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L-lysine high-yield strain and its construction method and application

A construction method, lysine technology, applied in the field of L-lysine high-yield strains, can solve the problems of unknowable influence of strains, huge difficulty, and difficulty in small increase

Active Publication Date: 2022-04-22
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

[0003] As we all know, cells are a complex collection of metabolic networks. During the synthesis of a certain compound, people usually modify the pathways directly related to its metabolism (including substrate transportation, metabolic pathways, product transportation, etc.) instead of modifying them. Other unrelated pathways in cells, because the impact of changes in unrelated pathways on strains is unknown, and there are many metabolic pathways in cells, it is necessary to dig out possible lysine production-related pathways from many pathways. It is very difficult to modify the point, especially when the current lysine industrial strain has reached a high level, it is very difficult to further improve, even a small increase

Method used

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  • L-lysine high-yield strain and its construction method and application
  • L-lysine high-yield strain and its construction method and application
  • L-lysine high-yield strain and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1. Attenuation and inactivation of the hisB gene in Escherichia coli BW25113

[0099] Escherichia coli genome rapid manipulation technology using CRISPR / Cas9 【Zhao, D., et al. (2017). Science Rep 7(1):16624.], using the primers HisB-F and HisB-Ra in Table 1, adding the N20PAM fragment to the chloramphenicol resistance gene [Zhao, D., et al. (2017). Sci Rep 7(1):16624.] was used as a template and amplified by PCR to obtain the recombinant fragment hisBgtg for replacing the start codon atg of the hisB gene (SEQ ID NO: 1) of BW25113 with gtg. The plasmid pCAGO [Zhao, D., et al. (2017). Sci Rep 7(1):16624.] Transformed into BW25113 to obtain strain BW25113 (pCAGO). Prepare BW25113 (pCAGO) competent cells for electrotransformation. The medium used for the preparation of competent cells is: 10g / L peptone, 10g / L sodium chloride, 5g / L yeast powder, 10g / L glucose, and the final concentration is 0.1 mM IPTG. Transform the above-mentioned recombinant fragment hi...

Embodiment 2

[0103] Example 2. Attenuation and inactivation of the hisD gene in E. coli

[0104] Using the CRISPR / Cas9-based Escherichia coli genome rapid manipulation technology mentioned in Example 1, the hisD gene (SEQ ID NO:2) start codon of Escherichia coli BW25113 was replaced from atg to gtg, and the primers used were listed in Table 2 In HisD-F and HisD-Ra, the constructed strain was named BW25113-hisDgtg; in addition, using HisD-F and HisD-Rn in Table 2, using the same genome rapid operation technology, the hisD gene (SEQ ID NO :2) to edit the 8th to 14th bases, the 7th to 9th bases of the hisD gene after editing are TAA, forming a stop codon, so that the hisD gene cannot be expressed normally, thereby causing the inactivation of hisD, and the obtained The strain was named BW25113ΔhisD.

[0105] Table 2. Primers for constructing the recombinant fragments used for the weakened expression and inactivation of the hisD gene in Escherichia coli

[0106]

[0107]

Embodiment 3

[0108] Example 3. Corynebacterium glutamicum B253 (GeneBank accession number CP010451) and 13032 (lysC T311I Knockout of hisB, hisN, hisD genes in )

[0109] First, using the method reported in the literature, the threonine codon at position 311 of the aspartokinase (lysC) gene of Corynebacterium glutamicum 13032 was replaced with an isoleucine codon by two-step recombination [ Ohnishi, J., et al. (2002). Appl Microbiol Biotechnol 58 (2): 217-223], obtained the strain 13032 (lysC T311I ).

[0110] Using CRISPR / Cas9 and activation-induced cytidine deaminase (AID) reported in the literature [Wang, Y., et al. (2018). Metab Eng47:200-210.], for B253 and 13032 (lysC T311I ) hisB, hisN, hisD genes were inactivated respectively.

[0111] The hisB gene (SEQ ID NO: 3) and 13032 (lysC) in B253 were synthesized in Table 3 respectively T311I ) in the hisB gene (SEQ ID NO:6) the 238th base C is mutated to T primers hisBF and hisBR, which are used to convert the hisN gene in B253 (SEQ...

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Abstract

The invention discloses a method for constructing an L-lysine producing bacterial strain, the method comprising making imidazole glycerol phosphate dehydratase, histidinol phosphorylase, histidinaldehyde / group in the lysine producing bacterial strain One or more of the amino alcohol dehydrogenases cannot function normally in the cell. The invention also discloses an L-lysine producing strain constructed by the method and a method for preparing L-lysine by using the strain. The lysine production of the L-lysine producing bacterial strain constructed by the invention is significantly improved, so that the cost can be significantly reduced.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a high-producing strain of L-lysine, a construction method and application of the high-producing strain. Background technique [0002] L-Lysine is an important essential amino acid in human and animal nutrition and plays a very important role in industries such as medicine, health, food, animal feed and cosmetics. The global lysine market will reach $6 billion by 2018, according to Global Industry Analysis (GIA)'s Amino Acids: A Global Strategic Business Report. L-lysine is mainly produced by microbial fermentation, and known microorganisms that can produce amino acid include Escherichia, Corynebavterium, Brevibacterium and the like. In recent years, the continuous improvement of raw material processing, fermentation process and separation and extraction process has reduced the cost of amino acid fermentation production to a certain extent, and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/74C12P13/08C12R1/19C12R1/15C12R1/13C12R1/07C12R1/425C12R1/63
CPCC12P13/08C12N15/70C12N15/74C12N9/88C12N9/16C12N9/0006C12Y402/01019C12Y301/03015
Inventor 孙际宾李庆刚周文娟郑平马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI