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Corynebacterium glutamicum for high yield of L-proline and method for high yield of 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: 2022-03-01
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 for high yield of L-proline and method for high yield of L-proline
  • Corynebacterium glutamicum for high yield of L-proline and method for high yield of L-proline
  • Corynebacterium glutamicum for high yield of L-proline and method for high yield of L-proline

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Experimental program
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Effect test

Embodiment 1

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

[0050] Based on Corynebacterium glutamicum ATCC13032 (Gene ID: 2830649), the SLCgP54 (CN112111469B, ProB introduces the V150N mutation) bacterial strain that releases the L-proline feedback inhibition of γ-glutamyl kinase ProB is the starting strain, and the present invention is defined as PRO-1 strain. At the same time, the inventors mutated the pyc promoter of the endogenous pyruvate carboxylase gene pyc in Corynebacterium glutamicum in the early stage, and obtained a series of promoter mutants with significantly improved promoter activity, among which P pyc-20 (The sequence is shown as 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 L-proline synthesis by enhancing the expression of three key enzymes ProB...

Embodiment 2

[0067] Embodiment 2, enhancing the expression of glutamate dehydrogenase gdh gene improves L-proline output

[0068] L-glutamic acid is the precursor of L-proline synthesis, in order to enhance the activity of glutamate dehydrogenase and then enhance the supply of glutamic acid precursor to increase the production of L-proline, the present invention adopts the enhanced activity of the gene itself The promoter mutation is directly modified in situ on the chromosome to achieve 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 as shown in SEQ ID NO: 1...

Embodiment 3

[0078] Example 3, Enhancing 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 oxaloacetate to increase the production of L-proline, the present invention adopts the enhanced activity promoter mutation of the gene itself to directly modify the expression of the target gene in situ on the chromosome, that is, Promoter mutants with increased expression strength were achieved by enhancing 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 expression intensity of the promoter (sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8) is 8.9, 12.1, and 16.1 times higher than that of th...

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Abstract

The invention provides corynebacterium glutamicum for producing L-proline and a method for producing the L-proline by using the strain. According to the corynebacterium glutamicum for producing the L-proline, proline dehydrogenase / pyrrole-5-carboxylate dehydrogenase PutA is inactivated, activity of glutamate kinase ProB, glutamic acid-5-semialdehyde dehydrogenase ProA, pyrrole-5-carboxylate dehydrogenase ProC, pyruvate carboxylase Pyc, glyceraldehyde-3-phosphate dehydrogenase GapN, L-proline efflux protein ThrE or SerE is enhanced, and activity of L-proline is enhanced. The L-glutamic acid efflux protein MscCG is inactivated, the L-proline yield, the conversion rate and the production intensity of the obtained strain are remarkably improved compared with those of an original strain, and the production cost of the L-proline can be reduced.

Description

technical field [0001] The invention belongs to the fields of molecular biology and bioengineering, and in particular relates to a Corynebacterium glutamicum producing L-proline and a method for producing L-proline and derivatives thereof by using the strain. Background technique [0002] L-proline is a naturally occurring non-essential amino acid in the human body, which has a wide range of applications in clinical, biomaterials 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. Small and other advantages and become the most widely used method in industry today. At present, the commonly used industrial fermentation strains include Corynebacterium and Escherichia, commonly used Escherichi...

Claims

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Application Information

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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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