Method for producing eriodictyol by reforming escherichia coli in metabolic engineering

A technology of Escherichia coli and eriodictyol, which is applied in the field of metabolic engineering, can solve problems such as poorly soluble expression, achieve the effect of reducing production costs and improving economic benefits

Active Publication Date: 2014-06-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is mainly due to the lack of cofactors of the P450 monooxygenase family in Escherichia coli - P

Method used

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  • Method for producing eriodictyol by reforming escherichia coli in metabolic engineering
  • Method for producing eriodictyol by reforming escherichia coli in metabolic engineering
  • Method for producing eriodictyol by reforming escherichia coli in metabolic engineering

Examples

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Embodiment 1

[0027] Example 1 Construction of tF3'H-tCPR expression vector

[0028] The MLRC secondary structure prediction method analyzed the protein secondary structure of flavone 3' hydroxylase (F3'H), and determined that the N-terminal 25aa may be the membrane binding site of F3'H. Primers were designed using pUC57-F3'H (F3'H was codon-optimized and synthesized by Nanjing GenScript Co., Ltd. and cloned into the pUC57 vector. The optimized F3'H sequence is shown in SEQ ID NO.5) as a template, and 1.47kb was amplified by PCR The target gene tF3'H with truncated membrane binding domain, NdeI restriction site at the 5' end, Gly-Ser-Thr linker sequence at the 3' end and 18bp CPR homology arm. Using pUC57-CPR as a template (CPR was codon-optimized and synthesized by Nanjing GenScript Co., Ltd. and cloned into the pUC57 vector, the optimized CPR sequence is shown in SEQ ID NO.6) PCR amplification to obtain a truncated membrane-binding domain with a size of 1.94kb Gene, the target gene tCPR ...

Embodiment 2

[0029] Embodiment 2 strengthens the construction of malonyl-CoA pathway

[0030] Using the Red homologous recombination knockout technology, the acetate kinase gene (ackA) in the E.coli genome is knocked out to block the acetate by-product pathway of acetyl-CoA and increase the flux of malonyl-CoA conversion. With plasmid pKD13 (purchased from CGSC http: / / cgsc.biology.yale.edu / ) as a template, the 1.40kb target fragment ackA::kan with 50bp upstream and downstream homology arms of the acetate kinase gene (ackA) at both ends was obtained by PCR (see SEQ ID NO.7 for the sequence). The ackA::kan fragment was electrotransformed carrying pKD46 (purchased from CGSC http: / / cgsc.biology.yale.edu / ) E.coli BL21(DE3) competent cells, because the successful knockout strain homologously recombined the ackA::kan fragment, and the recombinant bacteria had certain resistance to kanamycin. The knockout strains that can grow on the Kana plate were obtained after screening. The genome of t...

Embodiment 3

[0033] The construction of embodiment 3 eriodictymol engineering bacteria

[0034] The resulting recombinant expression vectors pACYC-tF3'H-tCPR and pRSF-acs-ACC were combined with pCDF-TAL-4CL and pET-CHS-CHI constructed in our laboratory (see literature for construction method: WuJ, Du G, Zhou J, Chen J.2012.Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy.Metab.Eng.16:48-55.) E.coli BL21 transformed together with the knockout acetate kinase ackA gene (DE3), through resistance plate screening and colony PCR identification, the eriodictyol engineering strain was obtained.

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Abstract

The invention discloses a method for producing eriodictyol by reforming escherichia coli in metabolic engineering, belonging to the field of metabolic engineering. The step of transforming naringenin into the eriodictyol is successfully realized through a gene engineering technology by carrying out fusion expression on two genes, namely a flavone 3' hydroxylase gene tF3'H with a membrane binding sequence cut and a P450 reductase gene tCPR, in escherichia coli. A tyrosine ammonia lyase gene TAL from R.glutinis, 4-coumarate:coenzyme A ligase gene 4CL from P.crispum, a chalcone synthase gene CHS from P.hybrida and a chalcone isomerase gene CHI from M.sativa are simultaneously expressed to realize that naringenin is directly produced through tyrosine, so that an engineered strain for directly producing eriodictyol through tyrosine is established. According to the method, in order to increase the output of eriodictyol, an acetokinase ackA gene contained in an escherichia coli genome is removed by being knocked, an acetyl coenzyme A synthetase gene acs and an acetyl coenzyme A carboxylase gene ACC are excessively expressed, and the output of eriodictyol can finally reach 106.7 mg/L.

Description

technical field [0001] The invention relates to a method for metabolic engineering transformation of Escherichia coli producing eriodictyol, belonging to the field of metabolic engineering. Background technique [0002] In recent years, the anti-oxidation, anti-inflammation and anti-cancer effects of flavonoids on human health and medicinal value have attracted more and more attention from researchers. Taking eriodictyol as an example, eriodictymol has antibacterial and anti-inflammatory effects, plays a vital role in the pathogenesis of diabetes, can inhibit IgE / Ag-induced type I allergic reactions, and also has analgesic and warming effects. [0003] Despite the numerous health benefits of flavonoids, the source of flavonoids is a major obstacle to their use in healthcare. Chemical synthesis is often accompanied by unfavorable factors such as toxic by-products and extreme reaction conditions, which are not conducive to large-scale production of flavonoids. It is expected...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P17/06C12R1/19
Inventor 陈坚周景文朱赛杰堵国成
Owner JIANGNAN UNIV
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