Chondroitin producing genetic engineering bacterium, method for constructing same and application of chondroitin producing genetic engineering bacterium

A technology of genetically engineered bacteria and chondroitin, applied in the field of genetic engineering

Active Publication Date: 2019-03-19
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no reports of using Corynebacterium glutamicum as a host to prepare chondroitin and its derivatives

Method used

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  • Chondroitin producing genetic engineering bacterium, method for constructing same and application of chondroitin producing genetic engineering bacterium
  • Chondroitin producing genetic engineering bacterium, method for constructing same and application of chondroitin producing genetic engineering bacterium
  • Chondroitin producing genetic engineering bacterium, method for constructing same and application of chondroitin producing genetic engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1 is used for the situation of target gene overexpression in corynebacterium glutamicum each promoter

[0080] In order to use strong promoters to introduce chondroitin synthesis system in recombinant Corynebacterium glutamicum, the strength of each promoter was first evaluated by using green fluorescent protein as a reporter gene. Constructed the promoters Pddh (promoter of diaminopimelate dehydrogenase gene), PdapA (promoter of dihydropicoline synthase gene), Psod (promoter of superoxide dismutase gene), Pfba (promoter of fructose-1,6-bisphosphate lyase gene), Ptuf (promoter of tuf transcription elongation factor gene, super promoter in Corynebacterium glutamicum transcriptome) and the E. coli hybrid carried by pXMJ19 The expression vector of green fluorescent protein with the promoter Ptac.

[0081] Construction methods of green fluorescent protein expression vectors containing different promoters:

[0082] First construct the plasmid pXMJ19-Ptac-GFPuv. U...

Embodiment 2

[0106] Example 2 Construction of Corynebacterium glutamicum-Escherichia coli shuttle plasmid carrying chondroitin synthase KfoC and UDP-glucosamine isomerase KfoA genes and recombinant bacteria C.glu-CA, C.glu-Δldh-CA

[0107] Construction of plasmid pXMJ19-CgKfoCA: Based on the codon preference of Corynebacterium glutamicum, a new chondroitin synthase KfoC gene CgkfoC (Wuxi Qinglan Biotechnology Co., Ltd.) was designed and chemically synthesized. Its sequence is shown in SEQ ID No: 1 and a new UDP-glucosamine KfoA gene CgkfoA, the sequence of which is shown in SEQ ID No.2.

[0108] The preferred ribosome recognition sequence (RBS) (AAAGGAGGACACAT) of Corynebacterium glutamicum was added before each gene, and an XbaI restriction site was added before CgkfoC, and a KpnI restriction site was added after CgkfoA. Using C-s and A-as as upstream and downstream primers, PCR reaction was carried out to amplify the gene RBS-CgkfoC-RBS-CgkfoA fragment. Primers were synthesized by Plati...

Embodiment 3

[0113] Example 3 Construction of recombinant bacteria C.glu-CAU and C.glu-Δldh-CAU overexpressing RBS-udgA gene and verification of gene expression effect

[0114] The construction of plasmid pXMJ19-CgKfoCAU: U-s and U-as were used as upstream and downstream primers, and the genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template to obtain UDP-glucose containing the preferred ribosome recognition sequence (RBS, AAAGGAGGACACAT) of Corynebacterium glutamicum Dehydrogenase gene fragment RBS-ugdA gene. Primers were synthesized by Platinum Biotechnology (Shanghai) Co., Ltd., dissolved in sterile water and diluted to 10 μM for use. The pre-made buffer used in PCR amplification was purchased from Vazayme Company.

[0115] The PCR amplification reaction system is:

[0116]

[0117]

[0118]Thermal cycle conditions were 94°C for 3min; 94°C for 15s, 60°C for 15s, 72°C for 50s, 35 cycles; 72°C for 10min. The amplified product and plasmid pXMJ19-CgKfoCA were di...

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Abstract

The invention discloses a chondroitin producing genetic engineering bacterium, a method for constructing the same and application of the chondroitin producing genetic engineering bacterium, and belongs to the technical field of genetic engineering. Chondroitin synthetase genes and UDP (uridine diphosphate)-glucosamine isomerase genes are transferred into corynebacterium glutamicum to obtain the chondroitin producing genetic engineering bacterium. The chondroitin producing genetic engineering bacterium, the method and the application have the advantages that the corynebacterium glutamicum is used as a host for the chondroitin producing genetic engineering bacterium, is a GRAS (generally recognized as safe) strain affirmed by the Food and Drug Administration of the America, does not secreteoptional endotoxin or exotoxin, is safe and can be applied to producing amino acid or food additives and the like for a long term, and chondroitin produced by the chondroitin producing genetic engineering bacterium is high in molecular weight and good in yield; the chondroitin yield of preferable recombinant bacteria of the chondroitin producing genetic engineering bacterium can reach 3.7 g / L, chondroitin products with high molecular weights can be produced by the chondroitin producing genetic engineering bacterium, and accordingly the chondroitin producing genetic engineering bacterium has anexcellent industrial prospect and can be effectively applied to medicines and health protection.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium producing chondroitin, a construction method and application thereof. Background technique [0002] Chondroitin sulfate (CS) is a sulfated mucopolysaccharide widely found in mammals and invertebrates. Its backbone is a linear polysaccharide chondroitin that is alternately linked by D-glucuronic acid (GlcA) and N-acetyl-galactosamine (N-acetylgalactosamine, GalNAc). According to the different sulfation sites, it can be divided into different types such as CS-O, A, B, C, D, and E. Due to the excellent biocompatibility of CS and the important role it plays in human physiological activities, its application range is quite wide, mainly including the fields of medical health and food cosmetics. In the field of clinical medicine, CS is mainly used in the prevention and treatment of osteoarthritis. In addition, new research sh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P19/26C12R1/15
CPCC12N9/88C12N9/92C12N15/77C12P19/26C12Y402/02004C12Y503/01005
Inventor 于慧敏成方宇罗榆盛王苗苗
Owner TSINGHUA UNIV
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