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Corynebacterium glutamicum high-producing l-proline and method for high-producing l-proline

A technology of Corynebacterium glutamicum and proline, applied in the fields of molecular biology and bioengineering, can solve the problems of low L-proline yield and the like, and achieve the effects of facilitating popularization and application, and improving transformation rate and production intensity

Active Publication Date: 2022-07-12
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently engineered strains have low L-proline production

Method used

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  • Corynebacterium glutamicum high-producing l-proline and method for high-producing l-proline
  • Corynebacterium glutamicum high-producing l-proline and method for high-producing l-proline
  • Corynebacterium glutamicum high-producing l-proline and method for high-producing l-proline

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, insert the proB of artificial copy V150N The AC operon and blocking L-proline degradation increase L-proline production

[0050] On the basis of Corynebacterium glutamicum ATCC13032 (Gene ID: 2830649), the SLCgP54 (CN112111469B, ProB introduces V150N mutation) strain that relieves the L-proline feedback inhibition of γ-glutamyl kinase ProB as the starting strain, the present invention is defined as PRO-1 strain. At the same time, the inventor mutated the pyc promoter of the endogenous pyruvate carboxylase gene of Corynebacterium glutamicum in the early stage, and obtained a series of promoter mutants with significantly improved promoter activity, wherein P pyc-20 (the sequence is shown in SEQ ID NO: 8) is the most active promoter, and the expression intensity is 16.1 times higher than that of the wild-type promoter.

[0051] The present invention further enhances the L-proline synthesis by enhancing the expression of the three key enzymes ProB, ProA and ...

Embodiment 2

[0067] Example 2. Enhance the expression of glutamate dehydrogenase gdh gene to improve L-proline production

[0068] L-glutamic acid is the precursor of L-proline synthesis, in order to enhance the supply of glutamic acid precursor to increase the production of L-proline in order to increase the activity of glutamate dehydrogenase, the present invention adopts the enhancing activity of the gene itself. Promoter mutation is directly modified in chromosomal in situ to achieve the enhanced expression of the target gene, that is, the promoter mutant with increased expression intensity enhances the expression of the glutamate dehydrogenase gdh (gene number Cgl2079) gene. The inventors mutated the endogenous glutamate dehydrogenase gdh promoter of Corynebacterium glutamicum in the early stage, and obtained a series of promoter mutants with significantly improved promoter activity, among which P gdh-16 , P gdh-23 , P gdh-26 , P gdh-29 Promoter mutants (sequences are shown in SEQ ...

Embodiment 3

[0078] Embodiment 3, enhance the expression of pyruvate carboxylase pyc gene to improve L-proline production

[0079] In order to enhance the activity of pyruvate carboxylase and then strengthen the supply of oxaloacetic acid to improve the L-proline production, the present invention adopts the gene's own enhanced activity promoter mutation to directly transform in situ on the chromosome to achieve the expression enhancement of the target gene, that is, Promoter mutants with increased expression strength enhance the expression of the pyruvate carboxylase pyc (Cgl0689) gene. The inventors mutated the endogenous pyruvate carboxylase pyc promoter of Corynebacterium glutamicum in the early stage, and obtained a series of promoter mutants with significantly improved promoter activity, among which P pyc-13 , P pyc-16 , P pyc-20 The promoter (sequences are shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8), the expression intensity was 8.9, 12.1, and 16.1 times higher than that of ...

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Abstract

The invention provides a Corynebacterium glutamicum for producing L-proline, and a method for producing L-proline by using the strain. The L-proline-producing Corynebacterium glutamicum constructed by the present invention, wherein proline dehydrogenase / pyrrole-5-carboxylate dehydrogenase PutA is inactivated, glutamate kinase ProB, glutamate-5- Enhanced activity of semialdehyde dehydrogenase ProA, pyrrole-5-carboxylate dehydrogenase ProC, pyruvate carboxylase Pyc, glyceraldehyde-3-phosphate dehydrogenase GapN, L-proline efflux protein ThrE or SerE, The L-glutamate efflux protein MscCG is inactivated, and the L-proline yield, transformation rate and production intensity of the obtained strain are significantly improved compared with the starting strain, which can reduce the production cost of L-proline.

Description

technical field [0001] The invention belongs to the fields of molecular biology and bioengineering, and in particular relates to a L-proline-producing Corynebacterium glutamicum and a method for producing L-proline and derivatives thereof by using the strain. Background technique [0002] L-proline, a naturally occurring non-essential amino acid in the human body, has a wide range of applications in clinical, biological materials and industry. The production methods of L-proline mainly include chemical method and fermentation method. Due to the serious pollution and high cost of chemical extraction method, it has gradually lost the market. Microbial fermentation method has low production cost, high production intensity, high specificity and environmental pollution. It has become the most widely used method in industry today. At present, commonly used industrial fermentation strains include Corynebacterium and Escherichia, commonly used Escherichia such as Escherichia coli, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P13/24C12R1/15
CPCC12N15/52C12N9/0026C12N9/1217C12N9/0008C12N9/0028C12N9/0016C12N9/93C07K14/34C12P13/24C12Y105/99008C12Y207/02011C12Y604/01001C12Y207/02013C12Y102/01041C12Y105/01012C12Y104/01002C12Y102/01009C12Y102/01012C12Y102/01013C12Y102/01059C12Y102/07006
Inventor 郑平刘娇孙际宾刘莫识周文娟孙冠男王钰石拓郭轩马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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