Escherichia coli gene engineering bacterium and method for producing L-threonine by using escherichia coli gene engineering bacterium

A technology of genetically engineered bacteria and Escherichia coli, applied in the field of genetic engineering, can solve problems such as affecting the synthesis and accumulation of L-threonine, affecting the normal metabolism and growth of cells, etc., to improve the conversion rate of sugar and acid, and enhance the yield and sugar acid. Conversion rate, ease of use effect

Active Publication Date: 2017-06-20
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some studies have shown that excessive expression of phosphoenolpyruvate carboxylase will affect the normal metabolism and growth of cells, thereby affecting the synthesis and accumulation of L-threonine

Method used

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  • Escherichia coli gene engineering bacterium and method for producing L-threonine by using escherichia coli gene engineering bacterium
  • Escherichia coli gene engineering bacterium and method for producing L-threonine by using escherichia coli gene engineering bacterium
  • Escherichia coli gene engineering bacterium and method for producing L-threonine by using escherichia coli gene engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Betaine enhances P zwf expression strength test

[0053] 1. Construction of a recombinant vector comprising the zwf gene promoter and the green fluorescent protein expression gene gfp

[0054] (1) Transformation of vector pUC19

[0055] Transform the vector pUC19 by designing reverse amplification primers pUC19-F / pUC19-R to remove the entire Lac operon (see Figure 4 ), the transformed vector fragment was named pUC19-L (SEQ ID No.1);

[0056] (2) Amplification of zwf gene promoter and gfp gene and construction of overlapping fragments

[0057] Using the zwf promoter sequence (SEQ ID No.2) in the Escherichia coli MG1655 genome as a template to design the amplification primer P zwf –up and P zwf -down; use the gfp (SEQ ID No.3) gene sequence on the pEGFP-N1 plasmid as a template to design amplification primers gfp–up and gfp–down, primer P zwf –down and gfp–up contain a 20bp overlapping sequence, primer P zwf The 5' ends of –up and gfp-down each contain ...

Embodiment 2

[0089] Example 2: P of E. coli THRD ppc replace with P zwf

[0090] 1. ppc gene promoter P ppc Upper and lower homology arms, Cm gene and zwf gene promoter P zwf Amplification of

[0091] The promoter P of the ppc gene (SEQ ID No.20) on the genome of Escherichia coli MG1655 ppc The upper and lower homology arm sequences are used as templates to design primers ppc-up-1 / 2 and ppc-down-1 / 2;

[0092]Primers zwf-1 and zwf-2 were designed using the zwf promoter sequence on the genome of Escherichia coli MG1655 as a template;

[0093] Cm fragment primers Cm-up and Cm-down were designed using plasmid pKD3 as a template.

[0094] The high-fidelity enzyme PrimeSTAR HS DNA Polymerase was used to amplify the upper and lower homology arm sequences of the ppc gene promoter and the zwf promoter sequence respectively, and the target band was obtained after electrophoresis verification (see Figure 5 ), using the OMEGA PCR product purification kit of Guangzhou Feiyang Bioengineering Co....

Embodiment 3

[0101] Embodiment 3: Shake flask fermentation

[0102] Test strain: the bacterial strain obtained in Example 2; Protobacterium THRD;

[0103] (1) The test strains were activated by slant culture, cultured at 37°C for 16h, transferred once (second-generation slope activation), and cultured at 37°C for 10h;

[0104] (2) Use an inoculation loop to draw the above-mentioned second-generation slant two-ring sludge into 30mL seed medium, and cultivate it to OD at 37°C and 200rpm 600 is 8;

[0105] (3) Inoculate the seed culture solution into the fermentation medium according to the inoculum size of 10%, cultivate at 37°C and 200rpm, start to add sugar after the lack of sugar, and betaine is added with sugar (80% glucose is added, containing 2.5 g / L betaine, add 1mL per 30mL fermentation system each time), and the fermentation ends for 28h;

[0106] Sugar deficiency standard: If there is no color change in the culture medium for more than 20 minutes (that is, the pH does not change...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an escherichia coli gene engineering bacterium and a method for producing L-threonine by using the escherichia coli gene engineering bacterium. The gene engineering bacterium is characterized in that a promoter P<ppc> of phosphoenolpyruvate carboxylase gene (ppc) of an initial strain is replaced into a promoter P<zwf> of 6-phosphate dehydrogenase gene (zwf), so that the goal of regulating the L-threonine production capacity by glycine betaine is achieved. In the fermentation process, the glycine betaine is added, so that the L-threonine yield through shaking flask fermentation can reach 50 to 55g / L; the 5L fermentation tank yield reaches 120 to 150g / L; the saccharic acid conversion rate reaches 59 to 61 percent.

Description

[0001] Technical field: [0002] The invention belongs to the technical field of genetic engineering, and in particular relates to a strain of Escherichia coli genetically engineered bacteria and a method for producing L-threonine using the same. [0003] Background technique: [0004] A promoter is a DNA sequence that RNA polymerase specifically recognizes and binds to. A promoter is an integral part of a gene that controls when and how much gene expression (transcription) begins. The promoter is like a "switch" that determines the activity of the gene, but the promoter does not control the activity of the gene itself, but controls the activity of the gene by binding to this protein called a transcription factor. The promoter is a DNA sequence located upstream of the 5' end of the structural gene, which can guide the holoenzyme to correctly combine with the template, activate RNA polymerase, and initiate gene transcription. [0005] The commonly used types of promoters are c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/08C12R1/19
Inventor 谢希贤马明利陈宁鄢芳清徐庆阳马倩刘涛王俊朋
Owner TIANJIN UNIV OF SCI & TECH
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