Construction of escherichia coli genetically engineered bacterium and application of escherichia coli genetically engineered bacterium to production of L-tryptophan

A technology of genetically engineered bacteria and Escherichia coli, which is applied in the field of microbial metabolism regulation and genetic engineering, can solve the problems of low sugar-acid conversion rate, complicated extraction process, easy loss of plasmids, etc., and achieve clear genetic background, broad application prospects, and convenient traits Improved effect

Active Publication Date: 2018-11-06
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, most of the Escherichia coli used to produce L-tryptophan in China are plasmid-carrying strains, and the plasmid is easily lost during the fermentation process. In addition, there are many precursors involved in the L-tryptophan synthesis pathway, which is easy to accumulate in the later stage of fermentation Due to the influence of many by-products and other factors, the fermentation process of the existing L-tryptophan Escherichia coli engineering strains has problems such as difficult control, low yield and sugar-acid conversion rate, and complex extraction process.

Method used

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  • Construction of escherichia coli genetically engineered bacterium and application of escherichia coli genetically engineered bacterium to production of L-tryptophan
  • Construction of escherichia coli genetically engineered bacterium and application of escherichia coli genetically engineered bacterium to production of L-tryptophan
  • Construction of escherichia coli genetically engineered bacterium and application of escherichia coli genetically engineered bacterium to production of L-tryptophan

Examples

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Effect test

Embodiment 1

[0051] Example 1: Construction of Escherichia coli L-tryptophan engineering strain E.coli TRP03

[0052] 1. Methods of gene editing

[0053] The gene editing method used in the present invention is carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR–Cas9 edited genome editing. Metabolic engineering, 2015, 31:13-21.), the method used The two plasmid maps of the attached figure 1 . Among them, pREDCas9 carries gRNA expression plasmid pGRB elimination system, bacteriophage λ Red recombination system and Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg / L), cultured at 32°C; pGRB uses pUC18 as the backbone, including the promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100mg / L), cultured at 37°C.

[0054] The concrete steps of this method are as follows:

[0055] 1.1 pGRB plasmid construction...

Embodiment 2

[0120] Embodiment 2: Introducing glutamine synthetase (glnA) from Lactobacillus acidophilus (Lactobacillus acidophilus) to L-tryptophan engineering bacteria

[0121] 1. glnA gene synthesis

[0122] According to the Lactobacillus acidophilus (Lactobacillus acidophilus) glutamine synthetase coding gene sequence published on GENBANK in NCBI, it is codon-optimized with codon software commonly used in Escherichia coli (sequences before and after codon optimization are respectively as SEQ ID NO: 8 and SEQ ID NO: 9), so that it can be efficiently transcribed in Escherichia coli. The optimized sequence was sent to Jinweizhi Company for synthesis, and the recombinant plasmid pUC57glnA with glnA gene was obtained, in which the enzyme cutting sites were Hind III and BamH I, and it was stored in Escherichia coli.

[0123] 2. Plac glnA is integrated into the genome ycjV pseudogene locus

[0124] Using the E.coli W3110 (ATCC27325) genome as a template, design upstream homology arm primers...

Embodiment 3

[0125] Example 3: Production of L-tryptophan by Escherichia coli genetic engineering bacteria shake flask fermentation

[0126] A kind of concrete operation that utilizes Escherichia coli genetically engineered bacteria to carry out shake flask fermentation to produce L-tryptophan is as follows:

[0127] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0128] Shake flask seed culture: Scrape a ring of slanted seeds with an inoculation loop and inoculate in a 500mL Erlenmeyer flask containing 30mL of seed medium, seal with nine layers of gauze, and incubate at 37°C and 200rpm for 8-10h;

[0129] Shake flask fermentation culture: Inoculate 10-15% (v / v) inoculum into a 500mL Erlenmeyer flask with fermentation medium (final volume is 30mL), seal with nine layers of gauze, culture at 37°C, 200r / min shaking, During the fermentation process, maintain the pH at 7.0-7.2 by adding ammonia water; add 6...

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Abstract

The invention relates to an escherichia coli genetically engineered bacterium and a method for efficiently producing L-tryptophan by utilizing the escherichia coli genetically engineered bacterium. The genetically engineered bacterium is obtained by correspondingly transforming and combining L-tryptophan synthesis-related genes in escherichia coli through a metabolic engineering method (includinggene integration, point mutation and promoter substitution) and introducing a glutamine synthetase coding gene glnA from lactobacillus acidophilus on genome of the escherichia coli. The strain is usedfor carrying out shake-flask fermentation; 10 to 12g/L of L-tryptophan can be accumulated within 24 hours, reaches the maximum value reported in China and is improved by 15.1 percent compared with that of an existing L-tryptophan industrial strain carrying plasmid, which shows that the strain has good potential for industrially producing the L-tryptophan.

Description

technical field [0001] The invention relates to an Escherichia coli genetically engineered bacterium and a construction method thereof, and a method for rapidly and efficiently producing L-tryptophan by using the engineered bacterium, belonging to the technical fields of microbial metabolism regulation and genetic engineering. Background technique [0002] L-tryptophan, an essential amino acid, has been widely used as a basic raw material for feed additives, medicines such as infusion solutions and health functional foods. Its production method includes chemical synthesis method, enzymatic reaction method, fermentation method etc., but mainly uses the direct fermentation method utilizing microorganism at present, and this method uses the low price raw material such as glucose as substrate to produce L-tryptophan by fermentation, and production cost is relatively low. Low, the production process is relatively simple and controllable. [0003] Because the synthetic metabolic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/22C12N1/21C12N15/70C12R1/19
CPCC07K14/245C12N9/0006C12N9/88C12N15/70C12P13/227C12Y101/01095C12Y401/02
Inventor 谢希贤鄢芳清徐庆阳陈宁韩亚昆门佳轩李燕军马倩张成林范晓光
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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