Escherichia coli for efficiently producing L-alanine by fermentation

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

Active Publication Date: 2015-07-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • Escherichia coli for efficiently producing L-alanine by fermentation
  • Escherichia coli for efficiently producing L-alanine by fermentation
  • Escherichia coli for efficiently producing L-alanine by fermentation

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, the gene deletion method reported by Datsenko et al. (Datsenko K.A.et al., Proc Natl Acad Sci USA, 2000, 97:6640-6645) was used to knock out the by-products acetate, formic acid, ethanol, The synthetic pathway of succinic acid and lactic acid encodes the genes ackA-pta, pflB, adhE, frdA, ldhA, and the strain E.coli B150 is obtained.

[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 escherichia coli for efficiently producing L-alanine by fermentation, and belongs to the technical field of microbial metabolic engineering. Coding genes for a synthesis route for byproducts acetic acid, formic acid, alcohol, succinic acid and lactic acid on a chromosome of escherichia coli CGMCC No.10628 provided by the invention are deleted, and the chromosome dadX gene is replaced as an alanine dehydrogenase gene, wherein the coding genes comprise ackA-pta, pflB, adhE, frdA and idhA. The recombinant bacterium is fermented for 26 hours to produce 106g/L or more than 106g/L L-alanine with high optical purity and high chemical purity at 28-45 DEG C; and in the whole fermentation process, the production strength reaches 4.27g/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 Applications(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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