Protein with L-proline efflux function and application thereof

A technology of proline and hydroxyproline, which is applied in the fields of molecular biology and bioengineering, can solve the problems of L-proline efflux protein and increase the production of L-proline, and achieves easy popularization and application and increased concentration Effect

Pending Publication Date: 2022-07-19
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, in the process of L-proline production, there is no report of using L-proline efflux proteins to increase the production of L-proline

Method used

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  • Protein with L-proline efflux function and application thereof
  • Protein with L-proline efflux function and application thereof
  • Protein with L-proline efflux function and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1: Screening of Corynebacterium glutamicum for L-proline efflux protein in Corynebacterium glutamicum ATCC13869 The inventors screened Corynebacterium glutamicum for L-proline through a genome-scale membrane transporter inhibition library efflux protein. The cas9 gene of pCas9 (LIU, Jiao, et al. Development of a CRISPR / Cas9 genome editing toolbox for Corynebacterium glutamicum. Microbial cell factories, 2017, 16.1:205.) was mutated by D10A and H840A, and the Bsa in the plasmid backbone was mutated at the same time. The I restriction site was removed to obtain the pdCas9 plasmid; then from pnCas9(D10A)-AID-gRNA-ccdB TS (WANG, Yu, et al. Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum. Biotechnology and bioengineering, 2019, 116: 3016-3029) The same as that of the amplified gRNA-ccdB expression cassette cloned into pdCas9 position, to obtain a CRISPRi plasmid pdCas9 gRNA-ccdB that can be effi...

Embodiment 2

[0096] Example 2. Identification of L-proline efflux proteins of Corynebacterium glutamicum in Corynebacterium glutamicum ATCC13869

[0097] (1) Construction of knockout, complement and overexpression strains of thrE gene in SZCgP1 strain

[0098] The inventors constructed knockout mutants based on CRISPR / Cas9 genome editing technology. The pCas9 (LIU, Jiao, et al. Development of a CRISPR / Cas9 genome editing toolbox for Corynebacterium glutamicum. Microbial cell factories, 2017, 16.1:205.) plasmid was used as the template, and cas9-1 / cas9-2 were used as primers. Introduced operator lacO mutation (TGTGTGGAATTGTGAGCG GATA ACAATTTCA CACA is mutated to TGTGTGGAATTGTGAGCG CTC ACAATTTCACACA) and RBS mutation (AA AGG) AGTTGAGA mutated to AAAGG CACCCGAT ), amplify the fragment containing cas9; then use pnCas9(D10A)-AID-gRNA-ccdB TS (WANG, Yu, et al. Expanding targeting scope, editing w indow, and base transition capability of base editing in Corynebacterium glutamicum. Biotec...

Embodiment 3

[0112] Example 3. Application of L-proline efflux protein in L-proline production in Corynebacterium glutamicum ATCC13032 strain

[0113] Firstly, an L-proline-producing strain SZCgP3 was constructed on the basis of Corynebacterium glutamicum ATCC13032 strain, that is, the G149K mutation of proB gene was introduced, and the codon was mutated from GGT to AAG (see CN101084312A). The construction of ThrE overexpression strain is similar to that of Corynebacterium glutamicum 13869 strain. The thrE was overexpressed on the pEC-ccdB plasmid derived from the pEC-XK99E plasmid, the pEC-ccdB plasmid was digested with Bsa I, and the thrE2 gene was amplified with thrE-5 / thrE-6 using the genome of Corynebacterium glutamicum ATCC13032 as a template Fragment, the amino acid sequence of the protein encoded by the thrE2 gene is shown in SEQ ID NO: 2, and the above two fragments are cloned and connected by Novozan's one-step recombination kit to obtain the pEC-thrE2 plasmid, and the plasmid ma...

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Abstract

The invention discloses a protein with an L-proline excretion function and application of the protein. The invention relates to a method for producing L-proline or hydroxyproline by using protein ThrE. The L-proline is produced by enhancing the activity of a polypeptide with an L-proline efflux function in a strain for producing L-proline. Alternatively, the activity of a polypeptide having an L-proline efflux function in an L-proline producing host cell is weakened and the activity of proline hydroxylase is enhanced to produce hydroxyproline.

Description

technical field [0001] The invention belongs to the fields of molecular biology and bioengineering, and in particular relates to a new use of the threonine efflux protein ThrE of Corynebacterium glutamicum as an L-proline efflux protein, and the production of L-proline by using the protein ThrE Methods of Acids and Derivatives thereof. 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, the commonly used industrial fermentation strains are Coryne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/34C12N1/21C12N15/70C12N15/77C12P13/24C12R1/19C12R1/15
CPCC07K14/34C12P13/24C12N15/70C12N15/77C12R2001/15C12R2001/19C12N15/74
Inventor 孙际宾刘娇郑平刘莫识王钰周文娟孙冠男
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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