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A kind of Escherichia coli for efficient fermentation and production of l-alanine

A technology of Escherichia coli and alanine, applied in the field of microbial metabolism engineering, can solve the problems of high production cost and application limitation of L-alanine, and achieve the effects of low production cost, high chemical purity and high optical purity

Active Publication Date: 2020-11-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high production cost of L-alanine limits its application

Method used

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  • A kind of Escherichia coli for efficient fermentation and production of l-alanine
  • A kind of Escherichia coli for efficient fermentation and production of l-alanine
  • A kind of Escherichia coli for efficient fermentation and production of l-alanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Acquisition of Escherichia coli CGMCC No.10628

[0027] Using E.coli B100 as the starting strain, use the gene deletion method reported by Datsenko et al. (Datsenko K.A.et al., Proc NatlAcad Sci USA, 2000, 97:6640-6645) to knock out the by-products acetate, formate, ethanol, and succinic acid on the chromosome Acid and lactic acid synthesis pathway encoding genes ackA-pta, pflB, adhE, frdA, ldhA, to obtain strain E.coliB150.

[0028] The alaD gene was amplified from Geobacillus stearothermophilus strain, and the kana resistance marker was cloned downstream of the gene. According to the method reported by Datsenko et al. (Datsenko K.A.et al., Proc Natl Acad Sci USA, 2000, 97:6640-6645), the promoter and alaD gene fragment were integrated into the dadX gene on the chromosome of strain B150, and the recombinant strain 62ALA was obtained by screening (Escherichia coli CGMCC No. 10628).

Embodiment 2

[0029] Example 2 Production of L-alanine by fermenting glucose in a 5L fermenter

[0030] Bacterial strain 62ALA carries out fermentation test in 5L fermenter, and its test process is:

[0031] Seed solution culture: Streak the strains stored in glycerol storage tubes at -80°C and culture them on LB plates for 24 hours, inoculate a single colony in 50 mL of LB liquid medium, and culture them at 28-35°C and 200 r / min for 10 hours. The thalline obtained in the LB liquid medium was collected by centrifugation, and inoculated in 150 mL M9-1 liquid medium containing 5 g / L glucose (500 mL triangular bottle), cultured on a shaker at 28-35°C and 200r / min for 9h. LB medium composition ( / L): 10g tryptone, 5g yeast powder, 10g NaCl.

[0032] Fermenter inoculation: the above seed liquid was inoculated into a 5L fermenter containing M9-1 medium (Winpact FS-02, Major Science, Saratoga, CA) with an inoculation amount of 0.062g / L (thalline dry weight / medium volume). , USA), the initial vol...

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Abstract

The invention discloses an Escherichia coli for efficiently fermenting and producing L-alanine, and belongs to the technical field of microbial metabolism engineering. The by-product acetic acid, formic acid, ethanol, succinic acid, lactic acid synthesis pathway encoding genes ackA-pta, pflB, adhE, frdA, ldhA on the chromosome of Escherichia coli (Escherichia coli) CGMCC No.10628 provided by the present invention are deleted, and its chromosome The dadX gene was replaced by the alanine dehydrogenase gene. Using the recombinant bacteria to ferment for 26 hours at 28-45°C, 106 g / L or more of L-alanine with high optical purity and high chemical purity is produced, and the production intensity of the whole fermentation process reaches 4.27 g / L·h or more.

Description

technical field [0001] The invention relates to an Escherichia coli for efficiently fermenting and producing L-alanine, belonging to the technical field of microbial metabolism engineering. Background technique [0002] L-alanine, one of the smallest chiral molecules, is used in medicine and veterinary medicine, and is used together with other L-type amino acids as a nutritional agent before and after surgery. Because L-alanine has a sweet taste, it is also used as a food additive. In addition, alanine can also be used to synthesize modified heat-resistant plastics, such as (PA)s, poly(ester-amide)s(PEA)s, poly(ester-amide-sulfide)s(PEAS)s and poly(ester -imide)s(PEI)s, etc. However, the high production cost of L-alanine limits its application. [0003] In recent years, using genetic engineering means, the exogenous alanine dehydrogenase gene was introduced into the E.coli strain, and the synthesis of L-alanine was realized in E.coli (Lee M.et al., Appl Microbiol Biotechn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/06C12R1/19
CPCC12P13/06C12N1/205C12R2001/19
Inventor 周哲敏周丽崔文璟刘中美
Owner JIANGNAN UNIV
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