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Gene engineering bacteria with high-yield malonyl coenzyme A and construction method and application thereof

A technology of malonyl coenzyme and genetically engineered bacteria, applied in the field of genetically engineered bacteria producing high malonyl coenzyme A and its construction, can solve the problems of lack of in-depth excavation of related genes, lack of integration of multiple pathways, etc., to achieve improved synthesis efficiency effect

Active Publication Date: 2020-01-21
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These studies have increased the content of malonyl-CoA in microorganisms to a certain extent, but there is a lack of in-depth excavation of the genes related to the pathway, and the lack of effective integration of multiple pathways

Method used

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  • Gene engineering bacteria with high-yield malonyl coenzyme A and construction method and application thereof
  • Gene engineering bacteria with high-yield malonyl coenzyme A and construction method and application thereof
  • Gene engineering bacteria with high-yield malonyl coenzyme A and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Base cell construction for knockout of five acetyl-CoA efflux pathway genes

[0047] Strains and plasmids

[0048] Escherichia coli (Escherichia coli JM109) was used for the replication of the plasmid, and Escherichia coli BL21 (DE3) was used for the expression of the pathway plasmid and strain fermentation. The strains were purchased from Novagen (Darmstadt, Germany). Plasmids pCDFDuet-1, pETDuet-1, pCOLADuet-1, pACYCDuet-1 were purchased from Novagen (Darmstadt, Germany).

[0049] Enzymes and other reagents

[0050] Various antibiotics including ampicillin and chloramphenicol were purchased from Shanghai Sangong Co., Ltd. All chemical reagents were of analytical grade and purchased from Shanghai Sangong Co., Ltd. Various restriction endonucleases and DNA ligases were purchased from Thermo. 1kb DNA Ladder, Plasmid Small Extraction Kit, Gel Recovery Kit, and Bacterial Genome Extraction Kit were purchased from Shanghai Sangong Co., Ltd.

[0051] Plasmid extraction w...

Embodiment 2-4

[0079] Fermentation:

[0080] The engineered bacteria were first cultivated in LB medium on a shaker (37°C, 200rpm) overnight, and then the initial OD 600 The inoculum size of 0.1 was retransferred to fresh MOPS fermentation medium, and carbon source glucose with a final concentration of 5g / L was added to the fermentation system at the same time, and the shaker culture (37°C, 200rpm) until the OD600 was 0.5. At this moment, induce with the IPTG (Isopropyl-β-D-thiogalactopyranoside) final concentration of 1mmol / L, and carry out the feed feeding that final concentration is 5g / L glucose, final concentration is the substrate sodium acetate of 1g / L and final concentration is 2.5 g / L substrate tyrosine was fed and fermented for 52 hours in a shaker culture (30° C., 200 rpm).

[0081] Acetyl-CoA content detection

[0082]Take 1 mL of the fermentation broth, centrifuge at 13,000 rpm, 4°C for 10 min, and discard the supernatant. Resuspend the lysed cells with 1 mL of 6% perchloric a...

Embodiment 2

[0086] Overexpression of acetyl-CoA synthetases from different sources

[0087] Escherichia coli (Escherichia coli JM109) was used for the replication of the plasmid, and Escherichia coli BL21 (DE3) was used for the expression of the pathway plasmid and strain fermentation. The strains were purchased from Novagen (Darmstadt, Germany). Plasmids pCDFDuet-1, pETDuet-1, pCOLADuet-1, pACYCDuet-1 were purchased from Novagen (Darmstadt, Germany). Saccharomyces cerevisiae ATCC9080 was purchased from Shanghai Qincheng Biotechnology Co., Ltd., Yarrowia lipolytica ATCC9773 was purchased from Shanghai Kewei Chemical Technology Co., Ltd., Salmonella enterica ATCC10723 was purchased from Beijing Biobowell Biotechnology Co., Ltd. and Corynebacterium glutamicum ATCC13032 was purchased from Beijing Qiangxin Borui Biotechnology Co., Ltd. Pseudomonas aeruginosa ATCC27853 was purchased from Shanghai Hengfei Biotechnology Co., Ltd.

[0088] In order to obtain the acs gene (Gene ID: 948572) encodi...

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Abstract

The invention discloses a gene engineering bacteria with high-yield malonyl coenzyme A and a construction method and application thereof. The gene engineering bacteria is constructed by: knocking outfive genes (ldhA, pta, frdA, poxB and adhE) in the Escherichia coli genome, and then introducing malonyl coenzyme A synthesis pathway genes including acetyl coenzyme A synthetase gene of the Escherichia coli, acetyl coenzyme A carboxylase gene of Salmonella enteritidis and biotin ligase gene of Corynebacterium glutamicum. According to the gene engineering bacteria of the invention, a highly-efficient accumulation of the malonyl coenzyme A can be realized by inhibiting internal acetyl coenzyme A outflow pathways of the engineering bacteria and constructing malonyl coenzyme A synthesis pathwaysinto different expression vectors to be transferred into the engineering bacteria; the engineering bacteria can efficiently synthesize a precursor, malonyl coenzyme A, of flavonoid compounds by takingacetic acid, a metabolite by-product of the Escherichia coli, as a substrate; and the engineering bacteria can be used to increase the yield of naringenin, a skeleton precursor of the flavonoid compounds, synthesized by a microbiological method.

Description

technical field [0001] The invention belongs to the technical field of gene recombination, and in particular relates to a strain of genetically engineered bacteria with high malonyl-CoA production and its construction method and application. Background technique [0002] Malonyl coenzyme A (Malonyl coenzyme A), also known as malonyl coenzyme A, is a derivative of coenzyme A and is a key compound in the metabolic pathway of microorganisms. In the metabolic network of Escherichia coli, glucose is used as carbon source to generate acetyl-CoA through glycolysis pathway, most of acetyl-CoA is metabolized into carbon dioxide and water through citric acid cycle, and a small part of acetyl-CoA is metabolized by fatty acid The pathway is converted to malonyl-CoA by acetyl-CoA carboxylase, and is widely involved in the metabolic activities of many important substances such as lipids, carbohydrates and amino acids. At the same time, malonyl-CoA is also an important signaling molecule,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/52C12P17/06C12P19/32C12R1/19
CPCC12N9/93C12N9/00C12N15/70C12P17/06C12P19/32C12Y604/01002C12Y602/01003
Inventor 吴俊俊周朋包美娇董明盛
Owner NANJING AGRICULTURAL UNIVERSITY
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