CN117230101A

CN117230101A - Corynebacterium glutamicum production strain and construction method and application thereof

Info

Publication number: CN117230101A
Application number: CN202311083134.XA
Authority: CN
Inventors: 徐庆阳; 王锐麒
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2023-08-28
Filing date: 2023-08-28
Publication date: 2023-12-15

Abstract

The invention provides a corynebacterium glutamicum production strain, a construction method and application thereof, which takes corynebacterium glutamicum as a chassis strain, weakens the expression of alpha-ketoglutarate dehydrogenase cgl1129, acyl-coa carboxylase cgl0700, fatty acid synthase cgl0836, cgl2495 and malate dehydrogenase cgl2380 in the chassis strain, enhances the expression of glutamate dehydrogenase cgl2079, glutamate synthase cgl0184, cgl0185 and phosphoenolpyruvate carboxykinase cgl2863, and knocks out isocitrate lyase cgl2331 genes to obtain a target strain after transformation success.

Description

Corynebacterium glutamicum production strain and construction method and application thereof

Technical Field

The invention relates to the field of biotechnology production, in particular to corynebacterium glutamicum production bacteria, a construction method and application thereof.

Background

Glutamic acid is an alpha-amino acid constituting a protein, and is present in a large amount in cereal proteins and animal brains, and plays an important role in the metabolic process of organisms. The glutamic acid has wide application, becomes the largest amino acid product in the world, has annual output of approximately 300 ten thousand tons, has a yield value of over 400 hundred million, and has wide market prospect and huge potential.

Glutamate can be synthesized in a variety of ways, the two most predominant of which are: firstly, directly generating the alpha-ketoglutarate into the glutamic acid through the catalysis of the glutamic acid dehydrogenase; secondly, alpha-ketoglutarate is combined with glutamine to generate glutamic acid through the catalysis of glutamate synthase. In addition, it can be produced by other metabolic pathways such as glucose or pyruvate. There are two main methods for producing glutamic acid: chemical synthesis and microbial fermentation. The chemical synthesis is to convert the related raw materials into glutamic acid through chemical reaction, but the method has the problems of high cost, complicated steps and the like. Microbial fermentation is the method currently mainly used, and glutamic acid is produced by fermentation using Corynebacterium glutamicum or mutant strains. The traditional glutamic acid production strain is complicated and unstable in molecular transformation, so that an efficient, convenient and stable engineering strain is urgently needed.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a corynebacterium glutamicum producing strain.

The technical problem to be solved by the invention is to provide a construction method of the corynebacterium glutamicum production strain.

The technical problem to be solved by the invention is to provide the application of the corynebacterium glutamicum producing strain.

In order to solve the technical problems, the technical scheme of the invention is as follows:

construction method of corynebacterium glutamicum production strain and collection methodThe strain is modified by using the homologous recombination gene editing technology and metabolic engineering, and the specific modification strategy is as follows: corynebacterium glutamicum was used as a chassis strain by attenuating the alpha-ketoglutarate dehydrogenase cgl1129, acyl-CoA carboxylase in the chassis strain _cgl 0700. The expression of fatty acid synthase cgl0836, cgl2495 and malate dehydrogenase cgl2380 enhances the expression of glutamate dehydrogenase cgl2079, glutamate synthase cgl0184, cgl0185 and phosphoenolpyruvate carboxykinase cgl2863, and simultaneously knocks out isocitrate lyase cgl2331 genes to obtain target strains after successful transformation.

Preferably, the method for constructing corynebacterium glutamicum comprises using P _sod The promoter weakens cgl1129 gene expression; using P _sod The promoter weakens the cgl0700 gene expression; using P _sod The promoter weakens the cgl0836 and cgl2495 gene expression; using P _sod The promoter weakens cgl2380 gene expression; using P _tuf The promoter enhances the overexpression of the cgl2079 gene; using P _tuf Promoters enhance the overexpression of cgl0184 and cgl0185 genes; using P _tuf The promoter enhances the overexpression of the cgl2863 gene; the cgl2331 gene was knocked out.

Preferably, the chassis strain is Corynebacterium glutamicum ATCC13032 (C.glutamicum) in the above construction method of Corynebacterium glutamicum producing strain.

Preferably, in the construction method of the corynebacterium glutamicum, the nucleotide sequence of the encoding gene of the alpha-ketoglutarate dehydrogenase cgl1129 is shown as SEQ ID NO. 1.

Preferably, in the construction method of the corynebacterium glutamicum, the nucleotide sequence of the encoding gene of the acyl-CoA carboxylase cgl0700 is shown as SEQ ID NO. 2.

Preferably, in the construction method of the corynebacterium glutamicum, the nucleotide sequence of the fatty acid synthase cgl0836 encoding gene is shown as SEQ ID NO. 3; the nucleotide sequence of the fatty acid synthase cgl2495 coding gene is shown in SEQ ID NO. 4.

Preferably, in the construction method of the corynebacterium glutamicum, the nucleotide sequence of the coding gene of the malate dehydrogenase cgl2380 is shown as SEQ ID No. 5.

Preferably, the nucleotide sequence of the coding gene of the glutamate dehydrogenase cgl2079 is shown as SEQ ID NO. 6.

Preferably, in the construction method of the corynebacterium glutamicum, the nucleotide sequence of the gene encoding the glutamate synthase cgl0184 is shown as SEQ ID NO. 7; the nucleotide sequence of the coding gene of the glutamate synthase cgl0185 is shown as SEQ ID NO. 8.

Preferably, in the construction method of the corynebacterium glutamicum, the nucleotide sequence of the encoding gene of phosphoenolpyruvate carboxykinase cgl2863 is shown as SEQ ID NO. 9.

Preferably, in the construction method of the corynebacterium glutamicum, the nucleotide sequence of the coding gene of the isocitrate lyase cgl2331 is shown as SEQ ID No. 10.

All the genes are Corynebacterium glutamicum self genes.

Preferably, the method for constructing the corynebacterium glutamicum producer weakens the expression of the alpha-ketoglutarate dehydrogenase by: using P _sod The promoter weakens the expression of the cgl1129 gene and weakens the downward carbon flow of alpha-ketoglutarate in the tricarboxylic acid cycle; the P is _sod The nucleotide sequence of the promoter is shown as SEQ ID NO. 11.

Preferably, the method for constructing the corynebacterium glutamicum comprises the following steps of: using P _sod The promoter weakens the cgl0700 gene expression and weakens the flow of the competition branch of acetyl coenzyme A; the P is _sod The nucleotide sequence of the promoter is shown as SEQ ID NO. 11.

Preferably, the method for constructing the corynebacterium glutamicum producer, which weakens the expression of the fatty acid synthase, comprises the following steps: using P _sod The promoter weakens the expression of the cgl0836 and cgl2495 genes and weakens the flow of the competition branch of acetyl coenzyme A; the P is _sod The nucleotide sequence of the promoter is shown as SEQ ID NO. 11.

Preferably, the glutamic acid stickThe method for constructing the bacillus thuringiensis production strain weakens the expression of the malate dehydrogenase by the following steps: using P _sod The promoter weakens the expression of the cgl2380 gene and weakens the flow of a competitive branch of oxaloacetate; the P is _sod The nucleotide sequence of the promoter is shown as SEQ ID NO. 11.

Preferably, the method for constructing the corynebacterium glutamicum producer, the mode for enhancing the expression of glutamate dehydrogenase is as follows: using P _tuf The promoter strengthens the overexpression of the cgl2079 gene and enhances the carbon flux to glutamic acid; the P is _tuf The nucleotide sequence of the promoter is shown as SEQ ID NO. 12.

Preferably, the method for constructing the corynebacterium glutamicum producer, the mode for enhancing the expression of glutamate synthase is as follows: using P _tuf The promoters strengthen the overexpression of cgl0184 and cgl0185 genes and enhance the carbon flux to glutamate; the P is _tuf The nucleotide sequence of the promoter is shown as SEQ ID NO. 12.

Preferably, the method for constructing the corynebacterium glutamicum production strain comprises the following steps of: using P _tuf The promoter strengthens the overexpression of the cgl2863 gene and weakens the flow of a competitive branch of oxaloacetate; the P is _tuf The nucleotide sequence of the promoter is shown as SEQ ID NO. 12.

Preferably, the method for constructing the corynebacterium glutamicum producer comprises the following steps of: knockout of the cgl2331 gene reduces the carbon flux in the glyoxylate cycle.

A corynebacterium glutamicum, strain Glu01, uses P at the original cgl1129 gene locus _sod The promoter controls the weakening expression of the cgl1129 gene.

A corynebacterium glutamicum strain Glu02 is prepared from Glu01 as primary strain and P at primary cgl0700 gene locus _sod The promoter controls the weakened expression of the cgl0700 gene.

A corynebacterium glutamicum is a strain Glu03, which is a starting strain of Glu02 at the original cgl0836 and original cgl2495 gene loci P _sod Start-upThe genes cgl0836 and cgl2495 are attenuated and expressed by the promoters.

A corynebacterium glutamicum is a strain Glu04, which is a starting strain Glu03 at the original cgl2380 gene locus P _sod The promoter controls the weakening expression of the cgl2380 gene.

A corynebacterium glutamicum producing strain, strain Glu05, is prepared from strain Glu04 as initial strain and P at original cgl2079 gene locus _tuf The promoter controls the intensified expression of the cgl2079 gene.

A corynebacterium glutamicum is a strain Glu06, which is a starting strain Glu05, and uses a promoter for primary cgl0184 and primary cgl0185 and P at primary cgl0184 and primary cgl0185 gene sites _tuf Promoters control the enhanced expression of the cgl0185 and cgl0185 genes.

A corynebacterium glutamicum producing strain, strain Glu07, is prepared from strain Glu06 as starting strain by using P at original cgl2863 gene locus _tuf The promoter controls the intensified expression of the cgl2863 gene.

The corynebacterium glutamicum producing strain is strain Glu08, which is obtained by the construction method, and the strain Glu07 is taken as an original strain, the original cgl2331 gene is knocked out to obtain the strain Glu08, and the strain Glu08 is the target strain after the transformation is successful.

The corynebacterium glutamicum producing strain is applied to the production of glutamic acid.

Preferably, the corynebacterium glutamicum is used for producing glutamic acid by fermentation culture.

Preferably, the application of the corynebacterium glutamicum comprises the following specific steps of fermentation culture:

(1) Activating strains: inoculating the genetically engineered bacterium Glu08 with a loop to obtain 3-loop bacterial liquid from a glycerol bacterial retaining tube, uniformly dividing the liquid on a test tube inclined plane solid culture medium, and culturing the liquid in a 32 ℃ incubator for 16 hours;

(2) Seed culture: after the strain is activated, the strain is resuspended by using sterile normal saline, and the obtained strain solution is inoculated into a 5L stirring type bioreactor, and the culture temperature is 3The pH value of the culture is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution at 4 ℃, the dissolved oxygen value of the culture is maintained at 45% by adjusting the stirring speed or ventilation quantity, and the bacterial liquid OD is obtained _600nm The inoculation requirement is met when the time is 25;

(3) Fermentation culture: A5L mechanical stirring type fermentation tank is used, the inoculation amount is 20%, the culture temperature is 34 ℃, the culture pH is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 50% by adjusting the stirring rotation speed or ventilation, the glucose concentration in the tank is controlled to be less than or equal to 3g/L by feeding 80% glucose solution, and the fermentation period is less than or equal to 34h.

Preferably, in the application of the corynebacterium glutamicum, the test tube slant solid medium is LB solid medium.

Preferably, in the application of the corynebacterium glutamicum, the culture medium used for seed culture is: 25g/L glucose, 15g/L corn steep liquor dry powder, 15ml/L soybean meal hydrolysate and K ₂ HPO ₄ ·3H ₂ O 1.0g/L，MgSO ₄ ·7H ₂ O1.0g/L, and the balance of water.

Preferably, the culture medium used for the fermentation culture is: glucose 60g/L, KH ₂ PO ₄ 3g/L，MgSO ₄ ·7H ₂ O 2g/L，MnSO ₄ ·H ₂ O 20mg/L，FeSO ₄ 20mg/L，V _B1 0.5mg/L，V _H 0.1mg/L, 7g/L of yeast powder, 0.5g/L of methionine, 20g/L of corn steep liquor dry powder, 30mL/L of soybean meal hydrolysate, 2 drops of defoamer and the balance of water.

The above culture medium can be prepared by standard method.

The beneficial effects are that:

the corynebacterium glutamicum has clear background knowledge in the aspects of basic biology, molecular genetics and the like of the strain Glu08, has clear molecular mechanism of gene expression regulation and control, has better stability, mild fermentation condition, simple product separation and extraction, short fermentation period, strong product discharge capacity and obvious improvement of production efficiency in unit time, provides a new thought for industrial production of glutamic acid, and has practical application value compared with the existing corynebacterium glutamicum.

Drawings

FIG. 1 is a diagram of a construction method for preparing glutamic acid engineering bacteria.

FIG. 2 is a graph showing the productivity of strains Glu01 to Glu 08.

FIG. 3 is a graph showing the sugar acid conversion during fermentation of strain Glu 08.

FIG. 4 is a graph showing the cell mass and yield of Glu08 strain during fermentation.

Detailed Description

In order to enable those skilled in the art to better understand the technical scheme of the present invention, the technical scheme of the present invention will be further described in detail below with reference to the specific embodiments.

The percentage "%" referred to in the examples is the mass percentage, the percentage of the solution is the gram of the solute contained in 100mL, and the percentage between the liquids is the volume ratio of the solution at 25 ℃.

The chassis strain used in the construction of the genetically engineered bacterium for high glutamic acid production in the following examples is Corynebacterium glutamicum (C.glutamicum) ATCC13032; primers used in the construction process of the strain are shown in table 1, the strain and the plasmid are shown in table 2, the PCR amplification system of the target fragment is shown in table 3, and the overlapping PCR system is shown in table 4.

TABLE 1 primer sequences for construction of strains

TABLE 2 strains and plasmids

TABLE 3 PCR amplification System for fragments of interest

TABLE 4 overlap PCR amplification System

PCR reaction conditions (Prime STAR HS enzyme) of the systems shown in tables 3 and 4: pre-denaturation (95 ℃) for 5min; denaturation (98 ℃) for 10s, annealing ((Tm-3/5) ℃for 15s, extension at 72℃for 1min for about 1 kb) for 30 cycles; continuing to extend for 10min at 72 ℃; maintained (4 ℃ C.).

Example 1

Construction of Strain Glu01

Corynebacterium glutamicum (C.glutamicum) ATCC13032 was used as Chaetomium, and P was used at the genomic Cgl1129 locus _sod The promoter weakens the Cgl1129 gene, and Glu01 strain is constructed.

(1) Construction of pEC-XK99E-Cgl1129 plasmid

The target fragment Cgl1129 was obtained by PCR amplification using the Corynebacterium glutamicum (C.glutamicum) ATCC13032 genome as a template, designing the upstream primer Cgl1129-u and the downstream primer Cgl1129-d (both ends of the upstream and downstream primers contain homologous sequences at both ends of the ScaI and XbaI cleavage sites of about 20 bp).

pEC-XK99E plasmid (nucleotide sequence shown as SEQ ID NO. 13) was digested with ScaI and XbaI, recombined with fragment Cgl1129, transformed into E.coli DH 5. Alpha. Competent cells, plated on neomycin resistance and kana resistance plates at a concentration of 0.05mg/mL, and verified to screen single colonies carrying the plasmid. BHI medium was shake-cultured and plasmid pEC-XK99E-Cgl1129 was proposed.

(2)pK18mobsacB-Cgl1129*::P _sod cgl1129 vector construction

The genome of corynebacterium glutamicum (C.glutamicum) ATCC13032 is used as a template, cgl1129 upstream homology arm amplification primers Cgl1129 x-u-s and Cgl1129 x-u-a, downstream homology arm amplification primers Cgl1129 x-d-s and Cgl1129 x-d-a are used as amplification primers, upstream and downstream homology arm fragments are amplified, and plasmid pEC-XK99E-Cgl1129 is used as a template, P _sod Cgl1129-u and P _sod Cgl1120-d(P _sod The promoter on plasmid pEC-XK 99E) as an amplification primer, amplified with P _sod The cgl1129 gene fragment of the promoter was recovered.

With homology arms upstream and downstream of amplified Cgl1129 gene and with P _sod The Cgl1129 gene fragment of the promoter was used as template with Cgl1129 x-u-s and P _sod Cgl1120-d as primer, overlap PCR was performed to obtain overlap fragment Cgl 1129:: P _sod cgl1129。

The pK18mobsacB plasmid (nucleotide sequence shown in SEQ ID NO. 14) was digested with XbaI and HindIII and was double digested with the overlapping fragment Cgl 1129:: P _sod cgl1129 was recombined and transformed into E.coli DH 5. Alpha. Competent cells, plated on kanamycin resistance plates at a concentration of 0.05mg/mL, and verified to select single colonies harboring plasmids. BHI culture medium shake tube culture and plasmid extraction

pK18mobsacB-Cgl1129*::P _sod cgl1129。

(3) Construction of a Glu01 Strain by integration of a copy at the Cgl1129 site

The constructed plasmid pK18mobsacB-Cgl 1129:: P _sod cgl1129 was shock transformed into competent cells of ATCC13032, plated on kanamycin resistance plates at a concentration of 0.01mg/mL, and incubated at 32℃for 24 hours. And screening to obtain positive transformants, namely single colonies subjected to single exchange.

The single colony with single exchange is inoculated into a shaking tube and cultured at 32 ℃. 50. Mu.L of fermentation broth was applied to BHI plates containing 15% sucrose at 2h, 4h and 6h, respectively, and incubated at 32℃for 24h. The single colony is inoculated on a BHI plate containing 15% sucrose and a kanamycin resistance plate with the concentration of 0.01mg/mL, the single colony which grows on the BHI plate containing 15% sucrose and does not grow on the kanamycin resistance plate with the concentration of 0.01mg/mL is picked, and a positive transformant after colony PCR verification is the target strain.

Example 2

Construction of Strain Glu02

Glu01 was used as Chaetomium, and P was used at the genomic Cgl0700 locus _sod The promoter weakens the Cgl0700 gene, and a Glu02 strain is constructed.

(1) Construction of pEC-XK99E-Cgl0700 plasmid

The Glu01 genome is used as a template, an upstream primer Cgl0700-u and a downstream primer Cgl0700-d are designed, and a target fragment Cgl0700 (two ends of the upstream primer and the downstream primer comprise homologous sequences at two ends of a ScaI enzyme cutting site and an XbaI enzyme cutting site which are about 20 bp) is obtained through PCR amplification.

The pEC-XK99E plasmid was digested with ScaI and XbaI, recombined with fragment Cgl0700, transformed into E.coli DH 5. Alpha. Competent cells, plated on neomycin resistance and Cana resistance plates at a concentration of 0.05mg/mL, and verified to select single colonies carrying the plasmid. BHI medium was shake-incubated and plasmid pEC-XK99E-Cgl0700 was proposed.

(2)pK18mobsacB-Cgl0700*::P _sod cgl0700 vector construction

The Glu01 genome is used as a template, upstream homology arm amplification primers Cgl0700 x-u-s and Cgl0700 x-u-a of a Cgl0700 gene and downstream homology arm amplification primers Cgl0700 x-d-s and Cgl0700 x-d-a are used as amplification primers to amplify upstream homology arm fragments and downstream homology arm fragments, and a plasmid pEC-XK99E-Cgl0700 is used as a template,

P _sod cgl0700-u and P _sod Cgl0700-d(P _sod The promoter on plasmid pEC-XK 99E) as an amplification primer, amplified with P _sod The cgl0700 gene fragment of the promoter was recovered.

With homology arms upstream and downstream of amplified Cgl0700 gene and with P _sod The Cgl0700 gene fragment of the promoter was used as a template with Cgl0700 x-u-s and P _sod Cgl0700-d was used as primer, overlap PCR was performed to obtain overlap fragment Cgl 0700:: P _sod cgl0700。

The pK18mobsacB plasmid was digested with XbaI and HindIII and was combined with the overlap Cgl 0700:: P _sod cgl0700 was recombined and transformed into E.coli DH 5. Alpha. Competent cells, plated on kanamycin-resistant plates at a concentration of 0.05mg/mL, and verified to select single colonies carrying plasmids. BHI medium shake tube culture, and plasmid pK18mobsacB-Cgl 0700:: P _sod cgl0700。

(3) Construction of a single copy of Glu02 Strain at the Cgl0700 locus

The constructed plasmid pK18mobsacB-Cgl 0700:: P _sod The cgl0700 was transformed into Glu01 competent cells by electric shock, plated on kanamycin-resistant plates at a concentration of 0.01mg/mL, and incubated at 32℃for 24 hours. And screening to obtain positive transformants, namely single colonies subjected to single exchange.

Example 3

Construction of Strain Glu03

Glu02 was used as Chaetomium, and P was used at the genomic original Cgl0836 locus and original Cgl2495 _sod The promoter weakens the Cgl0836 gene and the Cgl2495 gene, and the Glu03 strain is constructed.

(1) Construction of pEC-XK99E-Cgl0836 plasmid

The Glu02 genome is used as a template, an upstream primer Cgl0836-u and a downstream primer Cgl0836-d are designed, and a target fragment Cgl0836 (the two ends of the upstream primer and the downstream primer comprise homologous sequences at two ends of ScaI and XbaI cleavage sites with about 20 bp) is obtained through PCR amplification.

The pEC-XK99E plasmid was digested with ScaI and XbaI, recombined with the fragment Cgl0836, transformed into E.coli DH 5. Alpha. Competent cells, plated on neomycin resistance and kana resistance plates at a concentration of 0.05mg/mL, and verified to select single colonies carrying the plasmid. BHI medium was shake-cultured and plasmid pEC-XK99E-Cgl0836 was proposed.

(2)pK18mobsacB-Cgl0836*::P _sod cgl0836 vector construction

The Glu02 genome is used as a template, cgl0836 upstream homology arm amplification primers Cgl 0836-u-s and Cgl 0836-u-a and downstream homology arm amplification primers Cgl 0836-d-s and Cgl 0836-d-a are used as amplification primers to amplify upstream and downstream homology arm fragments, plasmid pEC-XK99E-Cgl0836 is used as a template,

P _sod cgl0836-u and P _sod Cgl0836-d(P _sod The promoter on plasmid pEC-XK 99E) as an amplification primer, amplified with P _sod The cgl0836 gene fragment of the promoter was recovered.

With homology arms upstream and downstream of the amplified Cgl0836 gene and with P _sod The Cgl0836 gene fragment of the promoter was used as template with Cgl0836 x-u-s and P _sod Cgl0836-d was used as primer and overlap PCR was performed to obtain overlap fragment Cgl 0836:: P _sod cgl0836。

The pK18mobsacB plasmid was digested with XbaI and HindIII and was digested with the overlapping fragment Cgl 0836:: P _sod cgl0836 was recombined and transformed into E.coli DH 5. Alpha. Competent cells, plated on kanamycin resistance plates at a concentration of 0.05mg/mL, and verified to select single colonies carrying plasmids. BHI medium shake tube culture, and plasmid pK18mobsacB-Cgl 0836:: P _sod cgl0836。

(3) Construction of a Glu03 Strain at Cgl0836

The constructed plasmid pK18mobsacB-Cgl0836*::P _sod cgl0836 was transformed into Glu02 competent cells by electric shock, plated on kanamycin-resistant plates at a concentration of 0.01mg/mL, and incubated at 32℃for 24h. And screening to obtain positive transformants, namely single colonies subjected to single exchange.

As Cgl2495 and Cgl0836 are isozymes, the band P is integrated at the genomic original Cgl2495 position in accordance with the above procedure _sod The Cgl2495 gene of the promoter (the corresponding primer is replaced, and the primer is shown in table 1) is used for obtaining the target strain Glu03. The selected strain was simultaneously preserved with 20% by volume of glycerol.

Example 4

Construction of Strain Glu04

Glu03 was used as Chaetomium, and P was used at the genomic Cgl2380 locus _sod The promoter attenuated the Cgl2380 gene and constructed Glu04 strain.

(1) Construction of pEC-XK99E-Cgl2380 plasmid

The Glu03 genome is used as a template, an upstream primer Cgl2380-u and a downstream primer Cgl2380-d are designed, and a target fragment Cgl2380 (the two ends of the upstream primer and the downstream primer comprise homologous sequences at two ends of ScaI and XbaI cleavage sites with about 20 bp) is obtained through PCR amplification.

pEC-XK99E plasmid was digested with ScaI and XbaI, recombined with fragment Cgl2380, transformed into E.coli DH 5. Alpha. Competent cells, plated on neomycin resistance and kana resistance plates at a concentration of 0.05mg/mL, and verified to screen single colonies carrying the plasmid. BHI medium was shake-cultured and plasmid pEC-XK99E-Cgl2380 was proposed.

(2)pK18mobsacB-Cgl2380*::P _sod cgl2380 vector construction

The Glu03 genome is used as a template, cgl2380 upstream homology arm amplification primers Cgl 2380-u-s and Cgl 2380-u-a, downstream homology arm amplification primers Cgl 2380-d-s and Cgl 2380-d-a are used as amplification primers to amplify upstream and downstream homology arm fragments, plasmid pEC-XK99E-Cgl2380 is used as a template,

P _sod cgl2380-u and P _sod Cgl2380-d(P _sod The promoter on plasmid pEC-XK 99E) as an amplification primer, amplified with P _sod The cgl2380 gene fragment of the promoter was recovered.

With homology arms upstream and downstream of the amplified Cgl2380 gene and with P _sod The Cgl0836 gene fragment of the promoter was used as template with Cgl2380 x-u-s and P _sod Cgl2380-d was used as a primer to perform overlap PCR to obtain an overlap fragment Cgl 2380:: P _sod cgl2380。

The pK18mobsacB plasmid was digested with XbaI and HindIII and was digested with the overlapping fragment Cgl 2380:: P _sod cgl2380 was recombined and transformed into E.coli DH 5. Alpha. Competent cells, plated on kanamycin resistance plates at a concentration of 0.05mg/mL, and verified to select single colonies harboring plasmids. BHI medium shake tube culture, and plasmid pK18mobsacB-Cgl 2380:: P _sod cgl2380。

(3) Construction of a copy of Glu04 Strain at Cgl2380 site

The constructed plasmid pK18mobsacB-Cgl 2380:: P _sod cgl2380 was transformed into competent cells of Glu03 by electric shock, plated on kanamycin-resistant plates at a concentration of 0.01mg/mL, and incubated at 32℃for 24h. And screening to obtain positive transformants, namely single colonies subjected to single exchange.

Example 5

Construction of Strain Glu05

Glu04 was used as Chaetomium, and P was used at the genomic Cgl2079 locus _tuf The promoter strengthens the Cgl2079 gene to construct Glu05 strain.

(1) Construction of pXT01-Cgl2079 plasmid

The Glu04 genome is used as a template, an upstream primer Cgl2079-u and a downstream primer Cgl2079-d are designed, and a target fragment Cgl2079 (the two ends of the upstream primer and the downstream primer contain homologous sequences at the two ends of HindIII and XbaI enzyme cutting sites with about 20 bp) is obtained through PCR amplification.

The pXT01 plasmid (nucleotide sequence shown as SEQ ID NO. 15) is digested with HindIII and XbaI, recombined with the fragment Cgl2079, transformed into competent cells of E.coli DH5 alpha, coated on a chloramphenicol resistance plate with a concentration of 0.05mg/mL, verified, and single colonies carrying plasmids are screened. BHI medium was shake-cultured and plasmid pXT01-Cgl2079 was proposed.

(2)pK18mobsacB-Cgl2079*::P _tuf construction of cgl2079 vector

The Glu04 genome is used as a template, cgl2079 upstream homology arm amplification primers Cgl 2079-u-s and Cgl 2079-u-a, downstream homology arm amplification primers Cgl 2079-d-s and Cgl 2079-d-a are used as amplification primers, upstream homology arm fragments and downstream homology arm fragments are amplified, and plasmid pXT01-Cgl2079 is used as a template, P _tuf Cgl2079-u and P _tuf Cgl2079-d(P _tuf Promoter on plasmid pXT 01) as an amplification primer, amplified with P _tuf The cgl2079 gene fragment of the promoter was recovered.

With homology arms upstream and downstream of amplified Cgl2079 gene and with P _tuf The Cgl2079 gene fragment of the promoter was used as template with Cgl2079 x-u-s and P _tuf Cgl2079-d was used as primer, overlap PCR was performed to obtain overlap fragment Cgl 2079:: P _tuf cgl2079。

The pK18mobsacB plasmid was double digested with XbaI and HindIII, and overlapping fragments

Cgl2079*::P _tuf Recombinant cgl2079 was transformed into E.coli DH 5. Alpha. Competent cells, plated on kanamycin-resistant plates at a concentration of 0.05mg/mL, and verified to select single colonies harboring plasmids. BHI medium shake tube culture and plasmid pK18mobsacB-Cgl 2079:: P _tuf cgl2079。

(3) Construction of a Glu05 Strain integrated at the Cgl2079 site

The constructed plasmid pK18mobsacB-Cgl 2079::: P _tuf cgl2079 was electric shock transformed into Glu04 competent cells, plated on 0.01mg/mL kanamycin resistant plates, and incubated at 32℃for 24h. And screening to obtain positive transformants, namely single colonies subjected to single exchange.

Example 6

Construction of Strain Glu06

Glu05 was used as Chaetomium, and P was used at genomic original Cgl0184 and original Cgl0185 sites _tuf The promoters strengthen the Cgl0184 and Cgl0185 genes, creating Glu06 strain.

(1) Construction of pXT01-Cgl184185 plasmid

The Glu05 genome is used as a template, an upstream primer Cgl184185-u and a downstream primer Cgl184185-d are designed, and a target fragment Cgl184185 (the two ends of the upstream primer and the downstream primer comprise homologous sequences at the two ends of HindIII and XbaI enzyme cutting sites with about 20 bp) is obtained through PCR amplification.

The pXT01 plasmid was double digested with HindIII and XbaI, recombined with the fragment Cgl184185, transformed into E.coli DH 5. Alpha. Competent cells, plated on chloramphenicol resistant plates at a concentration of 0.05mg/mL, and verified to select single colonies carrying the plasmid. BHI medium was shake-cultured and plasmid pXT01-Cgl184185 was proposed.

(2)pK18mobsacB-Cgl184185*::P _tuf cgl184185 vector construction

Glu05 genome is used as template, cgl184185 gene upstream homology is usedArm amplification primers Cgl184185 x-u-s and Cgl184185 x-u-a, downstream homology arm amplification primers Cgl184185 x-d-s and Cgl184185 x-d-a as amplification primers, upstream and downstream homology arm fragments were amplified, and plasmid pXT01-Cgl184185 was used as template, P _tuf Cgl184185-u and P _tuf Cgl184185-d(P _tuf Promoter on plasmid pXT 01) as an amplification primer, amplified with P _tuf The cgl184185 gene fragment of the promoter was recovered.

By using the homologous arm at the upstream and downstream of amplified Cgl184185 gene and carrying P _tuf The Cgl184185 gene fragment of the promoter was used as template with Cgl184185 x-u-s and P _tuf Cgl184185-d was used as primer and overlap PCR was performed to obtain overlap fragment Cgl 184185:: P _tuf cgl184185。

The pK18mobsacB plasmid was digested with XbaI and HindIII and was digested with the overlapping fragment Cgl 184185:: P _tuf cgl184185 was recombined and transformed into E.coli DH 5. Alpha. Competent cells, plated on kanamycin resistance plates at a concentration of 0.05mg/mL, and verified to select single colonies harboring plasmids. BHI medium shake tube culture and plasmid pK18mobsacB-Cgl 184185:: P _tuf cgl184185。

(3) Construction of a copy of Glu06 Strain at Cgl184185 site

The constructed plasmid pK18mobsacB-Cgl 184185:: P _tuf cgl184185 was transformed into competent cells of Glu05 by electric shock, plated on kanamycin-resistant plates at a concentration of 0.01mg/mL, and incubated at 32℃for 24h. And screening to obtain positive transformants, namely single colonies subjected to single exchange.

The single colony with single exchange is inoculated into a shaking tube and cultured at 32 ℃. 50. Mu.L of fermentation broth was applied to BHI plates containing 15% sucrose at 2h, 4h and 6h, respectively, and incubated at 32℃for 24h. The single colony is inoculated on a BHI plate containing 15% sucrose and a kanamycin resistance plate with the concentration of 0.01mg/mL, the single colony which grows on the BHI plate containing 15% sucrose and does not grow on the chloramphenicol resistance plate with the concentration of 0.01mg/mL is picked, and a positive transformant after colony PCR verification is the target strain.

Example 7

Construction of Strain Glu07

Glu06 was used as Chaetomium, and P was used at the genomic Cgl2863 locus _tuf The promoter strengthens the Cgl0184 gene to construct Glu07 strain.

(1) Construction of pXT01-Cgl2863 plasmid

The Glu06 genome is used as a template, an upstream primer Cgl2863-u and a downstream primer Cgl2863-d are designed, and a target fragment Cgl2863 (the two ends of the upstream primer and the downstream primer contain homologous sequences at the two ends of HindIII and XbaI enzyme cutting sites with about 20 bp) is obtained through PCR amplification.

The pXT01 plasmid was double digested with HindIII and XbaI, recombined with the fragment Cgl2863, transformed into E.coli DH 5. Alpha. Competent cells, plated on chloramphenicol resistant plates at a concentration of 0.05mg/mL, and verified to select single colonies carrying the plasmid. BHI medium was shake-cultured and plasmid pXT01-Cgl2863 was proposed.

(2)pK18mobsacB-Cgl2863*::P _tuf cgl2863 vector construction

The Glu06 genome is used as a template, cgl2863 upstream homology arm amplification primers Cgl 2863-u-s and Cgl 2863-u-a, downstream homology arm amplification primers Cgl 2863-d-s and Cgl 2863-d-a are used as amplification primers, upstream homology arm fragments and downstream homology arm fragments are amplified, and plasmid pXT01-Cgl2863 is used as a template, P _tuf Cgl2863-u and P _tuf Cgl2863-d(P _tuf Promoter on plasmid pXT 01) as an amplification primer, amplified with P _tuf The cgl2863 gene fragment of the promoter was recovered.

By using the homologous arm at the upstream and downstream of the amplified Cgl2863 gene and carrying P _tuf The Cgl2863 gene fragment of the promoter was used as template with Cgl2863 x-u-s and P _tuf Cgl2863-d was used as primer and overlap PCR was performed to obtain overlap fragment Cgl 2863:: P _tuf cgl2863。

The pK18mobsacB plasmid was digested with XbaI and HindIII and was digested with the overlapping fragment Cgl 2863:: P _tuf cgl2863 was recombined and transformed into E.coli DH 5. Alpha. Competent cells, plated on kanamycin resistance plates at a concentration of 0.05mg/mL, and verified to select single colonies carrying plasmids. BHI medium shake tube culture and plasmid pK18mobsacB-Cgl 184185:: P _tuf cgl184185。

(3) Construction of a Glu07 Strain by integration of a copy at the Cgl2863 site

The constructed plasmid pK18mobsacB-Cgl 2863:: P _tuf cgl2863 was shocked into competent cells of Glu06, plated on kanamycin-resistant plates at a concentration of 0.01mg/mL, and incubated at 32℃for 24h. And screening to obtain positive transformants, namely single colonies subjected to single exchange.

Example 8

Construction of Strain Glu08

Glu07 is taken as chassis bacteria, and a Cgl2331 gene is knocked out at a genomic original Cgl2331 site to construct Glu08 strain.

(1) Construction of pK18mobsacB- Δcgl2331 vector

With Glu07 genome as template, the upstream and downstream homology arm fragments are amplified and recovered by using Cgl2331-u-s and Cgl2331-u-a as amplification primers. Overlapping PCR was performed using Cgl2331-u-s and Cgl2331-d-a as primers to obtain an overlapping fragment Δcgl2331.

The pK18mobsacB plasmid was digested with XbaI and HindIII, recombined with the overlapping fragment Δcgl2331, transformed into E.coli DH 5. Alpha. Competent cells, plated on kanamycin resistance plates at a concentration of 0.05mg/mL, and verified to select single colonies carrying the plasmid. BHI medium was shake-cultured and plasmid pK18mobsacB- Δcgl2331 was set forth.

(2) Construction of a copy of Glu08 Strain integrated at the Cgl2331 site

The constructed plasmid pK18 mobsacB-. DELTA.cgl 2331 was transformed into Glu07 competent cells by electric shock, plated on 0.01mg/mL kanamycin resistance plates, and cultured at 32℃for 24 hours. And screening to obtain positive transformants, namely single colonies subjected to single exchange.

Example 9

Glutamic acid production by fermentation in 5L stirred bioreactor using each genetically engineered bacterium constructed in examples 1-8

1. Culture medium

(1) The seed culture medium is as follows: 25g/L glucose, 15g/L corn steep liquor dry powder, 15ml/L soybean meal hydrolysate and K ₂ HPO ₄ ·3H ₂ O 1g/L，MgSO ₄ ·7H ₂ O 1g/L。

(2) The fermentation medium is as follows:

glucose 60g/L, KH ₂ PO ₄ 3g/L，MgSO ₄ ·7H ₂ O 2g/L，MnSO ₄ ·H ₂ O 20mg/L，FeSO ₄ 20mg/L，VB ₁ 0.5mg/L, 0.1mg/L VH, 7g/L yeast powder, 0.5g/L methionine, 20g/L corn steep liquor dry powder, 30mL/L soybean meal hydrolysate, 2 drops of defoamer, and the balance of water, wherein pH=7.0.

2. Fermentation culture scheme

(1) Activating strains: c, inoculating 20% glycerol bacteria-retaining tube of Corynebacterium glutamicum Glu 01-Glu 08 strain at-80 ℃ into slant for activating culture under the culture condition of 32 ℃ for 16h;

(2) Seed culture: after the strain is activated, the strain is resuspended by using sterile normal saline, the obtained strain solution is inoculated into a 5L stirring type bioreactor, the culture temperature is 34 ℃, the culture pH is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 45% by adjusting the stirring rotation speed or ventilation quantity, and the inoculation requirement is met when the OD600nm is 25.

(3) Fermentation culture: the mature seed liquid obtained in the seed culture step is inoculated into a pre-prepared fermentation culture medium, a 5L mechanical stirring type fermentation tank is used, the inoculation amount is 20%, the fermentation culture temperature is 34 ℃, the culture pH is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 50% by adjusting the stirring rotation speed or ventilation amount, the glucose concentration in the tank is controlled to be less than or equal to 3g/L by feeding 80% glucose solution, and the fermentation period is less than or equal to 34h.

Example 10

This example was conducted to demonstrate glutamic acid productivity of Glu08, and the strains obtained in examples 1 to 8 were fermented by the application method of example 9, respectively, and the experimental results are shown in Table 5.

TABLE 5 comparison of yield and conversion during fermentation of different strains

The result shows that the strain Glu08 can accumulate 190g of glutamic acid after 34h fermentation culture, and the sugar acid conversion rate is 72%.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make several modifications and alterations without departing from the principles of the present invention, and the steps of constructing the strain of the present invention are not sequential, and those skilled in the art should consider the scope of the present invention as modifications and alterations of the strain according to the method of the present invention or based on the method.

Claims

1. A construction method of corynebacterium glutamicum production bacteria is characterized in that: the specific transformation strategy is as follows: corynebacterium glutamicum was used as a chassis strain by attenuating the alpha-ketoglutarate dehydrogenase cgl1129, acyl-CoA carboxylase in the chassis strain _cgl 0700. Expression of fatty acid synthases cgl0836 and cgl2495, malate dehydrogenase cgl2380, enhanced glutamate dehydrogenase cgl2079, glutamate synthases cgl0184 and cgl0185. And (3) expressing phosphoenolpyruvate carboxykinase cgl2863, and knocking out the isocitrate lyase cgl2331 gene to obtain the target strain after successful transformation.

2. The method for constructing corynebacterium glutamicum according to claim 1, wherein: the chassis strain is corynebacterium glutamicum ATCC13032; the nucleotide sequence of the alpha-ketoglutarate dehydrogenase cgl1129 encoding gene is shown in SEQ ID NO. 1; the nucleotide sequence of the encoding gene of the acyl-CoA carboxylase cgl0700 is shown as SEQ ID NO. 2; the nucleotide sequence of the fatty acid synthase cgl0836 encoding gene is shown in SEQ ID NO. 3; the nucleotide sequence of the fatty acid synthase cgl2495 encoding gene is shown in SEQ ID NO. 4; the nucleotide sequence of the coding gene of the malate dehydrogenase cgl2380 is shown in SEQ ID NO. 5; the nucleotide sequence of the coding gene of the glutamate dehydrogenase cgl2079 is shown in SEQ ID NO. 6; the nucleotide sequence of the coding gene of the glutamate synthase cgl0184 is shown as SEQ ID NO. 7; the nucleotide sequence of the coding gene of the glutamate synthase cgl0185 is shown as SEQ ID NO. 8; the nucleotide sequence of the phosphoenolpyruvate carboxykinase cgl2863 encoding gene is shown as SEQ ID NO. 9; the nucleotide sequence of the coding gene of the isocitrate lyase cgl2331 is shown as SEQ ID NO. 10.

3. The method for constructing corynebacterium glutamicum according to claim 1, wherein: the manner of attenuating the expression of alpha-ketoglutarate dehydrogenase is: using P _sod The promoter weakens the expression of the cgl1129 gene and weakens the downward carbon flow of alpha-ketoglutarate in the tricarboxylic acid cycle; the P is _sod The nucleotide sequence of the promoter is shown as SEQ ID NO. 11; the manner of attenuating expression of acyl-coa carboxylase is: using P _sod The promoter weakens the cgl0700 gene expression and weakens the flow of the competition branch of acetyl coenzyme A; the P is _sod The nucleotide sequence of the promoter is shown as SEQ ID NO. 11; the manner of attenuating fatty acid synthase expression is: using P _sod The promoter weakens the expression of the cgl0836 and cgl2495 genes and weakens the flow of the competition branch of acetyl coenzyme A; the saidP _sod The nucleotide sequence of the promoter is shown as SEQ ID NO. 11; the way to attenuate malate dehydrogenase expression is: using P _sod The promoter weakens the expression of the cgl2380 gene and weakens the flow of a competitive branch of oxaloacetate; the P is _sod The nucleotide sequence of the promoter is shown as SEQ ID NO. 11.

4. The method for constructing corynebacterium glutamicum according to claim 1, wherein: the mode of enhancing the expression of glutamate dehydrogenase is as follows: using P _tuf The promoter strengthens the overexpression of the cgl2079 gene and enhances the carbon flux to glutamic acid; the P is _tuf The nucleotide sequence of the promoter is shown as SEQ ID NO. 12; the mode of enhancing the expression of glutamate synthase is as follows: using P _tuf The promoters strengthen the overexpression of cgl0184 and cgl0185 genes and enhance the carbon flux to glutamate; the P is _tuf The nucleotide sequence of the promoter is shown as SEQ ID NO. 12; the mode for enhancing the expression of the phosphoenolpyruvate carboxykinase is as follows: using P _tuf The promoter strengthens the overexpression of the cgl2863 gene and weakens the flow of a competitive branch of oxaloacetate; the P is _tuf The nucleotide sequence of the promoter is shown as SEQ ID NO. 12; the mode of knocking out isocitrate lyase expression is: knockout of the cgl2331 gene reduces the carbon flux in the glyoxylate cycle.

5. A corynebacterium glutamicum producer, characterized in that: obtained by the construction method according to any one of claims 1 to 4, wherein,

strain Glu01 uses P at the original cgl1129 gene locus _sod The promoter controls the weakening expression of the cgl1129 gene;

the strain Glu02 is a starting strain with the strain Glu01, and P is used at the original cgl0700 gene locus _sod The promoter controls the weakening expression of the cgl0700 gene;

the strain Glu03 is a starting strain with the strain Glu02 at the original cgl0836 and the original cgl2495 gene locus P _sod The promoters control the weakened expression of the cgl0836 and cgl2495 genes;

strain Glu04 is the same as strain Glu03 is the original strain, and is located at the original cgl2380 gene locus P _sod The promoter controls the weakening expression of the cgl2380 gene;

the strain Glu05 is a starting strain with strain Glu04, and P is used at the original cgl2079 gene locus _tuf The promoter controls the enhancement expression of the cgl2079 gene;

the strain Glu06 is a starting strain of the strain Glu05, the primary cgl0184 and the primary cgl0185 share a promoter, and P is used at the primary cgl0184 and primary cgl0185 gene loci _tuf Promoters control the enhanced expression of cgl0185 and cgl0185 genes;

strain Glu07 is a starting strain with strain Glu06, P is used at the original cgl2863 gene locus _tuf The promoter controls the intensified expression of the cgl2863 gene;

the strain Glu08 is obtained by taking the strain Glu07 as an original strain and knocking out the original cgl2331 gene.

6. The use of the corynebacterium glutamicum according to claim 5 for the production of glutamic acid.

7. The use of Corynebacterium glutamicum as claimed in claim 6, characterized in that: glutamic acid is produced by a fermentation culture method.

8. The use of corynebacterium glutamicum according to claim 7, wherein: the specific steps of fermentation culture are as follows:

(2) Seed culture: after the strain is activated, the strain is resuspended by using sterile normal saline, the obtained strain solution is inoculated into a 5L stirring type bioreactor, the culture temperature is 34 ℃, the culture pH is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 45% by adjusting the stirring rotation speed or the ventilation quantity, and the strain solution OD is obtained _600nm The inoculation requirement is met when the time is 25;

9. The use of corynebacterium glutamicum according to claim 8, wherein: the test tube slant solid culture medium is LB solid culture medium.

10. The use of corynebacterium glutamicum according to claim 8, wherein: the culture medium used for seed culture is as follows: 25g/L glucose, 15g/L corn steep liquor dry powder, 15ml/L soybean meal hydrolysate and K ₂ HPO ₄ ·3H ₂ O 1.0g/L，MgSO ₄ ·7H ₂ O1.0g/L, and the balance of water; the culture medium used for the fermentation culture is as follows: glucose 60g/L, KH ₂ PO ₄ 3g/L，MgSO ₄ ·7H ₂ O 2g/L，MnSO ₄ ·H ₂ O 20mg/L，FeSO ₄ 20mg/L，V _B1 0.5mg/L，V _H 0.1mg/L, 7g/L of yeast powder, 0.5g/L of methionine, 20g/L of corn steep liquor dry powder, 30mL/L of soybean meal hydrolysate, 2 drops of defoamer and the balance of water.