Genetic engineering strain taking tyrosine as substrate to synthesize naringenin and construction method thereof

A technology of genetically engineered bacteria and tyrosine, applied in the field of synthetic biology or metabolic engineering, can solve the problems of low solubility and high price of coumaric acid

Active Publication Date: 2014-01-01
湖南鸿健生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the industrial production of naringenin mainly adopts the method of plant extraction, but how to obtain the required high-purity naringenin from the complex plant extract is still a technical problem that cannot be solved by human beings, and high pollution, use of toxic and Factors such as harmful chemical reagents and the complex structure of naringenin limit the use of chemical methods. For this reason, microbial fermentation methods are favored by the use of cheap and non-polluting raw materials, lower pollution emissions, and lower energy requirements. more and more attention
At present, domestic and foreign st

Method used

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  • Genetic engineering strain taking tyrosine as substrate to synthesize naringenin and construction method thereof
  • Genetic engineering strain taking tyrosine as substrate to synthesize naringenin and construction method thereof
  • Genetic engineering strain taking tyrosine as substrate to synthesize naringenin and construction method thereof

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Embodiment 1

[0029] The selection of embodiment 1 pathway gene

[0030] Phenylalanine deaminase (TAL) widely exists in higher plants, fungi, yeast and a kind of prokaryotic streptomyces. Human beings have not found this enzyme in eubacteria or animal tissues so far. Although TALs are widely distributed, only R. glutinis is used commercially, and R. glutinis has a higher catalytic activity for tyrosine than other species. In recent studies, 4-coumaric acid: Coenzyme A ligase (4CL) from P. hybrida, chalcone synthase (CHS) from P. hybrida, Combination of chalcone isomerase (CHI) to obtain various unnatural flavonoid skeleton substances. Therefore, the present invention selects TAL of Rhodotorula glutinis, 4CL of parsley (Petroselinum crispum), CHS of petunia (Petunia X hybrida), and CHI (M91079) of alfalfa (Medicago sativa).

[0031]The development of natural product production platforms is usually limited by precursor substances or cofactors, because the amount of these substances in the h...

Embodiment 2

[0033] Example 2 Synthetic route optimization based on modular transformation theory

[0034] For the constructed synthetic pathway, this study intends to divide it into three modules, mainly based on the following principles: i) The product of 4CL, coumaroyl-CoA / cinnamoyl-CoA, will inhibit the TAL gene in the pathway, so TAL and 4CL is divided into one module, and CHS and CHI are divided into one module. It is intended to alleviate this feedback inhibition by strengthening the downstream module and weakening the upstream module; Low content usually limits the production of flavonoids in bacteria, so the malonyl-CoA synthesis pathway (matB and matC) is placed in a separate module, and the optimal malonyl-CoA content is found by changing the metabolic flux of this module . Therefore, the total approach is divided into three modules: module one is composed of TAL and 4CL; module two is composed of CHS and CHI; module three is composed of matB and matC. The metabolic flux of ea...

Embodiment 3

[0036] Embodiment 3 contains the construction of optimized module engineering bacteria

[0037] The six genes needed for the pathway were obtained by using the whole gene synthesis method, and the sizes were 2.1Kb, 1.7Kb, 1.5Kb, 0.9Kb, 1.5Kb, and 1.3Kb, respectively. Ligate the TAL, 4CL genes with the plasmids digested with NcoI and HindIII, NdeI and BlnI respectively, connect the CHS and CHI genes with the plasmids digested with NcoI and HindIII, NdeI and BlnI, and connect the matB The matC gene was connected with the plasmid digested with EcoRI, HindIII, NdeI and KpnI, thus obtaining three co-expression plasmids pCDF-RgTAL-Pc4CL, pET-PhCHS-MsCHI, and pACYCD-matB-matC. The constructed recombinant plasmid was digested and analyzed, and DNA sequencing was carried out. The results of gene sequencing were consistent with expectations, indicating that the recombinant plasmid was constructed correctly. The three co-expression plasmids were chemically transformed into Escherichia ...

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Abstract

The invention discloses a genetic engineering stain taking tyrosine as a substrate to synthesize naringenin and a construction method thereof, belonging to the field of synthetic biology or metabolic engineering. According to the construction method disclosed by the invention, a synthesis route constituted by six genes, namely a gene matB encoding a malonic acid transporting enzyme, a gene matC encoding a malonic acid absorption route, a gene encoding a tyrosine ammonialyase (TAL), the gene encoding a 4-cinnamic acid: coenzyme A ligase (4CL), the gene encoding a chalcone synthase (CHS) and the gene encoding a chalcone isomerase (CHI) is introduced into Escherichia coli BL21 to obtain a recombinant strain capable of taking the tyrosine as the substrate to synthesize the naringenin, the synthesis route is further optimized through a modular transformation theory, the strain is finally fermented in a shaking flask for 72 hours by taking the tyrosine as the substrate, and the yield of the naringenin achieves 90mg/L. A strategy adopted by the construction method disclosed by the invention provides certain reference significance for future production of natural small molecular substances by a microbiological method.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for synthesizing naringenin with tyrosine as a substrate and a construction method thereof, belonging to the field of synthetic biology or metabolic engineering. 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 for flavonoids has grown by more than 30% every year, and has a broad market in the field of pharmaceuticals and nutritional chemicals. application prospects. [0003] Flav...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P17/06C12R1/19
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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