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Method for preparing D-alanine by microbial fermentation method

A technology of Corynebacterium alanine and glutamic acid, applied in the field of microbial fermentation, can solve the problems of high price of substrate keto acid, restricting application, etc., and achieve the effects of low cost and simple technology

Active Publication Date: 2021-10-01
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] When using these two D-stereoselective enzymes to synthesize D-type amino acids, the substrate ketoacids are expensive
Moreover, meso-DAPDH needs the coenzyme NADPH, which restricts its application
At present, there are few reports on the preparation of D-amino acids by fermentation. Another reason is that for most microorganisms, D-amino acids inhibit cell growth, and it is particularly important to select starting strains with better tolerance.

Method used

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  • Method for preparing D-alanine by microbial fermentation method
  • Method for preparing D-alanine by microbial fermentation method
  • Method for preparing D-alanine by microbial fermentation method

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Screening of D-amino acid tolerant strains

[0037] Four strains were selected from the three commonly used industrial fermentation strains of Bacillus subtilis, Escherichia coli and Corynebacterium glutamicum, specifically: Bacillus subtilis 168, E. coli BL21, E. coli (E. coli) W3110, Corynebacterium glutamicum (C. glutamicum) ATCC 13032.

[0038] Inoculate the four strains on gradient plates containing D-alanine respectively, draw a line from low concentration to high concentration (0-50g / L), and observe the growth after 24h as follows: figure 1 (a). After that, the highest tolerated concentration was investigated, and the shake tube culture was carried out with CGXII basic medium, and the concentration of D-alanine was set to 1g / L, 5g / L, 10g / L, 20g / L, and 50g respectively. / L, 100g / L, 24h results such as figure 1 (b).

[0039] According to the gradient plate display, the tolerance concentration of Bacillus subtilis to D-alanine is about 8g / L, the tolera...

Embodiment 2

[0040] Example 2 D-alanine production strain construction

[0041] Using C.glutamicum ATCC 13032 as the starting bacteria, rpsL K43R The point mutation made the strain resistant to streptomycin, and the suicide plasmid pK18mobrpsL was used (for the construction method, see: An update of the suicide plasma-mediated genome editing system in Corynebacterium glutamicum[J]. Microbial Biotechnology, 2019, 12(5): 907-919 .) mediated genome editing system of Corynebacterium glutamicum for genome editing.

[0042] (1) Firstly, the alanine racemase gene alr is knocked out on the genome, and the upper and lower homology arms of the knockout gene alr are subjected to homologous recombination with the double-digestion (XbaI and KpnI) vector of pK18mobrpsL to obtain pK18rpsL -The Δalr plasmid was electrotransformed into the competent state of the starting bacteria, and the double crossover was identified by primers alr-jd-1 and alr-jd-2, and strain S-1 was obtained;

[0043] (2) Continue ...

Embodiment 35

[0056] Example 3 Preparation of D-alanine by 5L tank fermentation method

[0057] Using the S-4 / pXtuf-St* prepared in Example 2 as the production strain to ferment and produce D-alanine:

[0058] Put the activated strain of the second-generation eggplant bottle into a fermenter equipped with seed medium, and cultivate it at 32°C until the OD is about 30, leaving 600mL of seed liquid, adding 20% ​​of the inoculum to the fermentation medium, and continuing to It is fermented and cultivated under the conditions of pH6.7-7.2, temperature 32-34°C, and dissolved oxygen 10-40%. After 36 hours of fermentation, the yield reaches 50g / L.

[0059] Among them, the formula of D-alanine upper tank culture medium is as follows:

[0060] The composition of the seed medium is: glucose 60g / L, KH 2 PO 4 3g / L, MgSO 4 ·7H 2 O 2g / L, MnSO 4 ·H 2 O20mg / L, FeSO 4 20mg / L, V B1 0.5mg / L, VH 0.1mg / L, yeast powder 7g / L, methionine 0.5g / L, corn steep liquor 30g / L, soybean meal hydrolyzate 20-30mL...

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Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing D-alanine by a microbial fermentation method. The method comprises the following steps: firstly, constructing a genetically engineered bacterium for producing D-alanine, taking corynebacterium glutamicum ATCC 13032 as a starting strain of the genetically engineered bacterium, and knocking out an alanine racemase gene alr and L-alanine transaminase alaT; allowing meso-diaminopimelate dehydrogenase coding gene to be subjected to overexpression; knocking out an iolR repressor protein gene and simultaneously performing genome integration expression on a glucokinase gene glk1; and integrating edd and eda genes from Escherichia coli to obtain the gene. The strain uses glucose as a substrate to produce D-type amino acid through a direct fermentation method, the cost is low, the technology is simple, and the yield of D-alanine in a 5L fermentation tank can reach 50-60g / L. Besides, efficient synthesis of the D-type amino acid by a fermentation method belongs to creative research, and by means of the method, the fermentation method can be infinitely expanded, and various D-type amino acids can be efficiently synthesized at low cost.

Description

Technical field: [0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for preparing D-alanine and other D-type amino acids through microbial fermentation. Background technique: [0002] The basic chiral units of natural proteins are all L-type amino acids. It is generally believed that D-type amino acids rarely exist in nature. However, with the development of analytical methods, people have discovered D-type amino acids in various organisms. Taking microorganisms as an example, more than 20 kinds of D-type amino acids have been found, which exist in the components of peptidoglycan, polypeptide antibiotics and ergot alkaloids in the cell wall. [0003] Due to its remarkable biochemical properties, D-amino acids have been widely used in the synthesis of antibiotics and physiologically active peptides, and are playing an increasingly important role in food, pesticides, cosmetics, and especially medicine. D-amino ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/61C12N15/54C12N15/53C12N15/31C12P13/06C12R1/15
CPCC12N9/90C12N9/1096C12N9/0016C12N9/1205C07K14/34C07K14/245C12P13/06C12Y501/01001C12Y206/01002C12Y104/01016C12Y207/01002
Inventor 李燕军赵桂红张成林徐庆阳马洪坤苏蕊袁梦姜灏吴晨薄泰东
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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