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A method, bacterial strain and application for reducing by-product succinic acid in L-malic acid fermentation process

A fermentation process, malic acid technology, applied in the field of bioengineering, can solve the problems of rapid equipment depreciation, high content, low product conversion rate or production efficiency, and achieve the effect of reducing costs

Active Publication Date: 2022-03-29
南京昊禾生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The chemical synthesis method uses the petroleum-based chemical benzene as a raw material to obtain racemic DL-malic acid under high temperature and high pressure conditions. As early as 1970, the US FDA banned the addition of DL-malic acid to infant foods. In addition, chemical synthesis The law has high requirements on the equipment and the depreciation of the equipment is fast, which limits its application in the field of food and medicine
In addition, the source of raw materials for this method is petroleum-based chemicals, which is also a great challenge to the ever-decreasing petroleum energy and environmental problems
The biocatalytic method is mainly immobilized enzyme or immobilized cell transformation method. Because of the high cost of extraction, separation, purification and immobilized enzyme, the immobilized enzyme method has certain limitations on the income; and the shortcomings of the immobilized cell transformation method The reason is that living cells themselves contain complex enzyme systems, which are prone to the formation of many by-products, resulting in an increase in the downstream purification and purification costs of the product
In summary, it is difficult for malic acid prepared by chemical synthesis and biocatalysis to meet the growing demand of the malic acid market.
[0004] Compared with the above two methods, the microbial fermentation method has been paid more and more attention due to its environmental friendliness and the availability of renewable carbon sources. Low, heteroacid by-products are many and high in content, which seriously limits the industrialization process of fermentation to produce L-malic acid
[0005] Through the search, no patent publications related to the patent application of the present invention have been found

Method used

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  • A method, bacterial strain and application for reducing by-product succinic acid in L-malic acid fermentation process
  • A method, bacterial strain and application for reducing by-product succinic acid in L-malic acid fermentation process
  • A method, bacterial strain and application for reducing by-product succinic acid in L-malic acid fermentation process

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Embodiment 1

[0075] Example 1: frd A gene and frd Construction of B gene knockout vector

[0076] This embodiment includes the following steps:

[0077] (1) frd Construction of A gene knockout vector

[0078] for amplification frd The downstream sequence fragment of A gene, using the Aspergillus niger ATCC1015 genome as a template to design amplification primers frd A-F-F and frd A-F-R, recovered by PCR amplification frd A gene downstream sequence fragment, via Xba I and Speech 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 c...

Embodiment 2

[0094] Embodiment 2: Aspergillus niger frd A and frd Obtaining of B gene knockout strain

[0095] This embodiment is realized through the following steps:

[0096] (1) frd Construction of A Gene Knockout Strain K1

[0097] The vector pLH1067 was transferred to Agrobacterium, and then the Agrobacterium was co-cultured with Aspergillus niger host strain S489 on IM medium for Agrobacterium-mediated transformation. After 2.5 days of culture, the culture product was evenly spread on CM medium for cultivation Until the transformants grow out, the transformants are transferred to different media for screening, and their phenotypes on different media should be resistant to hygromycin and sensitive to glufosinate-ammonium. Genome verification of such transformants is carried out. The primers for verification were designed (Table 1), and the amplification results met the negative amplification of the left and right homology arms ( Figure 12 (P1 / P2), Figure 13 (P3 / P4)), the le...

Embodiment 3

[0108] Example 3: Application of engineering strains to produce L-malic acid by fermentation

[0109] Aspergillus niger constructed by the present invention frd A gene and frd The method for producing malic acid by fermenting B gene knockout engineering strains K1 and K2 in shake flasks, its concrete steps are as follows:

[0110] First, the obtained engineering strains K1 and K2 were inoculated on PDA medium and placed in an incubator at 28°C for upside-down culture for 5 days until sufficient conidia were produced;

[0111] Then, the conidia of strains K1 and K2 were collected and inoculated in malic acid fermentation medium, wherein the final concentration of spores was 1*10 8 cells / mL, the shake flask was placed at 28°C, 200 rpm and cultured for 5 days.

[0112] The composition of the malic acid fermentation medium is: glucose 100g / L, bacto-peptone 6 g / L, anhydrous potassium dihydrogen phosphate 0.15 g / L, anhydrous dipotassium hydrogen phosphate 0.15 g / L, calcium chlo...

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Abstract

The invention discloses a strain of Aspergillus niger ( Aspergillus niger ) engineering strain, the Aspergillus niger engineering strain is simultaneously knocked out of fumarate reductase frd A and fumarate reductase flavoprotein subunits frd B's engineered strain of Aspergillus niger. The present invention provides a frd A and frd An engineering strain of Aspergillus niger with double knockout of the B gene, and a method for greatly reducing the by-product succinic acid in the fermentation process of Aspergillus niger. The invention significantly reduces the by-product succinic acid accumulated in the process of fermenting and producing malic acid by Aspergillus niger, reduces the cost in the process of downstream separation and purification of malic acid, and provides excellent strains for industrial fermentation and production of 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 succinic acid, a by-product 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12N15/80C12N15/53C12N9/02C12P7/46C12R1/685
CPCC12N9/001C12N15/80C12P7/46C12Y103/01006C12Y103/00C07K14/38
Inventor 刘浩徐晴
Owner 南京昊禾生物科技有限公司
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