Strain producing dynamic controlling recombinant strain and method for preparing D-lactic acid with recombinant strain

A technology of recombinant bacteria and lactic acid, applied in the fields of biochemical engineering, microbial fermentation engineering, and microbial genetic engineering, can solve the problems of affecting bacterial cells, increased lactic acid accumulation, lack of three-carbon intermediate metabolites, etc., and achieve the effect of ensuring polymer-grade quality.

Active Publication Date: 2012-08-01
SHANDONG BAISHENG BIOTECH
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Problems solved by technology

However, Bunch et al. (Bunch P.K. et al., Microbiology, 1997, 143: 187~195) showed that the D-lactate dehydrogenase gene was highly expressed in E. coli, and the excess lactate dehydrogenase converted the pyruvate pool into lactate , resulting in the lack of three-carbon intermediate metabolites, and the growth of bacteria is inhibited
In addition, the production of lactic acid in the growth stage of the bacteria will consume a large amount of carbon sources, so that a high bacterial concentration cannot be obtained in the aerobic stage, so that the volume production intensity of lactic acid in the second stage will decrease
For example, our previously reported strain B0013-070 (Zhou L. et al., Current Microbiology, 2011, 62: 981~989) synthesized 7 g / L lactic acid during the growth stage of the bacteria, and competed with cell synthesis for substrates. During the large-scale fermentation process, the oxygen supply capacity will decrease significantly, and the accumulation of lactic acid will further increase in the aerobic stage, which will affect the accumulation of bacterial cells and reduce the volume production intensity of lactic acid in the lactic acid accumulation stage.

Method used

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  • Strain producing dynamic controlling recombinant strain and method for preparing D-lactic acid with recombinant strain
  • Strain producing dynamic controlling recombinant strain and method for preparing D-lactic acid with recombinant strain
  • Strain producing dynamic controlling recombinant strain and method for preparing D-lactic acid with recombinant strain

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1 ： Replacement of the Chromosomal Lactate Dehydrogenase Promoter in Escherichia coli by a Temperature-Regulated Promoter

[0050] Using primers Ec-lA3-5 and YldhA3 PCR amplification of B0013-070 chromosome wxya Gene fragment wxya ’, cloned into the pMD18-T simple vector to obtain the recombinant plasmid T- wxya '. Plasmid pPL451 (Gene, 1996, 176: 49~53) was amplified by reverse PCR with primers PPL1 and PPL2, and combined with the kanamycin resistance gene fragment (plasmid pSK sym Km (Applied Microbiology and Biotechnology, 1999, 52: 820~828) use restriction enzymes, such as Sma I was digested, and the gel recovered a 966 bp fragment) for ligation to obtain the recombinant plasmid pPL- Kan . Plasmid pPL- Kan Carry out PCR amplification, and the products are treated with restriction endonucleases, such as Eco RI and Stu I carry out double digestion, and with T- wxya ' is used as the template to carry out inverse PCR amplification with primers Ec-...

Embodiment 2

[0051] Example 2 ： Strain B0013-070B p R -p L Determination of promoter activity

[0052] The strains B0013-070 and B0013-070B were respectively at 25~36 ° C and 37~50 ° C was cultured for 2~10 h, and its medium was (g / L): yeast extract 15, peptone 0.5, anhydrous MgSO 4 0.25, glucose 5. And determine their lactate dehydrogenase (LDH) specific enzyme activity, the typical results are as follows Figure 4 shown. Strain B0013-070B in 30 ° C produced only very low amounts of LDH activity. at 42 ° C culture, the LDH activity of the strain B0013-070B is 2.2 times that of the starting strain B0013-070, which is sufficient for lactic acid synthesis. Strain B0013-070 in 30 ° The specific enzyme activity value of LDH cultivated in C was calibrated as 100%, compared with this, the strain was at 42 ° LDH enzyme activity decreased when C grew. Explain that the temperature change is used to control the strain B0013-070B p R - p L The promoter effectively controls the wx...

Embodiment 3

[0053] Example 3 ： Production of lactic acid by fermenting glucose in a 7 L fermenter

[0054] Strain B0013-070B was subjected to lactic acid fermentation in a 7 L fermenter to test the effect of temperature-regulated LDH expression under controlled production conditions. Strain B0013-070B at 25~36 ° C for aerobic culture to OD 600 The value is about 15~40, set the fermenter temperature to 37~50 ° C continued aerobic culture for 0~120 min, and then set the ventilation rate to 0~0.2vvm for oxygen-limited fermentation, and the fermentation temperature in the oxygen-limited stage was 37~50 ° c. Its fermentation medium is (g / L): yeast extract 0~25, peptone 0~2.5, MgSO 4 0~0. 5, CaCO 3 0~175, pH 6.0~7.5. The concentration of lactic acid, by-products and bacteria in the fermentation process such as Figure 5 shown.

[0055] The conversion rate of aerobic cells of the strain B0013-070B was 9% higher than that of the fermentation tank of the strain B0013-070, indicating tha...

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Abstract

A Strain producing dynamic controlling recombinant strain and a method for preparing D-lactic acid with the recombinant strain belong to the technical field of genetically engineered agricultural microorganisms. The recombinant strain is named (Escherichia coli)B0013-070B, and is preserved in the China center for type culture collection, and the preservation number is CCTCCNO:M2012071. A lactate dehydrogenase gene promoter ldhAp in the genome of the strain is replaced with a culture environment/nutritional factors control-type promoter. Through the utilization of the recombinant strain, fermentation is conducted for 28 to 40 hours in stages at 25 to 50 DEG C, and the level of producing D-lactic acid reaches 12.5%; the optical purity lives up to 99.9%; and the chemical purity reaches to 98.4%. The dynamic controlling to the D-lactic acid dehydrogenase encoding gene expression on D-lactic acid high-producing strain B0013-070 chromosomes is conducted, so that the gene expression is controlled only through changing the fermentation temperature during the D-lactic acid production process to achieve the purpose of efficiently synthesizing D-lactic acid through taking glucose as a raw material. After simple modification, the invention can be used for producing important microbial metabolites in other industries.

Description

technical field [0001] The present invention relates to a fermentation process for the efficient preparation of D-lactic acid by a microbial method and a fermentation production strain thereof, in particular to a fermentation process capable of efficiently synthesizing and producing D-lactic acid only through simple regulation of fermentation process conditions such as temperature factors in an industrialized production scale. The low-cost fermentation production process belongs to the technical fields of microbial genetic engineering, biochemical engineering and microbial fermentation engineering. Background technique [0002] Lactic acid (alpha-hydroxypropionic acid, molecular formula C 2 h 5 OCOOH) is a naturally occurring organic acid widely found in humans, animals, plants and microorganisms. Lactic acid is one of the three recognized organic acids in the world. Lactic acid and its derivatives are widely used in brewing, food, agriculture, medicine, chemical industry,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/56C12R1/19
Inventor 王正祥周丽田康明沈微
Owner SHANDONG BAISHENG BIOTECH
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