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Genetically engineered bacterium for producing L-threonine as well as construction method and application of genetically engineered bacterium

A genetically engineered bacteria and genetic engineering technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problem of the conversion rate of cofactor biotin-deficient strains, etc.

Inactive Publication Date: 2021-03-12
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the expression of pyc gene is further increased, the lack of cofactor biotin of pyruvate carboxylase will greatly limit the further improvement of the transformation rate of the transformed strain

Method used

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  • Genetically engineered bacterium for producing L-threonine as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing L-threonine as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing L-threonine as well as construction method and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Embodiment 1 prepares the bacterial strain MHZ-0215-3 (promoter strengthening) of strengthening bioABFCD gene

[0152] (1) Construction of pTargetF-N20(tac-bioABFCD) plasmid and Donor DNA-1

[0153] Step 1: Using the pTargetF plasmid as a template (see Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al.Appl.EnvironMicrobiol, 2015), select pTF-sgRNA-F1 / pTF-sgRNA - R1 primer pair, amplify the pTF linear plasmid with N20, use the seamless assembly ClonExpress kit to assemble the linear plasmid at 37°C, and then transform Trans1-T1 competent cells to obtain pTargetF-N20(tac-bioABFCD) , and carry out PCR identification and sequencing verification; Step 2: Using the W3110 genome as a template, select bioAtac-UF / bioAtac-UR1 and bioAtac-UF / bioAtac-UR2 primer pairs to amplify the upstream homology arm containing the tac promoter① ; Step 3: Using the W3110 genome as a template, select the PtacI-F / PtacI-R primer pair to amplify the ...

Embodiment 2

[0160] Example 2 Preparation of bioABFCD gene RBS enhanced strain MHZ-0215-4 (RBS-bioABFCD)

[0161] (1) Construction of pTargetF-N20 (RBS bioABFCD) plasmid and Donor DNA-2 construction

[0162] Step 1: Using the pTargetF plasmid as a template (see Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al.Appl.EnvironMicrobiol, 2015), select pTF-sgRNA-F1 / pTF-sgRNA - R1 primer pair to amplify the pTF linear plasmid with N20, use the seamless assembly ClonExpress kit to assemble the linear plasmid at 37°C, and then transform Trans1-T1 competent cells to obtain pTargetF-N20 (RBS bioABFCD), And carry out PCR identification and sequencing verification; Step 2: Using the W3110 genome as a template, select the bioA-RBS-UF / bioA-RBS-UR primer pair to amplify the upstream homology arm containing RBS ①; Step 3: Using the W3110 genome As a template, use bioAp-F / bioAp-R, bioAp-F / bioABp-R, bioAp-F / bioBp-R1, bioAp-F / bioBp-R2 primer pairs to amplify t...

Embodiment 3

[0169] Example 3 Preparation of the strain MHZ-0215-5 (multiple copies) of the enhanced bioABFCD gene

[0170] (1) Construction of pTargetF-N20(IS1::bioABFCD) plasmid and Donor DNA-3

[0171] Step 1: Using the pTargetF plasmid as a template (see Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al.Appl.EnvironMicrobiol, 2015), select pTF-sgRNA-F2 / pTF-sgRNA -R2 primer pair to amplify the pTF linear plasmid with N20, use the seamless assembly ClonExpress kit to assemble the linear plasmid at 37°C, and then transform Trans1-T1 competent cells to obtain pTargetF-N20 (IS1::bioABFCD ), and carry out PCR identification and sequencing verification; Step 2: Using the W3110 genome as a template, select the bioABFCD2-UF / bioABFCD2-UR primer pair to amplify the upstream homology arm ①; Step 3: Using the W3110 genome as a template, select bioABFCD2 -F1 / bioABFCD2-R1 primer pair to amplify the upper half of the bioABFCD operon ②; Step 4: Using th...

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Abstract

The invention discloses a genetically engineered bacterium for producing L-threonine as well as a construction method and application of the genetically engineered bacterium. The biotin synthesis of the L-threonine producing strain is enhanced, especially the pyc gene of the strain is enhanced, and the adopted specific means is to enhance the bioABFCD gene (including promoter enhancement, RBS sequence enhancement and multi-copy), release the transcription repression of the brA gene, and release the transcription repression of the brA while enhancing the bioABFCD. According to the constructed genetically engineered bacterium for producing the L-threonine, through fermentation culture, the yield of the L-threonine can reach 20.2 g / L, and the conversion rate of saccharic acid can reach 23.8%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a genetic engineering bacterium producing L-threonine and its construction method and application. Background technique [0002] L-threonine is one of the 8 kinds of amino acids necessary for the growth of humans and animals. It is widely used in feed, food additives and preparation of pharmaceutical auxiliary materials. At present, L-threonine is mainly produced by microbial fermentation, and various bacteria can be used for L-threonine production, such as wild-type induced mutants of Escherichia coli, Corynebacterium, Serratia, etc. as production strains. Specific examples include mutants resistant to amino acid analogs or various auxotrophs such as methionine, lysine, and isoleucine (Japanese Patent Application Laid-Open No. 224684 / 83; Korean Patent Application Laid-Open No. 8022 / 87). However, traditional mutation breeding is difficult to obtain high-yield strains due to the slow...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/64C12P13/08C12R1/19
CPCC12N15/70C07K14/245C12P13/08
Inventor 刘慧敏尹春筱康培
Owner MEIHUA BIOTECH LANGFANG CO LTD
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