Genetic engineering bacteria for producing D-lactic acid and constructon method and application thereof

A technology of genetically engineered bacteria and lactic acid, applied in the field of genetic engineering, can solve problems such as shortage, and achieve the effects of improving production capacity, broad application prospects and reducing costs

Inactive Publication Date: 2012-11-14
FUSHUN RES INST OF PETROLEUM & PETROCHEMICALS SINOPEC CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, due to the limitations of related technologies and the lack of means, there are few domestic reports on the genetic engineering of D-lactic acid producing bacteria. Compared with traditional strain transformation technologies such as mutagenesis and evolution, genetic engineering methods have good visibility and directionality

Method used

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  • Genetic engineering bacteria for producing D-lactic acid and constructon method and application thereof
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  • Genetic engineering bacteria for producing D-lactic acid and constructon method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: Construction of gene knockout vector:

[0036] 1. Cloning of D-lactate dehydrogenase gene in Lactobacillus bulgaricus

[0037] (1) According to the DNA sequence of the ldhA gene in Lactobacillus bulgaricus ATCC 11842 (NCBI-GI: 4085369), design primers to amplify its structural gene by PCR. The primer sequences are as follows:

[0038] primer1: 5′-GGCTAG GAGCTC TGTAAGAAAATCTGTAGGT-3′ (the underline is the introduced Sac I restriction site)

[0039] primer2: 5′-TGAGC TCTAGA AAAGGAGGAGGGACAArTAATGACT-3′ (the underline is the introduced Xba I restriction site)

[0040] Using the genomic DNA of Lactobacillus bulgaricus ATCC 11842 as a template, under the guidance of primers primer1 and primer2, the ldhA gene sequence was cloned by PCR. The amplification conditions and system of PCR were as follows:

[0041]

[0042] The PCR product obtained is detected by 1% agarose gel electrophoresis, and the electrophoresis band with a size of about 1000bp is obtaine...

Embodiment 2

[0047] Embodiment 2: Screening of ldhA gene recombinant strain

[0048] The constructed D-lactate dehydrogenase gene expression vector pXJM 19 was electrotransformed into competent cells of Corynebacterium glutamicum Res 167Δldh, the transformation conditions were: 25δF, 600Ω, 2.5k V electric shock, pulse time 10-12ms, The bacterial solution was spread on a BHIS plate with 10 μg / ml chloramphenicol and cultured for 24 hours, the positive colonies were selected, the plasmid was extracted, and PCR amplification was performed with primers primer1 and primer2. The amplification results were consistent with the expected values, indicating that the picked positive colony was the strain introduced with the recombinant vector pXJM19-ldhA, which was named C. glutamicum Res 167Δldh / ldhA.

Embodiment 3

[0049] Embodiment 3: the fermentation test of recombinant bacterial strain

[0050] (1) Seed medium formula (g / L): glucose 40, urea 2, casamino acid 7, yeast extract 2, dipotassium hydrogen phosphate 0.5, potassium dihydrogen phosphate 0.5, ferrous sulfate heptahydrate 6mg, seven Magnesium sulfate hydrate 0.5, manganese sulfate tetrahydrate 0.25, vitamin B 10.2mg, biotin 0.2mg, water 1000mL. Adjust pH 7.5.

[0051] (2) Fermentation medium (g / L): glucose 40, dipotassium hydrogen phosphate 0.5, potassium dihydrogen phosphate 0.5, ferrous sulfate heptahydrate 6 mg, magnesium sulfate heptahydrate 0.5, manganese sulfate tetrahydrate 0.25, vitamin B10.2 mg , biotin 0.2mg, water 1000mL. Adjust pH 7.5.

[0052] 1. Preparation of seeds:

[0053] With seed medium, each 1L Erlenmeyer flask is filled with 300mL medium, wrapped and sterilized. Take out the slant with bacteria stored in the refrigerator, add 1mL sterile water to each test tube, and scrape off the bacterium on the slant...

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Abstract

The invention relates to genetic engineering bacteria for producing D-lactic acid and a construction method and application thereof. The strain of C.glutamicum Res 167 delta ldh deleted in L-lactate dehydrogenase gene (ldh) is used as a starting strain to over express exogenous D-lactate dehydrogenase gene so as to obtain C.glutamicum Res 167 delta ldh / ldhA. The genetic engineering bacteria are preserved in China General Microbiological Culture Collection Center with the preserving registration number of CGMCC No.4041. By utilizing the genetic engineering means, the expression of the exogenous D-lactate dehydrogenase gene is realized in the corynebacterium glutamicum deleted in L-lactic acid metabolic pathway, and the genetic engineering bacteria producing high optical purity D-lactic acid are successfully constructed. When the engineering bacteria are used for producing lactic acid through fermentation, the yield of the D-lactic acid is over 40g / L, and the purity is over 99 percent. The invention has important significance for the industrial production of the D-lactic acid, and has wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a D-lactic acid genetically engineered bacterium, a construction method thereof and an application in the production process of D-lactic acid. Background technique [0002] As one of the three major organic acids, lactic acid can be divided into D-lactic acid, L-lactic acid and D, L lactic acid according to its optical activity. At present, lactic acid is mainly produced by microbial fermentation in the world. Among them, L-lactic acid has been developed and produced earlier because of its wide biocompatibility, and its production technology and products have tended to mature. With the discovery of new functions of D-lactic acid, the research on its production and application has attracted more and more attention. [0003] D-lactic acid is widely used in food, medicine, chemical industry, agriculture, etc., especially the polymer of lactic acid - polylact...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/63C12P7/56C12R1/15
Inventor 闻建平李爽贾晓强
Owner FUSHUN RES INST OF PETROLEUM & PETROCHEMICALS SINOPEC CORP
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