Recombinant D-lactic acid produced lactobacillus plantarum strain and method for producing D-lactic acid by utilizing same

A technology for producing Lactobacillus plantarum and strains, which is applied in the field of genetic engineering and microbial fermentation, can solve problems such as limited strains, and achieve the effects of increasing the rate of acid production, saving cooling water, and reducing energy consumption

Active Publication Date: 2019-03-22
JILIN COFCO BIOCHEM +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in the large-scale industrial production of D-lactic acid through microbial fermentation, the selectable strains are still very limited, and it is necessary to fur

Method used

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  • Recombinant D-lactic acid produced lactobacillus plantarum strain and method for producing D-lactic acid by utilizing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: strain isolation and identification

[0053] Isolation and screening of strains: mash 1g of Daqu sample and dissolve it in 10mL of sterile normal saline, mix well for 30min; take the above diluted solution 10-fold gradient dilution and spread it on a solution containing 1% (w / v) CaCO 3(neutralizing agent) MRS solid medium, cultured at 37°C for 24 hours, picked colonies with larger transparent circles to inoculate MRS medium, cultured at 150rpm at 37°C for 1 hour, took 5 μl of culture solution and spot on MRS+CaCO 3 On a solid plate, place the plate in an incubator at 45°C and incubate for 40 hours; select a single colony on the plate that grows faster and has a larger calcium-dissolving circle and transfer it to MRS medium again, culture at 37°C and 150rpm for 1 hour, and take 5 μl for culture Liquid point at MRS+CaCO 3 On a solid plate, place the plate in a 48°C incubator for culture. Among them, the candidate strains with obvious calcium-dissolving circle...

Embodiment 2

[0057] Embodiment 2: Recombinant plasmid construction

[0058] Gene knockout plasmid construction: amplify the Escherichia coli replicon p15Aori (sequence amplified from commercial vector pACYC), erythromycin resistance gene (sequence from commercial vector pMG36e), and 1000bp sequence upstream of the CDS of the gene to be knocked out (sequence amplified from Lp43 #genome), chloramphenicol resistance gene (sequence from commercial vector pNZ8148) and gene CDS downstream 1000bp sequence to be knocked out (sequence amplified from Lp43#genome), using DNA Assembly recombination kit (purchased from Transgen company) to assemble into Gene knockout plasmids. The gene knockout plasmid can replicate in Escherichia coli but cannot replicate in lactic acid bacteria, and has erythromycin and chloramphenicol resistance genes that can be used in Escherichia coli and plantarum lactobacillus. To knock out the ldh1 gene (Gene ID: 1061886), ldh2 gene (Gene ID: 1063343) and larA gene (Gene ID: ...

Embodiment 3

[0088] Example 3: Strain Homologous Recombination

[0089] Lactobacillus plantarum electrotransformation scheme: inoculate a single colony of Lactobacillus plantarum Lp43# obtained in Example 1 activated on the plate and culture overnight at 37°C in 4mL MRS medium, according to the initial OD 600 =0.2 was transferred to 100mL MRS medium containing 1% glycine, cultured on a shaker at 37°C until OD 600 =0.6; Bacterial liquid was ice-bathed for 20min, and the bacterial cells were collected by centrifugation at 4℃, 8000×g for 15min; 100mL 1mM MgCl 2 30% PEG1000 and 30% PEG1000 were used to wash the cells once respectively; resuspended cells were resuspended with 1 mL of 30% PEG1000 pre-cooled at 4°C, and each aliquot was filled with 100 μL of competent cells. Add 5 μg of the corresponding recombinant plasmid prepared in Example 2 to each competent cell, ice-bath for 10 minutes, use a 0.2cm electric cup, 1.5kV, 25F, 200Ω electric shock, quickly add 0.8mL MRS-SM medium (in MRS medi...

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Abstract

The invention relates to a D-lactic acid produced strain and a method for producing D-lactic acid by using the same. When the D-lactic acid is produced by using the D-lactic acid produced strain Lp-DAprovided by the invention, the acid production speed and the optical purity of a product are obviously improved, namely, the D-lactic acid produced strain Lp-DA has obviously improved acid productionperformance for producing the D-lactic acid. Correspondingly, the production of the D-lactic acid can be improved greatly by using the D-lactic acid produced strain Lp-DA to produce the D-lactic acidthrough fermentation. Moreover, the D-lactic acid produced strain Lp-DA also has the advantages that the fermentation cost is low, the production cycle can be shortened, energy consumption required for temperature control in fermentation production is reduced, cooling water is saved, living contaminants are reduced and the method is environmentally-friendly.

Description

technical field [0001] The invention relates to the fields of genetic engineering and microbial fermentation. Specifically, the present invention relates to a recombinant D-lactic acid producing Lactobacillus plantarum strain capable of rapidly producing D-lactic acid with high optical purity at relatively high temperature and a method for producing D-lactic acid using the D-lactic acid producing bacterial strain. Background technique [0002] Polylactic acid (Polylactic acid, PLA) is a biodegradable polymer formed by polycondensation of lactic acid monomers. PLA is unanimously recognized by the industry as the most promising new "bio-based material" in the new century because its raw material is a renewable biological resource. In addition, products made of PLA have good gloss, transparency, feel and heat resistance, and also have certain bacteria resistance, flame retardancy and UV resistance, and have high gloss and processing performance; PLA also has It has the charac...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P7/56C12R1/25
CPCC12N1/20C12P7/56C12R2001/25C12N1/205
Inventor 佟毅张媛王小艳王燕陈博李义焦琳郑晓卫刘志刚
Owner JILIN COFCO BIOCHEM
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