Genetic engineering bacteria for producing L-lactic acid from acetic acid, construction method thereof and application thereof

A construction method, gene technology, applied in the field of genetic engineering, fermentation engineering, and biotechnology, can solve the problems of restricting wide application and high market price of L-lactic acid

Active Publication Date: 2019-02-12
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there have been some studies on the fermentation of lactic acid from non-grain and cheap raw materials such as cellulose, cassava, molasses, and Jerusalem artichoke, cornstarch is still the main ra

Method used

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  • Genetic engineering bacteria for producing L-lactic acid from acetic acid, construction method thereof and application thereof
  • Genetic engineering bacteria for producing L-lactic acid from acetic acid, construction method thereof and application thereof
  • Genetic engineering bacteria for producing L-lactic acid from acetic acid, construction method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Example 1. Construction of recombinant bacteria E.coli LA5 (pMCS-LA2+pUC19-LA4)

[0147] 1. Construction of recombinant expression vectors pMCS2-LA1 and pMCS2-LA2

[0148] 1. The artificially synthesized DNA shown in Sequence 1 in the sequence list contains the acs expression cassette, the upstream is the SacI site, and the downstream is the XbaI site, wherein the 9th-51st nucleotide is the promoter sequence, and the 70th-2028th nucleotide Nucleotides are acs gene sequences.

[0149] 2. The artificially synthesized DNA shown in Sequence 2 in the sequence list contains ackA and pta expression cassettes, the upstream is the XbaI site, and the downstream is the EcoRI site, wherein the 9th-51st nucleotides are the promoter sequence, and the 70th- Nucleotide 1272 is the ackA gene sequence, and 1347-3491 nucleotides are the pta gene sequence.

[0150]3. Use SacI and XbaI to double digest the DNA sequence synthesized in sequence 1, and recover a DNA fragment with a size of a...

Embodiment 2

[0233] Embodiment 2, the application of recombinant bacteria E.coli LA5 (pMCS-LA2+pUC19-LA4) in the production of L-lactic acid

[0234] 1. Shake flask culture experiment of recombinant bacteria E.coli LA5 (pMCS-LA2+pUC19-LA4)

[0235] 1. Cultivate the E.coli LA5 (pMCS-LA2+pUC19-LA4) prepared in step 7 of Example 1 in LB liquid medium containing ampicillin and kanamycin at 37°C and 200rpm for 16h , as a seed solution.

[0236] 2. Inoculate the seed liquid into the MM liquid medium containing ampicillin and kanamycin according to the inoculation amount of 4% by volume, and the liquid amount in a 250 ml shake flask is 50 ml, and cultivate under the condition of 37 ° C and 100 rpm At 24h, the fermentation broth was collected at 12h and 24h of cultivation, respectively.

[0237] 3. Quantitative detection of L-lactic acid produced by genetically engineered bacteria and acetic acid consumed by high performance liquid chromatography. The specific conditions are as follows:

[023...

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Abstract

The invention discloses genetic engineering bacteria for producing L-lactic acid from acetic acid, a construction method thereof and application thereof. The genetic engineering bacteria for producingL-lactic acid from acetic acid are obtained by modifying the recipient bacteria as follows: knocking out the poxB gene, pflB gene, aceEF gene, ldhA gene and lldD gene of the recipient bacteria; and increasing the content of protein encoded by the acs gene, pta gene, ackA gene, aceA gene, aceK gene, aceB gene, maeA gene, maeB gene, and ldh2 gene in the recipient bacteria. According to the engineering bacteria obtained by the invention, the desired level of the yield of L-lactic acid obtained by using acetic acid in shake flask culture is achieved, the use of carbon sources such as glucose is avoided, the reduction of the raw material cost of L-lactic acid fermentation is facilitated, and good application prospects are realized.

Description

technical field [0001] The invention belongs to the fields of biotechnology, genetic engineering and fermentation engineering, and specifically relates to a genetically engineered bacterium for producing L-lactic acid using acetic acid, a construction method and application thereof. Background technique [0002] Human society consumes a large amount of fossil resources such as oil and coal every year, and the resulting emission of a large amount of greenhouse gases has caused concerns about climate change. Therefore, the development and utilization of renewable biomass resources has become one of the hot spots in the current scientific and industrial circles. At present, glucose, xylose, starch, and cellulose derived from agriculture and planting are carbon sources and energy materials commonly used by microorganisms in the fermentation industry. my country is a big country in the bio-fermentation industry. The output of many products ranks first in the world, and consumes ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P7/56C12R1/19
CPCC12N15/70C12P7/56C07K14/245
Inventor 李正军笪央央普楠史理陇
Owner BEIJING UNIV OF CHEM TECH
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