organisms and is widely used in food, medicine and chemical industries. In the food industry, malic acid is widely used as a food sour taste regulator and citric acid because of its natural flavor of apples. It can directly participate in the metabolism of the human body, and is often used to treat abnormal liver function and high blood ammonia. It is also commonly used in amino acid injection drugs to help the utilization of amino acids, etc.; in the chemical industry, it is often used in metal cleaning, printing and dyeing industries, and elect...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12N15/60C12N9/88C12P7/46C12R1/685
CPCC12N9/88C12N15/80C12P7/46C12Y402/01002C12R2001/685C12N1/145C12N2800/10
Inventor 刘浩徐晴
Owner 南京昊禾生物科技有限公司
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