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Recombinant corynebacterium glutamicum strengthening capability of synthesizing L-leucine from pyruvic acid and application of recombinant corynebacterium glutamicum

A technology encoding Corynebacterium glutamicum and alanine aminotransferase, which is applied in the field of genetic engineering, can solve problems such as high leucine yield, and achieve the effect of improving yield and product conversion rate

Active Publication Date: 2020-04-10
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The results of shake flask fermentation showed that the yield and conversion rate of L-isoleucine of this strain increased by 14.8% and 18.6% respectively compared with the original strain, but the highest yield of leucine was only about 7g / L

Method used

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  • Recombinant corynebacterium glutamicum strengthening capability of synthesizing L-leucine from pyruvic acid and application of recombinant corynebacterium glutamicum
  • Recombinant corynebacterium glutamicum strengthening capability of synthesizing L-leucine from pyruvic acid and application of recombinant corynebacterium glutamicum
  • Recombinant corynebacterium glutamicum strengthening capability of synthesizing L-leucine from pyruvic acid and application of recombinant corynebacterium glutamicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of Corynebacterium glutamicum C. glutamicum XQ-9ΔltbRΔavtAT3-alaT (WL-8)

[0033] Using the genome of Corynebacterium glutamicum ATCC XQ-9ΔltbR as a template, and using pyc-U-F / pyc-U-R and pyc-D-F / pyc-D-R as primers respectively (Table 1), pyc-U and pyc were obtained after PCR amplification -D PCR product, the obtained pyc-U and pyc-D fragments were subjected to fusion PCR to obtain the homology arm fragment Δpyc. The above PCR product was enzyme-ligated with the linearized plasmid pK18mobsacB after double digestion with XbaI and HindIII to construct the plasmid pK18mobsacB-Δpyc.

[0034] Using the genome of Corynebacterium glutamicum ATCC XQ-9ΔltbR as a template, using avtA-U-F / avtA-U-R and avtA-D-F / avtA-D-R as primers respectively (Table 1), avtA-U and avtA were obtained after PCR amplification -D fragment, the obtained avtA-U and avtA-D fragments were seamlessly cloned with the linearized plasmid pK18mobsacB after double digestion with SmaI an...

Embodiment 2

[0043] Construction of embodiment 2 promoter replacement plasmid

[0044] The strain WL-8 genome and pDXW-8 plasmid were used as templates respectively, and P tac -AU-F / P tac -AU-R,P tac- AD-F / P tac -AD-R and A-P tac -F / A-P tac -R is primer PCR (table 2), obtains leuA-U and leuA-D and P tac DNA fragment, seamlessly cloned with linearized plasmid pK18mobsacB to construct recombinant plasmid pK18mobsacB-P tac -leuA.

[0045] Using the strain WL-8 genome as a template, P tuf -AU-F / P tuf -AU-R,P tuf- AD-F / P tuf -AD-R and A-P tuf -F / A-P tuf -R is primer PCR (table 2), obtains leuA-U and leuA-D and P tuf DNA fragment, seamlessly cloned with linearized plasmid pK18mobsacB to construct recombinant plasmid pK18mobsacB-P tuf -leuA.

[0046] The strain WL-8 genome and pDXW-8 plasmid were used as templates respectively, and P tac -BU-F / P tac -BU-R,P tac- BD-F / P tac -BD-R and B-P tac -F / B-P tac -R is primer PCR (table 2), obtains ilvBNC-U and ilvBNC-D and P tac DNA fr...

Embodiment 3

[0053] Construction of embodiment 3 recombinant bacterial strain WL-10

[0054] The correct promoter was verified in Example 3 to replace the plasmid pK18mobsacB-P tac -leuA and pK18mobsacB-P tac -ilvBNC transforms WL-8 competent cells by electroporation in turn, and replaces the leuA gene and ilvBNC gene promoters in turn with P tac Promoter, with primers A-P tac -F / P tac AD-R, B-P tac -F / P tac BD-R colony PCR was used to verify a single colony and screen for correct transformants. The recombinant strain obtained by screening was named WL-10.

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Abstract

The invention relates to a method for strengthening capability of synthesizing L-leucine from pyruvic acid in corynebacterium glutamicum, and belongs to the field of genetic engineering. According tothe invention, a gene engineering method is applied, and in order to regulate and control an L-leucine anabolic flux, sequence sites of an ilvBNC operon and an leuA gene promoter are replaced by a Ptuf promoter; and meanwhile, the key enzyme gene isopropyl malate synthetase IPMS is overexpressed to further strengthen the anabolic flux of synthesizing leucine from pyruvic acid. An efficient L-leucine synthesis pathway is constructed, and the synthesis flux of L-valine in L-leucine producing bacteria of corynebacterium glutamicum is weakened. A recombinant strain WL-14 produces 28.47+ / -0.36 g / Lof leucine, which is 56.8% higher than leucine yield of an original strain WL-8; and the valine accumulation amount in the strain WL-14 is reduced to 1.78+ / -0.21 g / L.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant Corynebacterium glutamicum that strengthens the ability of pyruvate to synthesize L-leucine and its application. Background technique [0002] As one of the eight essential amino acids, L-leucine, together with L-valine and L-isoleucine, has a branched methyl side chain structure and is collectively referred to as branched-chain amino acids. L-leucine has a variety of physiological functions and is widely used in food industry, feed industry and pharmaceutical industries. At the same time, the amount of L-leucine in amino acid intravenous infusion is increasing day by day. It is one of the indispensable raw materials used in clinical amino acid compound infusion, and it plays an active role in maintaining the nutritional needs of critically ill patients and saving their lives. [0003] The production methods of L-leucine mainly include extraction method, chemical...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12N15/90C12N15/54C12N15/52C12P13/06C12R1/15
CPCC12N15/52C12N9/1025C12N9/93C12N9/1096C12N15/77C12N15/902C12P13/06C12Y203/03013C12Y604/01001C12Y206/01002
Inventor 张伟国史可王颖妤徐建中章洁颖朱晗
Owner JIANGNAN UNIV
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