A strain of genetically engineered bacteria that produces l-aspartic acid by fermentation

A technology of genetically engineered bacteria and aspartic acid, applied in fermentation, bacteria, enzymes, etc., can solve problems such as deviations in the growth stability of strains

Active Publication Date: 2019-05-21
CHANGMAO BIOCHEMICAL ENG CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A strain (CN105296411A) that can produce L-aspartic acid by monosaccharide fermentation under anaerobic conditions was also obtained earlier, but the growth stability of the strain was deviated under anaerobic conditions, and the product concentration was only 10 g / L, so further Construction screening obtained a strain with excellent growth performance and higher concentration under aerobic conditions

Method used

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  • A strain of genetically engineered bacteria that produces l-aspartic acid by fermentation
  • A strain of genetically engineered bacteria that produces l-aspartic acid by fermentation
  • A strain of genetically engineered bacteria that produces l-aspartic acid by fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] This example illustrates the use of homologous recombination to knock out the gene encoding citrate dehydrogenase ( icdA ), the gene encoding malate dehydrogenase ( Mdh ), the gene encoding malic enzyme ( sfcA and maeB ) and the gene encoding fumarase that functions under aerobic conditions ( fum AC ) five genes, the knockout process is basically the same, with fum AC Taking gene knockout as an example, the specific steps include:

[0046] 1. Using LB medium, cultivate Escherichia coli W1485 under aerobic conditions at 37°C to OD600=0.4-0.6, and make it competent for electroporation.

[0047] 2. Electrotransform the recombinant plasmid into competent Escherichia coli W1485. The electric shock conditions were: 200 Ω, 25 μF, electric shock voltage 2.3 kv, electric shock time 4-5 ms. Immediately after electric shock, the cells were added to pre-cooled 1 mL SOC medium, cultured at 150 r / min, 30°C for 1 h, and then spread on LB medium plate with ampicillin (amp) ...

Embodiment 2

[0060] This example illustrates that mutant strains with good cell growth performance under aerobic conditions are obtained through evolutionary metabolic selection.

[0061] Evolutionary metabolism is the adaptation process of the bacteria itself to the environment. When a certain microorganism mutates during continuous culture, the mutant strain will compete with the original strain. If the mutant strain has an advantage over the original strain, the mutant strain will be in the reactor get reserved.

[0062] Such as image 3 As shown, the initial stage of continuous culture starts at 0.015 h -1 The dilution rate is continuously fed with fresh medium containing glucose. The density of the initial strain in the evolutionary metabolism device is 0.6. When the bulk density reaches a steady state and remains unchanged for a period of time, increase the dilution rate to 0.03 h -1 , the concentration of bacteria in the reactor drops rapidly, which indicates that the growth rate...

Embodiment 3

[0064] This example illustrates the construction of an overexpressed phosphoenolpyruvate carboxylase encoding gene ( ppc ) and the aspartase-encoding gene ( aspA ) expression plasmid, and the recombinant plasmid was introduced into the mutant strain CM-AS-105 to increase the concentration and yield of L-aspartic acid in the strain.

[0065] 1. Construction of overexpressed phosphoenolpyruvate carboxylase encoding gene ( ppc ) and the aspartase-encoding gene ( aspA ) expression plasmid, the process includes:

[0066] (1) Artificially designed and synthesized with Nco I and Hind The III restriction site contains the operon of two genes, the specific sequence is shown in SEQ ID NO:1.

[0067] (2) Expression plasmid pTrc99a was used respectively Nco I and Hind III double digestion, and ligated with the synthetic gene to obtain the recombinant plasmid pTrc99a- ppc-aspA .

[0068] 2. The plasmid pTrc99a- ppc-aspA Introduce the mutant strain CM-AS-105 competen...

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Abstract

The invention discloses a genetically engineered bacterium for directly producing L-aspartic acid through fermentation. The classification naming of the genetically engineered bacterium is Escherichia coli CM-AS-115, and the preservation number of the genetically engineered bacterium is CCTCC NO: M 2016457. The bacterial strain relates to inactivation of multiple genes, evolution, metabolism and domestication are simultaneously carried out on the bacterial strain which knockouts the multiple genes, and a mutant strain, namely, CM-AS-105, which has a lower respiratory quotient under the aerobic condition and of which the highest dry cell weight is 60-70% of dry weight of an original strain W1485 is obtained; meanwhile, the bacterial strain further relates to over expressions of two genes, wherein the two genes comprise an enol phosphate type pyruvate carboxylase encoding gene (ppc) and an aspartase encoding gene (aspA), and the obtained bacterial strain is CM-AS-115. The genetically engineered bacterium for directly producing L-aspartic acid through fermentation achieves a way of completely adopting renewable biomass resources such as starch and cellulose as raw materials to ferment and prepare the L-aspartic acid, and the way is green and environmentally friendly.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, and in particular relates to a genetically engineered bacterium for directly producing L-aspartic acid by fermentation of glucose or xylose. Background technique [0002] L-Aspartic acid is widely used in medicine, food and chemical industry. In medicine, it is the main component of amino acid preparations; in chemical industry, it can be used as a raw material for the manufacture of synthetic resins, and is widely used in the synthesis of polyaspartic acid, an environmentally friendly material; especially in the food industry, L-aspartic acid is a It is a good nutritional supplement and is also the main raw material for the production of sugar substitute aspartame. Has a good market prospect. [0003] At present, L-aspartic acid is mainly synthesized from fumaric acid by biological enzyme method, while fumaric acid is mainly prepared by chemical method. Therefore, from the analysi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P13/20C12R1/19
CPCC12N9/0006C12N9/88C12P13/20C12Y101/01037C12Y101/01041C12Y401/01031C12Y402/01002C12Y403/01001
Inventor 马江锋高有军陈阳万屹东潘春芮新生
Owner CHANGMAO BIOCHEMICAL ENG CO LTD
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