Escherichia coli engineering bacterium for synthesizing pinocembrin by using glucose as substrate and application thereof

A technology of Escherichia coli and engineering bacteria, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of high price, inability to apply large-scale industrialization, and commercial application of cinnamic acid

Active Publication Date: 2013-02-20
湖南鸿健生物科技有限公司
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the industrial production of pinocin mainly adopts methods such as plant extraction and chemical synthesis, but how to obtain the required high-purity flavonoids from the complex extracts of plants is still a technical problem that cannot be solved by human beings. Factors such as harmful chemical reagents and the complex structure of flavonoids limit the use of chemical methods. For this reason, microbial fermentation is favored by the use of cheap and non-polluting raw materials, low pollution emissions, and low energy requirements. more and more attention
At present, domestic and foreign studies on the production of pinecone by microbial fermentation all need t

Method used

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Experimental program
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Effect test

Embodiment 1

[0028] The selection of embodiment 1 pathway gene

[0029]The L-Phe biosynthetic pathway with glucose as substrate consists of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) in 3-deoxy-D-arabinoheptonate 7-phosphate synthase ( The condensation of 3-deoxy-D-arabinoheptonate 7-phosphate (DAHP) under the catalysis of DAHP synthases (DS) is the first rate-limiting reaction. DS is three isozymes encoded by aroF, aroG and aroH genes respectively, and the composition ratio of enzyme activity is aroF:aroG:aroH=80:1:20. The expression and enzymatic activity of the three isozymes (aroF, aroG, and aroH) were subject to feedback repression and feedback inhibition by the products L-Tyr, L-Phe, and L-Trp, respectively. The second and third rate-limiting reactions are the conversion of chorismate to prephenate under the action of chorismate mutase (CM), followed by the action of prephenate dehydratase (Prephenate dehydratase, PDT). PPA is formed after dehydration and decarboxyla...

Embodiment 2

[0032] The synthesis of embodiment 2 pathway gene and the construction of expression vector

[0033] According to Genbank on the NCBI website: NC000913.2, GQ303716, AAC83455, AAC83457, DQ013364, X13325, AF233638, M91079, using the whole gene synthesis method to obtain the eight genes required for the pathway, the sizes are 1.5bp, 1.2bp, 2.1bp , 1.7bp, 1.5bp, 0.9bp, 1.5bp, 1.3bp. The aroF, pheA fbr The gene was connected with the plasmid pRSFDuet-1 (Novagen: 71363) digested with EcoRI and HindIII, NdeI and KpnI, and the PAL, 4CL gene was digested with NcoI and HindIII, NdeI and BlnI. The plasmid pETDuet-1 (Novagen: 71353) connection, the CHS, CHI genes were connected with the plasmid pCDFDuet-1 (Novagen: 71330) digested with NcoI and HindIII, NdeI and BlnI, and the matB and matC genes were digested with EcoRI and HindIII, NdeI and KpnI The plasmid pACYCDuet-1 (Novagen: 71430) was ligated to obtain four co-expression plasmids pRSFD-pheA-aroF, pET-RgPAL-Pc4CL, pCDF-PhCHS-MsCHI,...

Embodiment 3

[0038] LB+100mg ampicillin, 50mg kanamycin, 35mg chloramphenicol, 50mg streptomycin, add 20g agar to the solid medium. Embodiment 3 Fermentation produces the method for pinecone

[0039] The Escherichia coli engineering bacteria were used as the starting strain, inoculated into MOPS medium, 25 mL of liquid in a 500 mL shake flask, and cultivated at 37 ° C and 250 rpm for 12-20 h to strain OD 600 reach 1.0-2.0; then add 25ml of the same MOPS medium and IPTG with a final concentration of 1mM, and culture for 60h at 30°C and 250rpm.

[0040] Comparing the recombinant bacteria with the control bacteria, the production of pinocin was not detected in the control bacteria, while the production of pinocin in the recombinant bacteria reached 21mg / L.

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Abstract

The invention discloses an Escherichia coli engineering bacterium for synthesizing pinocembrin by using glucose as a substrate and application thereof and belongs to the field of genetic engineering or metabolic engineering. Synthetic biology and metabolic engineering means are adopted to respectively synthesize a synthesis route consisting of eight genes in pinocembrin metabolism to be introduced into Escherichia coli (BL21), accordingly the recombinant bacterium for synthesizing the flavone framework material pinocembrin by using the glucose as the substrate, and the pinocembrin yield reaches to 21mg/L after glucose serves as the unique substrate in a shake flask to be fermented for 60 hours. The synthesis route consisting of the eight genes is successfully built in the Escherichia coli, and synthesis from the glucose into the pinocembrin is achieved successfully. Certain reference significance is provided for flavonoid material production by means of a microbiological method in the future.

Description

technical field [0001] The present invention relates to a kind of Escherichia coli engineering bacterium producing pinesin, which is an overexpressed aroF and pheA by means of synthetic biology and metabolic engineering fbr , PAL, 4CL, CHS, CHI, matB, matC and other eight gene synthesis pathways to realize the Escherichia coli engineering bacteria that use glucose as a substrate to produce pinocin. Background technique [0002] Flavonoids (flavonoids) are a class of compounds that exist in nature and have a 2-phenylchromone structure. Most of their hydroxy derivatives are yellow, hence the name. Flavonoids have a variety of biological activities, mainly including cardiovascular system, antibacterial and antiviral, antitumor, antioxidative free radicals, anti-inflammation, etc. Prevention, treatment or auxiliary treatment effects, and the human body cannot synthesize flavonoids and can only be obtained from plant foods. Therefore, in recent years, the market of flavonoids ha...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P17/06C12R1/19
Inventor 陈坚周景文吴俊俊堵国成
Owner 湖南鸿健生物科技有限公司
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