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Glucosamine synthase producing bacterium based on CRISPR-Cas9 technology and construction method and application of glucosamine synthase producing bacterium

A technology for producing bacteria and ammonia sugar, which is applied in the field of building and producing bacteria for ammonia sugar synthase, which can solve the problems of high GlcN accumulation concentration and difficulty in obtaining it, and achieve the effects of reducing inhibition, low production cost and short fermentation time

Inactive Publication Date: 2019-10-29
YANGZHOU RIXING BIO TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Previous studies have shown that GlcN has a strong inhibitory effect on the growth of Escherichia coli cells, so it is difficult to obtain a higher cumulative concentration of GlcN in microbial fermentation with wild-type strains, and GlcNAc, as an N-acetylated derivative of GlcN, is stable to cells And without inhibition, it is easy to hydrolyze to generate GlcN under mild acidic conditions

Method used

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  • Glucosamine synthase producing bacterium based on CRISPR-Cas9 technology and construction method and application of glucosamine synthase producing bacterium
  • Glucosamine synthase producing bacterium based on CRISPR-Cas9 technology and construction method and application of glucosamine synthase producing bacterium
  • Glucosamine synthase producing bacterium based on CRISPR-Cas9 technology and construction method and application of glucosamine synthase producing bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: CRISPR-Cas9 gene editing technology to knock out the genes mannX and naggE

[0042] Using E.coli BL21(DE3) as the starting strain, the gene editing technology mediated by CRISPR-Cas9 was used to knock out the target gene of the starting strain. The specific operation steps are as follows:

[0043] (1) Add the pCas9 plasmid to a centrifuge tube containing 100 μL of E.coli BL21(DE3) competent, quickly maintain it in an ice bath for 30 minutes, then heat shock it in a water bath at 42°C for 90 seconds, and put it on ice immediately after the heat shock Cool in the bath for 5 min. Take out the centrifuge tube from ice, add 800 μL of LB medium to the centrifuge tube, place it on a shaker for activation and culture for 45 min. Take out the centrifuge tube and centrifuge it at 8000r / min for 5min. After centrifugation, suck off 800μL supernatant on the ultra-clean bench, blow and mix the bacteria and spread it on LB containing the corresponding antibiotic at a fina...

Embodiment 2

[0053] Example 2: Exploration of carbon sources available for Escherichia coli with double gene deletion

[0054] Two kinds of bacteria were inoculated in the M9+glucosamine Glc (final concentration 0.5%) of 50mL respectively with the same inoculum size, M9+glucosamine GlcN (final concentration 0.5%), in M9+acetylglucosamine GlcNAc (final concentration 0.5%) liquid medium, Cultivate at 220rpm, 37°C under the same conditions at the same time, take samples regularly, and measure OD 600 , draw the growth curves of the two bacteria in different liquid media to explore whether the double gene deletion Escherichia coli can utilize glucosamine and acetylglucosamine.

Embodiment 3

[0055] Embodiment 3: Construction of recombinant expression plasmid pET-28a / gnal / glms

[0056] The primer sequences needed to amplify the gene of interest in Table 3 Example 3

[0057]

[0058] The design of the primers for the glucosamine synthase gene in Table 3 is mainly based on the glms sequence of the glucosamine synthase gene in the NCBI genome of Escherichia colistr.K-12 substr.MG1655, and the design of the primers for the glucosamine acetylase gene is mainly based on the optimized The gnal sequence of the glucosamine acetylase gene derived from Saccharomyces cerevisiae was used as a standard, and was designed using SnapGene software.

[0059] (1) Primer STAR HS DNA polymerase has good fidelity, so it is used to amplify the target gene. The PCR reaction program was: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 0.5 min, annealing at 60°C for 0.75 min, extension at 72°C for 2 min, final extension at 72°C for 10 min, and repeated for 34 cycles. The P...

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Abstract

The invention discloses a glucosamine synthase producing bacterium based on a CRISPR-Cas9 technology and a construction method and application of the glucosamine synthase producing bacterium. The glucosamine synthase producing bacterium is obtained in the modes that a glucosamine acetylase gene gnal and a glucosamine synthase gene glms are connected to a carrier pET-28a, and then a recombinant plasmid is transferred into a double-gene deletion host bacterium obtained by knocking out a glucosamine metabolism gene naggE and a glucosamine metabolism gene mannX of an E.coli BL21(DE3) genome through the CRISPR-Cas9 gene editing technology. The recombinant bacterium is applied to fermentation culture, on a shake flask, 7.51 g / L of glucosamine can be accumulated, 3.73 g / L of acetylglucosamine canbe accumulated, and the total output is 11.24 g / L; and on a fermentor of 5 L, 7.30 g / L of the glucosamine can be accumulated, 10.53 g / L of the acetylglucosamine can be accumulated, and the total output is 17.83 g / L.

Description

technical field [0001] The invention relates to the technical field of gene knockout, in particular to a CRISPR-Cas9 technology-based ammonia sugar synthase-producing bacterium, a construction method and an application thereof. Background technique [0002] Glucosamine (GlcN) has a good curative effect in the prevention and treatment of arthritis. Arthritis is mainly caused by joint damage and abnormal articular cartilage, which causes inconvenience to patients in daily activities and even leads to loss of bone and joint function. , Glucosamine stimulates chondrocytes through a certain mechanism in the body, effectively promotes the synthesis of collagen, and gradually repairs damaged articular cartilage. Glucosamine supplementation can promote the synthesis of glycoproteins to repair damaged cartilage, increase the lubrication between joints, and reduce the wear and tear between joints. In addition, glucosamine can also regulate the body's endocrine and adsorb on the intes...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/90C12P19/26C12R1/19
CPCC12N9/1029C12N9/1096C12N15/70C12N15/902C12P19/26C12Y206/01016
Inventor 史劲松龚劲松李晓鹏许正宏李会张超丁振中
Owner YANGZHOU RIXING BIO TECH
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