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Engineered Escherichia coli and method for producing chondroitin

A technology of Escherichia coli and engineering bacteria, applied in the fields of fermentation engineering and genetic engineering, can solve the problems of low chondroitin yield, yield and production intensity, etc.

Inactive Publication Date: 2012-08-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the economic instinct of microbial metabolism and the huge difference between the industrial production environment and the natural environment, the yield, yield and production intensity of chondroitin are low, which has become a key bottleneck restricting the industrialization of chondroitin sulfate produced by fermentation.

Method used

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  • Engineered Escherichia coli and method for producing chondroitin
  • Engineered Escherichia coli and method for producing chondroitin
  • Engineered Escherichia coli and method for producing chondroitin

Examples

Experimental program
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Embodiment 1

[0022] Embodiment 1: Construction of SlyA expression vector

[0023] Using the Escherichia coli K4 (ATCC 20502) genome as a template, perform PCR according to the method described in the Molecular Cloning Experiment Guide (Third Edition), and then insert the obtained PCR product into pTrcHisA (histidine fusion protein expression vector, Invitrogen) , thus generating pTrcHisA-SlyA.

[0024] Amplification primers:

[0025] SlyAF: 5′-cccaagctttcaccctttggcctgtaactcaat-3′ SlyAR: 5′-cgggatccatgaaattggaatcgccactaggtt-3′

Embodiment 2

[0026] Embodiment 2: SlyA expression vector is expressed in Escherichia coli K4

[0027] The expression vector pTrcHisA-SlyA was introduced into Escherichia coli K4 by electroporation transformation method (cell 50 μL, expression vector 2 μL, 200Ω, 25F, 1.8kV, electric shock cup 0.1cm), thereby producing E. coli engineering bacteria. The clones were transferred to LB medium (containing 10 g / L peptone, 10 g / L sodium chloride and 5 g / L yeast powder, natural pH) containing 100 μg / mL ampicillin, and cultured overnight at 37°C. Transfer 1 mL of the culture to 50 mL of fresh LB medium containing 100 μg / mL ampicillin. Incubate at 37°C for 1-3 hours (OD 600 = 0.5-0.8), IPTG was added at a final concentration of 0.5-1.0 mM. Continue culturing for 3-8 hours to induce the expression of recombinant SlyA protein. The cells were collected by centrifugation of 1 mL of induction culture medium, followed by SDS-PAGE electrophoresis according to the Molecular Cloning Experiment Guide (Third ...

Embodiment 3

[0028] Example 3: Production of capsular polysaccharide by fermentation

[0029] Inoculate the Escherichia coli engineered bacteria in LB medium containing 100 μg / mL ampicillin, and perform seed culture at 37° C. for 10-14 hours. Then transfer to 100μg / mL GY medium (containing 10g / L glycerol, 1g / L peptone, 1g / L ammonium chloride, 0.5g / L trisodium citrate, 0.1g / L oxidized Magnesium, 9.7g / L dipotassium hydrogen phosphate and 2g / L potassium dihydrogen phosphate, pH 7.2~7.4), cultured at 37°C for 1-3 hours (OD 600 =0.5-0.8), add IPTG with a final concentration of 1 mM, and continue culturing for 24 hours. During the whole culture process, Escherichia coli K4 was used as the control, but no ampicillin was added.

[0030] The content of capsular polysaccharide in the fermentation broth was detected according to the carbazole method described in Microbial Cell Factories, 2010, 9: 34-43. Table 1 is the comparison of the fermentation results of Escherichia coli engineering bacteria ...

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Abstract

The invention discloses engineered Escherichia coli for high yielding capsular polysaccharide and a method for producing chondroitin by the aid of the capsular polysaccharide. Synthetic gene cluster transcriptional regulation factor slyA protein of the capsular polysaccharide is over expressed in the Escherichia coli, and expression of a synthetic gene cluster of the capsular polysaccharide in the Escherichia coli is promoted, so that the capsular polysaccharide produced by the Escherichia coli K4 is increased. The yield of the capsular polysaccharide after fermenting and culturing the engineered Escherichia coli is 112% higher than that of capsular polysaccharide produced by original bacteria, and the chondroitin with purity higher than 93% is obtained by means of collection and preparation. The engineered Escherichia coli and the method lay a certain foundation for developing fermentation production of chondroitin sulfate.

Description

technical field [0001] The invention relates to a genetically engineered bacterium producing high chondroitin and a construction method thereof, belonging to the field of genetic engineering. The invention relates to a method for producing chondroitin and belongs to the field of fermentation engineering. Background technique [0002] Chondroitin (Ch) is unsulfated chondroitin sulfate (Chondroitin Sulfate, CS), which is composed of glucuronic acid (D-GlcUA) and N-acetylgalactosamine (GalNAc) with β-1,3 bonds Polysaccharides linked alternately with β-1,4 bonds. Can be sulfated at its active site to form chondroitin sulfate. At present, the industrialized production of chondroitin sulfate is to use animal cartilage as raw material to produce chondroitin sulfate by extraction. However, due to the limited source of raw materials (the production cycle of animal cartilage is long), the industrial level is not high (the process is cumbersome, the yield is not high, and the qualit...

Claims

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Application Information

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IPC IPC(8): C12P19/04C12N15/31C12N1/21C12N15/63C07K14/245C12R1/19
Inventor 刘立明吴秋林杨爱华刘杰
Owner JIANGNAN UNIV
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