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Corynebacterium glutamicum recombinant strain capable of producing high-yield L-leucine, and construction method thereof

A technology of Corynebacterium glutamicum and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of high content of by-products, low yield of L-leucine-producing bacteria, etc., and achieve the effect of reducing the content of by-products

Inactive Publication Date: 2019-02-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing L-leucine producing bacteria modified through metabolic pathways still have the problems of low yield and high content of by-products

Method used

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  • Corynebacterium glutamicum recombinant strain capable of producing high-yield L-leucine, and construction method thereof
  • Corynebacterium glutamicum recombinant strain capable of producing high-yield L-leucine, and construction method thereof
  • Corynebacterium glutamicum recombinant strain capable of producing high-yield L-leucine, and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Obtaining of B. sphaericus LeuDH coding gene leuDH expression cassette

[0045] According to the B.sphaericus leuDH gene sequence in GenBank, the restriction endonuclease EcoR I and Kpn I restriction site sequences were added to the upstream and downstream of the gene, and the Corynebacterium glutamicum SD recognition sequence GAAAGGAGATATACC was added to the upstream of the gene, and The combined sequence was submitted to General Biosystems (Anhui) Co., Ltd. for synthesis, and the recombinant plasmid pUC57-leuDH containing the target gene was obtained. Subsequently, plasmid pUC57-leuDH was digested with restriction endonucleases EcoR I and Kpn I. The leuDH fragments were then recovered using a gel recovery kit. The leuDH fragment was ligated with the C.gluatmicum-E.coli shuttle expression plasmid pDXW-8 digested with the same restriction endonuclease to construct the recombinant plasmid pDXW-8-leuDH. Finally, using pDXW-8-leuDH as a template, P tac -F and...

Embodiment 2

[0048] Example 2: In C.gluatmicum XQ-9, the TA-encoding gene ilvE is replaced by the B.sphaericus LeuDH-encoding gene leuDH

[0049] Using the C.gluatmicum XQ-9 genome as a template, using ilvE-L-F, ilvE-L-R and ilvE-R-F, ilvE-R-R as primers for PCR, respectively, the 3' of ilvE-L and the 5' of ilvE-R have the same Restriction enzyme PCR products. The above PCR products were connected to the linearized pK18mobsacB vector to construct the recombinant plasmid pK18mobsacB-△ilvE; then the purified PCR product P tac -leuDH-rrnBT1T2 was ligated with pK18mobsacB-△ilvE, the two were digested with Xba I at the same time, and the digested products were connected through cohesive ends to obtain recombinant plasmid pK18mobsacB-△ilvE::leuDH.

[0050] The correct verified plasmid pK18mobsacB-△ilvE::leuDH was transformed into C.gluatmicum XQ-9 by electroporation, cultured on LBG+Km solid medium at 30°C, and the first homologous recombination transformant was obtained by screening. Subseque...

Embodiment 3

[0052] Example 3: Expression of LeuDH in C.gluatmicum XQ-9

[0053] The starting strain C.gluatmicum XQ-9 and the recombinant strain C.gluatmicum XQ-9-△ilvE::leuDH were inoculated into liquid LBG medium respectively. After IPTG induction, the bacteria were collected and subjected to ultrasonic crushing, and the supernatant was electrophoresed by SDS-PAGE , a specific band with a molecular weight of about 40kDa was detected ( figure 2 ), consistent with the reported target protein size, indicating that LeuDH derived from B. sphaericus can be correctly expressed in Corynebacterium glutamicum.

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Abstract

The invention discloses a Corynebacterium glutamicum recombinant strain capable of producing high-yield L-leucine, and a construction method thereof, and belongs to the field of genetic engineering. According to the present invention, the NADP-dependent branched-chain amino acid transaminase in Corynebacterium glutamicum is replaced with NAD-dependent leucine dehydrogenase (LeuDH) derived from B.sphaericus by using a genetic engineering method, such that the new efficient L-leucine synthesis pathway is constructed, the disadvantages of excessive accumulation of NADH in the Corynebacterium glutamicum L-leucine producing bacteria and insufficient supply of NADPH are solved, the L-leucine synthesis ability in the recombinant strain is enhanced, and the NADPH accumulation ability of the strainis improved; the shake fermentation experiment results show that the L-leucine accumulation of the recombinant strain achieves 16.7 g.L<-1> and is higher than the L-leucine accumulation of the starting stain of 13.2 g.L<-1>, and the maximum specific growth rate of the recombinant strain is 0.23 g.L<-1>.h<-1>, and is higher than the maximum specific growth rate of the recombinant strain of 0.18 g.L<-1>.h<-1>; and the L-leucine synthesis pathway in Corynebacterium glutamicum is successfully modified, the disadvantage of the intracellular cofactor imbalance is improved, and the new idea is provided for the breeding of high-yield L-leucine production bacteria.

Description

technical field [0001] The invention relates to a high-production L-leucine Corynebacterium glutamicum recombinant bacterium and a construction method thereof, belonging to the field of genetic engineering. Background technique [0002] L-leucine, also known as leucine, its chemical name is α-amino-γ-methylpentanoic acid, α-aminoisocaproic acid, which has the same methyl group as L-valine and L-isoleucine The side chain branching structure is collectively referred to as branched chain amino acids. Since humans and mammals cannot synthesize these three amino acids by themselves and must rely on external sources, all three are also essential amino acids. L-leucine has a variety of physiological functions and is widely used in food additives, pharmaceutical industry, cosmetic industry and animal feed additive industry. At the same time, it is also widely used clinically as a compound amino acid intravenous injection. Therefore, it is of great significance to breed production...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P13/06C12R1/15
CPCC12N9/0016C12N15/77C12P13/06C12Y104/01009
Inventor 张伟国钱和王颖妤
Owner JIANGNAN UNIV