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Genetic engineering strain for producing inosine as well as construction method and application thereof

A genetically engineered strain and gene technology, applied in the field of genetic engineering, can solve the problems of easily lost production of plasmids, increase of production costs, complex fermentation process, etc., and achieve good industrial application prospects, stable and efficient production, and clear genetic background effects

Pending Publication Date: 2022-04-12
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The specific performance is as follows: (1) The inosine production performance of rationally constructed strains is generally low at present, the highest yield is only 14g / L, and the production cycle is long, generally as long as 72h or even longer
(2) The strains carry a lot of plasmids, the burden of the bacteria is heavy, and the plasmids are easy to lose, which will cause unstable production
In order to maintain the stable existence of the plasmid, antibiotics need to be added, which not only increases the production cost, but also increases safety risks such as drug resistance
(3) In terms of construction strategies, most of the existing strains are limited to the transformation of individual nodes such as degradation pathways, de novo synthesis pathways, precursor synthesis pathways, and branched metabolic pathways, and lack the systematic combination of modules in the entire inosine metabolic network
(4) Most of the strains are adenine-deficient, and adenine needs to be added during the fermentation process, which leads to high raw material costs, complicated fermentation process, and reduced production stability

Method used

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  • Genetic engineering strain for producing inosine as well as construction method and application thereof
  • Genetic engineering strain for producing inosine as well as construction method and application thereof
  • Genetic engineering strain for producing inosine as well as construction method and application thereof

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Embodiment approach

[0040] According to a preferred embodiment of the present invention, the nucleoside transporter gene pbuE is connected with a promoter P trc and / or the purine operon mutant gene purEKBCSQLF K316Q MNHD-linked promoter P trc and / or the PRPP transamidase mutant gene purF K316Q linked with promoter P trc ; Preferably, the promoter P trc The nucleotide sequence of is shown in SEQ ID NO:5.

[0041] According to the present invention, the starting strain for constructing the E. coli genetic engineering strain may be any E. coli, and according to a preferred embodiment of the present invention, the starting strain is E. coli MG1655.

[0042] In a second aspect, the present invention provides a method for constructing the Escherichia coli genetically engineered strain as described above, comprising: introducing the nucleoside transporter gene pbuE into the starting strain Escherichia coli, the purine operon mutant gene purEKBCSQLF K316Q MNHD and PRPP transamidase mutant gene purF ...

Embodiment 1

[0059] Embodiment 1: the construction of Escherichia coli genetic engineering strain E.coli INO4

[0060] 1. Blocking inosine decomposition pathway

[0061] 1.1 Knockout of deoD gene:

[0062] Using the E.coli MG1655 genome as a template, the upstream homology arm primers (UP-deoD-S, UP-deoD-A) and the downstream homology arm primers ( DN-deoD-S, DN-deoD-A), and its upstream and downstream homology arm fragments were amplified by PCR. The above fragments were fused by overlapping PCR to obtain a knockout fragment of the deoD gene (upstream homology arm-downstream homology arm). The DNA fragment containing the target sequence used in the construction of pGRB-deoD was prepared by annealing the primers gRNA-deoD-S and gRNA-deoD-A. The recombinant fragment and plasmid pGRB-deoD were electrotransformed into competent cells of E.coliMG1655, and the positive strains were screened, and then the plasmid was eliminated to obtain strain INO1-1. The construction of the deoD knockout f...

Embodiment 2

[0106] Embodiment 2: strain INO2-6, INO2-7, INO2-8 and INO2-9 shake flask fermentation to produce inosine experimental shake flask culture method is as follows:

[0107] Slant activation culture: Streak inoculation of -80°C preserved strains on the activation slant, culture at 37°C for 12 hours, and then passage again;

[0108] 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 7-10h;

[0109] Fermentation culture: Inoculate 10-15% of the volume of seed culture solution into a 500mL Erlenmeyer flask with fermentation medium (final volume is 30mL), seal with nine layers of gauze, 37°C, 200r / min shaking culture, fermentation process Maintain the pH at 7.0-7.2 by adding ammonia water; add 60% (m / v) glucose solution to maintain the fermentation; the fermentation period is 24 hours.

[0110] Incline medium: glucose 1-5g / L,...

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Abstract

The invention belongs to the technical field of genetic engineering, and relates to breeding of industrial microorganisms, in particular to a genetic engineering strain for producing inosine as well as a construction method and application of the genetic engineering strain. According to the genetic engineering strain, a nucleoside transport protein gene pbuE, a purine operon mutant gene purEKBCSQLFK316QMNHD and a PRPP transamidase mutant gene purFK316Q are subjected to heterologous overexpression, a gene purA is replaced by a heterologous adenosine succinate synthetase mutant gene purAP242N, and purine nucleoside phosphorylase genes deoD and ppnP and nucleoside hydrolase genes rihA, rihB and rihC are not expressed. Starting from the genome level of Escherichia coli, modules of an inosine decomposition pathway, an inosine synthesis pathway, an inosine transport system and a branch metabolic pathway are systematically and comprehensively combined and optimized mainly through a metabolic engineering technical means, and the inosine fermentation performance of the strain is improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to the breeding of industrial microorganisms, in particular to a genetic engineering bacterial strain for producing inosine and its construction method and application. Background technique [0002] Inosine is a kind of purine nucleoside, which participates in the material metabolism and energy metabolism of the body, and has important application value. Microbial fermentation is currently the main method for industrial production of inosine. However, due to the poor fermentation performance of existing strains, the production level of inosine produced by fermentation is still low, and high-performance strains are urgently needed to meet the industrialization needs of inosine. The traditional breeding of inosine producing strains is obtained by mutagenesis to screen structural analogue resistant strains. However, due to the unclear genetic background and the accumulation of...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/32C12N15/52C12N15/54C12N15/56C12N9/10C12P19/40C12R1/19
CPCY02A50/30
Inventor 谢希贤朱彦凯刘铁重吴鹤云
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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