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Bacterial strain for producing L-proline as well as construction method and application of bacterial strain

A technology for the production of strains and construction methods, which is applied in the fields of molecular biology and bioengineering, can solve the problems of limited increase in acid production of strains and few targets for transformation, and achieve the effect of facilitating popularization and application and increasing production

Pending Publication Date: 2022-08-09
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

It can be seen that there are few existing transformation targets, and the improvement of the acid production level of the strain is limited. There is an urgent need to discover new transformation targets in this field in order to further increase the L-proline production of the strain and enhance the application value of the strain

Method used

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  • Bacterial strain for producing L-proline as well as construction method and application of bacterial strain
  • Bacterial strain for producing L-proline as well as construction method and application of bacterial strain
  • Bacterial strain for producing L-proline as well as construction method and application of bacterial strain

Examples

Experimental program
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Effect test

Embodiment 1

[0079] Example 1. Screening of membrane transporters whose expression weakening can improve L-proline production in Corynebacterium glutamicum

[0080] The invention selects the membrane transporter which can improve the L-proline production of Corynebacterium glutamicum after weakening the gene expression through the weakened library of the whole genome scale membrane transporter gene. The Cas9 gene of pCas9 (LIU, Jiao, et al. Development of a CRISPR / Cas9 genome editing toolbox for Corynebacterium glutamicum. Microbialcell factories, 2017, 16.1:205.) was mutated by D10A and H840A, and the Bsa I in the plasmid backbone was mutated at the same time. Removal of the restriction site to obtain pdCas9 plasmid; then from pnCas9(D10A)-AID-gRNA-ccdB TS (WANG, Yu, et al. Expanding targeting scope, editing window, and base transitioncapability of base editing in Corynebacterium glutamicum. Biotechnology and bioengineering, 2019, 116: 3016-3029) Amplification of the gRNA-ccdB expression ...

Embodiment 2

[0086] Example 2. Membrane transporter gene knockout for increased L-proline production

[0087] (1) Construction of Recombinant Vector for Knockout of Corynebacterium glutamicum Membrane Transporter Gene

[0088]According to the reported genome sequence of Corynebacterium glutamicum ATCC13869, using the ATCC13869 genome as a template, 060563805.1-UF / 060563805.1-UR and 060563805.1-DF / 060563805.1-DR, -DR is a primer, PCR amplification of WP_060563805.1, WP_060563806.1 encoding the upstream and downstream homology arms of gene knockout; at the same time, pK18-1 / 2 primer is used to amplify the plasmid backbone of pK18mobsacB (GenBank: FJ437239.1). After the above-mentioned corresponding three PCR fragments were recovered, cloned and ligated by Novozan's one-step recombination kit to obtain recombinant vectors pK18-1 and pK18-2 encoding gene knockout of WP_060563805.1 and WP_060563806.1, respectively. The primer sequences used above are shown in Table 3.

[0089] table 3

[009...

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PUM

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Abstract

The invention discloses a strain for producing L-proline as well as a construction method and application of the strain. Through screening, the inactivation of the two membrane transporters can improve the yield of the L-proline in the L-proline producing strain. The discovery of the invention can provide new thoughts and targets for technicians in the field to construct the L-proline production strain, that is, not only concern on the transformation of the L-proline synthesis route, but also increase the yield of the strain through inactivation of the membrane transporter, thereby providing reference for constructing the production strain with higher yield of L-proline.

Description

technical field [0001] The invention belongs to the field of molecular biology and bioengineering, in particular to the application of membrane transporter in the production of L-proline, a strain for producing L-proline, a construction method and application of 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. Chemical method has gradually lost the market due to serious pollution and high cost of chemical extraction method. It has become the most widely used method in industry today due to its advantages of low environmental pollution and so on. At present, commonly used industrial fermentation strains include Corynebacterium and Escherichia, commonly used Escherichia such as Escherichia coli, commonly used Corynebacterium such as Cory...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/77C12P13/24C12R1/15
CPCC07K14/34C12N15/77C12P13/24Y02E50/10
Inventor 郑平刘娇孙际宾刘莫识周文娟马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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