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Gene engineering bacterial strain for producing L-malic acid and construction method and application thereof

A technology of genetically engineered bacteria and malic acid, applied in the biological field, can solve the problems of narrow sources of raw materials, high extraction costs, and expensive equipment

Active Publication Date: 2010-02-17
ANHUI BBCA FERMENTATION TECH ENG RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conversion rate of the immobilized cell (enzyme) production process is relatively low, the production process is long, the product content in the conversion solution is not high, and the post-extraction cost is high
In addition, the amount of precursor substance fumarate used in the bioenzyme conversion method is large, and the source of raw materials is narrow
There are two optical isomers of D-type and L-type in chemically synthesized malic acid, which need to be separated, the cost is high, the process is complicated, the technology is difficult, the equipment is expensive, the yield is low, and there are waste pollution problems

Method used

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  • Gene engineering bacterial strain for producing L-malic acid and construction method and application thereof
  • Gene engineering bacterial strain for producing L-malic acid and construction method and application thereof
  • Gene engineering bacterial strain for producing L-malic acid and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0039] The screening of embodiment 1SMF resistant bacterial strain

[0040] The rumen of freshly slaughtered cattle was taken, and microorganisms were enriched by anaerobic culture method, and a succinic acid-producing bacterium was obtained through primary screening on a flat plate and secondary screening in a shaker flask. After the identification of morphology, physiology and biochemistry, and 16S rRNA, it was determined that the screened strain belonged to Actinobacillus succinogenes, and the actinomycete was named AS-FY10.

[0041] Pick a single colony of AS-FY10 from the plate, activate and enrich it for 1-2 times, and culture it in a specific liquid medium (MA) at 37°C in a CO 2Under the conditions, culture in a 250ml Erlenmeyer flask (with rubber stopper) with slight shaking for 1-4 days to the logarithmic phase, centrifuge the AS-FY10 cells in the logarithmic growth phase (8000g, 20min), wash with 0.1mol / L phosphate buffer ( pH7.0) was washed twice, then the cells we...

Embodiment 2

[0042] Transformation of embodiment 2 recombinant plasmids, screening of recombinants

[0043] 1 Construction of recombinant plasmid FY-fmr-Kan-fmr

[0044] Use pCR2.1 (invitrogen) as a template, use primers Kan F and Kan R (see Table 1) to amplify the kanamycin resistance gene, pfu as a polymerase, and the total volume of PCR reaction is 50 μl Reaction conditions: 94 ° C pre-denaturation 5min, denaturation at 94°C for 1min, annealing at 55°C for 40sec, extension at 72°C for 2min, a total of 30 cycles, and finally extension at 72°C for 10min, adding ordinary Taq enzyme and continuing to incubate for 10min. The size of the electrophoresis analysis band was 1.0kb, which was consistent with the expected size ( Figure 4 ) Purify the PCR product with a kit from Shanghai Bioengineering Co., Ltd. (see the instruction manual for specific methods), and ligate the product with pGEM-T vector (invitrogen) using T4 DNA ligase (product of Takara Company) overnight at 4°C. Transform Esche...

Embodiment 3

[0049] Embodiment 3PEPCE and MDH enzyme activity assay

[0050] 1 Preparation of crude enzyme solution

[0051] At 37°C, AS-FY10 and AS-FY-Rec in CO 2 Cultivate under anaerobic conditions for 24 hours, take 1mL of bacterial suspension, centrifuge to obtain bacterial cells, dilute to 10mL with pH 7.5, 0.1mmol / L phosphate buffer, sonicate in an ice bath for 10min, and then freeze and centrifuge at 10000rpm for 15min. The obtained supernatant is the crude enzyme solution.

[0052] 2 PCEPCK enzyme activity assay

[0053] Take two cuvettes, add 2.3mL pH 7.5, 0.1mol / L phosphate buffer to one of them, and then add 50mmol / L MnCl in sequence 2 , 0.5mmol / L NaHCO 3 , 0.5mmol / L sodium phosphoenolpyruvate, 50mmol / L ADP, 0.1ml each of 5mmol / L NADH solution, add 3mL phosphate buffer saline to the control cuvette. Place the two cuvettes at 25°C for 2 minutes, and quickly add 0.1ml 500u / mL malate dehydrogenase pure enzyme solution and an appropriate amount of crude enzyme solution to star...

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Abstract

The invention provides a gene engineering bacterial strain for producing L-malic acid as well as a construction method and application thereof. In the invention, key phosphoenolpyruvate carboxykinase(PEPCK) related to the L-malic acid, malate dehydrogenase (MDH) and fumarase (FumC) are transformed to obtain the gene engineering bacterial strain; a deactivation mutation is carried out on the fumCto cause the interruption of a metabolic flux at the converting position from the malic acid to the fumaric acid so as to accumulate the target product L-malic acid; before the mdh, the promoter of aninner source pepck gene is added to generate the metabolic flux biased towards the malic acid; in addition, through the inducement of monofluorine sodium acetate, the activity of the PEPCK is improved, and the product feedback inhibition is greatly reduced. The bacterial strain applied to the fermentative production of the L-malic acid so as to obviously improve the yield of L-malic acid. The invention has advantages of simple technology, obvious effect and low cost and can satisfy the demand of markets.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a genetically engineered bacterium producing L-malic acid, a construction method and application of the genetically engineered bacterium, in particular to the transformation of key enzymes involved in the production of L-malic acid by using genetic engineering technology , to increase the level of L-malic acid fermentation. Background technique [0002] L-malic acid is mainly used in the food industry, it is an excellent sour agent and preservative. Adding L-malic acid to various fruit wines, beverages fermented by lactic acid bacteria, and margarine can keep the flavor of natural fruit juice to the maximum extent. The sour taste of L-malic acid is relatively slow to stimulate and has a long retention time. The sour taste is 20% stronger than citric acid. It can also eliminate the aftertaste when used with synthetic sweeteners, so it is more and more popular among consum...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/21C12N15/01C12N15/76C12N15/70C12N15/74C12P7/46C12R1/19C12R1/01
Inventor 李荣杰徐斌薛培俭段绪果
Owner ANHUI BBCA FERMENTATION TECH ENG RES
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