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Related gene for high-yield L-leucine, construction method for engineering bacteria and application

A leucine and mutant gene technology, applied in the field of microorganisms, can solve the problems of failing to meet the industrial production requirements of leucine, long fermentation acid production cycle, low acid production level, etc., to achieve convenient trait improvement, good growth traits, Produces high-intensity effects

Active Publication Date: 2019-03-12
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the leucine-producing bacteria of Corynebacterium glutenum can be obtained through genetic engineering, the acid production level is low, and the fermentation acid production cycle is long, and the production efficiency is low. At present, it cannot meet the industrial production requirements of leucine.

Method used

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  • Related gene for high-yield L-leucine, construction method for engineering bacteria and application
  • Related gene for high-yield L-leucine, construction method for engineering bacteria and application
  • Related gene for high-yield L-leucine, construction method for engineering bacteria and application

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Experimental program
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Effect test

Embodiment 1

[0028] Construct L-leucine high-efficiency production strain AL03, the specific method is as follows:

[0029] 1 Integration of isopropylmalate synthase-encoding mutant gene leuA*:

[0030] (1) The leuA mutant gene was integrated into the cgl1135 pseudogene site. Site-directed mutagenesis of the isopropylmalate synthase gene leuA was performed, and the mutant gene leuA* was obtained by PCR; using the genome of Corynebacterium glutamicum ATCC 13032 as a template, the upstream and downstream homology arm fragments of cgl1135 and glutamine were amplified acid Corynebacterium strong promoter Ptuf. PCR reaction conditions (PrimeSTAR HS enzyme): pre-denaturation (95°C) for 5 minutes; then 30 cycles: denaturation (98°C) for 10s, annealing ((Tm-3 / 5)°C) for 15s, extension at 72°C (this Enzyme activity extends about 1kb for 1min; 72°C for 10min; maintain (4°C). The PCR amplification system is shown in Table 2; the primer sequences are shown in Table 1, and have the sequence shown in ...

Embodiment 2

[0054] A method for fermenting and producing L-leucine by using genetic engineering bacteria AL03 strain is as follows:

[0055] (1) Shake flask fermentation

[0056] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 32°C for 24 hours, and passage once;

[0057] Shake flask seed culture: Scrape a ring of slanted seeds with an inoculation loop and inoculate in a 500mL Erlenmeyer flask containing 30mL seed medium, seal with twelve layers of gauze, and incubate at 32°C and 200rpm for 7-10h;

[0058] Shake flask fermentation culture: Inoculate 10-15% inoculum into a 500 mL Erlenmeyer flask containing fermentation medium (final volume is 30 mL), seal with twelve layers of gauze, 32°C, 200r / min shaking culture, during fermentation Maintain the pH at 7.0-7.2 by adding ammonia water; add 60% (m / v) glucose solution to maintain the fermentation; the fermentation period is 24-30h;

[0059] The composition of the slant medium is: glucose 5g / ...

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Abstract

The invention relates to a related gene for high-yield L-leucine, a construction method of engineering bacteria and application. The construction method for the engineering bacteria comprises the following steps: (1) integrating a leuA mutant gene to a cgl1135 pseudogene locus, wherein the leuA mutant gene is as shown in sequence 1; (2) integrating an ilvBN mutant gene to a cgl1890 pseudogene locus, wherein the ilvBN mutant gene is as shown in sequence 2; and (3) replacing a citrate synthase primary promoter by a PCP_2928 specific promoter, wherein the PCP_2928 specific promoter is as shown insequence 3. By adopting the technical scheme provided by the invention, 32 g / L L-leucine can be produced by fermenting for 30 hours in a shaking flask, and 60 g / L L-leucine can be produced by fermenting for about 50 hours in a 5L fermenting tank; the maximum production intensity can reach 1.5 g / (L*h); the saccharic acid conversion rate is 30 percent; and the production level is the highest levelfor producing the L-leucine by the fermenting method reported at present.

Description

technical field [0001] The invention relates to the field of microorganisms and molecular biology, and relates to related genes in the synthetic pathways of L-leucine and L-valine and applications thereof. Background technique [0002] Corynebacterium glutamicum is a Gram-positive bacterium of the order Actinomycetes, and its cells are arranged in a short rod shape. Corynebacterium glutamicum plays a pivotal role in the field of amino acid fermentation and has been safely used for nearly 60 years. Currently, most amino acids including L-lysine, L-valine, L-threonine, L-leucine, L-isoleucine, L-alanine, L-aspartic acid, etc. Both began to use Corynebacterium glutamicum fermentation for production. [0003] The molecular structure of L-leucine (L-leucine), L-isoleucine (L-isoleucine) and L-valine (L-valine) contains a methyl side chain, which is called branched chain Amino acids (branched chain amino acids, BCAAs). L-leucine is one of the eight essential amino acids that h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/113C12N1/21C12P13/06C12R1/15
CPCC12N9/1022C12N9/1025C12P13/06C12Y202/01006C12Y203/03012
Inventor 谢希贤崔毅刘晓倩马跃超徐庆阳马倩陈宁李燕军
Owner TIANJIN UNIV OF SCI & TECH
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