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Recombinant Escherichia coli for efficiently producing clarified acid and application of recombinant Escherichia coli

A technology of recombinant Escherichia coli and claritic acid, applied in the fields of genetic engineering, fermentation engineering and material science, can solve problems that only focus on monosaccharide components, molecular weight and rheological properties

Active Publication Date: 2021-12-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there are few current studies on the properties of CA, and most of them only focus on the monosaccharide composition, molecular weight and rheological properties

Method used

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  • Recombinant Escherichia coli for efficiently producing clarified acid and application of recombinant Escherichia coli
  • Recombinant Escherichia coli for efficiently producing clarified acid and application of recombinant Escherichia coli
  • Recombinant Escherichia coli for efficiently producing clarified acid and application of recombinant Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Screening for high-yielding claric acid starting strains

[0088] CA production can be effectively promoted by knocking out the waaF gene in E. coli W3110 or the waaL-waaQ gene cluster in MG1655, resulting in truncation of the LPS core polysaccharide (e.g. figure 1 shown in A). In the present invention, the CA production rate of W3110ΔwaaF and MG1655Δ(L-Q) were compared, and a strain with high CA production was selected as the starting strain for further optimization and transformation. Specific steps are as follows:

[0089] (1) The MG1655Δ(L-Q) strain was constructed according to the method in the SCI paper (Wang C H, Zhang H L, Wang J L, et al. Colanic acid biosynthesis in Escherichia coli is dependent on lipopolysaccharide structure and glucose availability. Microbiological Research, 2021.243.), using The CRISPR / Cas9 knockout system was used to knock out the genes of E. coli. First, the pCas9 plasmid was electrotransfected into E. coli Escherichia coli ...

Embodiment 2

[0093] Embodiment 2: the construction of high-yielding claritic acid bacterial strain

[0094] The activator RcsA can be degraded and inhibited by Lon and HNS proteins in MG1655Δ(L-Q) (eg figure 1 C shown). Therefore, the lon and hns genes need to be knocked out from the MG1655Δ(L-Q) genome.

[0095] Specific steps are as follows:

[0096] 1. Construction of WQM001 strain

[0097]The lon gene on the genome of the MG1655Δ(L-Q) strain prepared in Example 1 was knocked out. The specific method was the same as that in Example 1. The Escherichia coli MG1655Δ(L-Q) strain was knocked out using the CRISPR / Cas9 knockout system. Bacillus MG1655Δ(L-Q) strain was electroporated into pCas9 plasmid, and L-arabinose was added to induce the expression of recombinase Gam, Bet, and Exo to obtain MG1655Δ(L-Q) / pCas9 strain

[0098] Using the Escherichia coli MG1655 genome as a template, use F1-lon / R1-lon and F2-lon / R2-lon to amplify the upstream and downstream homology arms of the knockout lo...

Embodiment 3

[0122] Embodiment 3: CA medium composition and fermentation condition optimization

[0123] 1. Single factor experiment to optimize medium composition

[0124] According to the results of exopolysaccharide staining in step 5 of Example 2, it can be seen that the composition of the medium has a significant impact on the production of exopolysaccharides. Compared with LB and LBG medium, WQM003 / pRAU was cultured in M9 medium at 30°C The exopolysaccharide production was the highest at this time. Therefore, media optimization was therefore performed in M9 media. The effects of carbon source, nitrogen source, phosphate, and NaCl on CA production were investigated using a single factor test to determine approximate variable ranges. The experiment studied 4g / L glucose, 4g / L fructose, 4g / L galactose, 4g / L mannose and 4g / L sucrose to select the best carbon source for CA biosynthesis; The nitrogen source is selected from 10g / L NH 4 Cl, peptone of 10g / L, yeast extract of 10g / L and cor...

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Abstract

The invention discloses recombinant escherichia coli for efficiently producing clarified acid and application of the recombinant escherichia coli, and belongs to the fields of genetic engineering, fermentation engineering and material science. According to the invention, by editing a key gene for CA synthesis, a mutant for excessive production of CA is constructed; and then an optimal culture medium is obtained through adoption of a single factor and response surface method, and fermentation conditions is optimized in a fermentation tank. The yield of the CA prepared by the method disclosed by the invention reaches 19.79 g / L, is higher than the known maximum yield (10.39 g / L), and is improved by 90.47%. Besides, the CA prepared by adopting the method disclosed by the invention is subjected to property characterization, so that the CA is proved to have excellent properties, such as solid form, molecular weight, space structure, chain conformation, thermal property, rheological property and the like. The high yield of CA provides a solid foundation for large-scale production and CA property research, and revelation of CA properties provides a solid theoretical foundation for development and utilization of CA.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli capable of producing claric acid efficiently and an application thereof, belonging to the fields of genetic engineering, fermentation engineering and material science. Background technique [0002] Natural polysaccharides, especially polysaccharides with triple-helical conformation, have unique properties and functions such as anti-tumor, immune regulation, etc., and have attracted more and more attention in the fields of food and medicine. Therefore, the research and development of polysaccharides is of great significance in the field of polymers. Claric acid (CA) is an extracellular protective barrier polysaccharide widely present in intestinal bacteria, which plays a vital role in the adaptation of bacteria to the external environment. CA in Escherichia coli K-12 strain MG1655 is composed of repeating units consisting of D-galactose, L-fucose, D-gluconic acid and D-glucose. It has been report...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P19/04C12N15/31C12N15/54A23L33/125A61K31/715A61P39/06C12R1/19
CPCC12N15/70C12P19/04C07K14/245C12Y207/07009C12N9/1022A23L33/125A61K31/715A61P39/06A23V2002/00A23V2200/30
Inventor 王小元乔君詹依檀昕胡晓清
Owner JIANGNAN UNIV
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