Fusion Expression Of The 4CL And RS Genes Of Anabolic Key Enzymes Of Resveratrol

With damage, fungal infection or ultraviolet radiation on some plants, the resveratrol, an important secondary metabolite, was produced as a phytoalexin. As an active ingredient in red wine, it had many benefits for human health, i.e., anti-coagulant, anti-oxidation, anti-inflammatory, anti-cancer, cardiovascular protection, and neuroprotection. However, the content of resveratrol in plants was generally very low, which made it difficult to meet market demands. Resveratrol biosynthesis is currently a hot topic. Based on this method, the 4-coumarate coenzyme A ligase(4CL) gene from Arabidopsis thaliana and resveratrol synthase(RS) gene from Arachis hypogaea were cloned, fused, and put into one expression box using gene fusion technology to construct a bifunctional enzyme. In this way, resveratrol can be synthesized by adding an appropriate substrate. Finally, the p-coumaric acid was added to detect the activity of the bifunctional enzyme, and to achieve the synthesis of resveratrol in microorganism. The main results were as following:1. Study on the 4CL gene from Arabidopsis thaliana: The 4CL gene was cloned from Arabidopsis thaliana with the 4CL-F1 and 4CL-R1. Subsequently, the 4CL gene was analysed and identified as an open reading frame of 1686 bp encoding a protein of 561 amino acids. A prokaryotic expression vector containing BamHⅠand Hind Ⅲ restriction sites, pET-30a/4CL, was constructed and transformed to Escherichia coli BL21(DE3). Then the recombinant bacteria were identified by bacteria liquid PCR, enzymes digestion and sequencing. After adding the 0.3 mM IPTG, the recombinant strains were induced to express the target protein that was approximate 66 kDa in molecular weight. Further analysis showed that the induced protein was a soluble protein. This provided a theoretical basis for the induce temperature in prokaryotic microbial fermentation.2. Study on the RS gene from Arachis hypogaea: The RS gene was cloned from luhua14(Arachis hypogaea) with the RS-F1 and RS-R1. Subsequently, the RS gene was analysed and identified as an open reading frame of 1170 bp encoding a protein of 389 amino acids. A prokaryotic expression vector containing BamHⅠand SalⅠrestriction sites, pET-30a/RS, was constructed and transformed to E. coli BL21(DE3). Then, the recombinant bacteria were identified. After adding the 0.3 mM IPTG, the recombinant strains were induced to express the object protein at 28℃. Further analysis showed that the molecular weight of the induced protein was approximate 43 kDa, and the induced protein was a soluble protein.3. Study on the 4CL:RS fusion gene: The flexible linker, 15-amimo-acids chain, was designed to connect 4CL gene and RS gene. The sequence of linker was designed as(GGGGS)3. The nucleotide sequences of the flexible linker were designed as follows: ggt gga ggc gga tcc ggc gga ggt ggc tct ggc ggt ggc gga tcg(sequences underlined are BamHⅠrestriction site). According to the sequences of the 4CL gene and RS gene, five specific primers were designed as 4CL::RS-F1, 4CL::RS-R1, 4CL::RS-F21, 4CL::RS-F22 and 4CL::RS-R2. The 4CL::RS fusion gene was constructed by PCR, BamHⅠdigestion and ligation. A prokaryotic expression vector containing Nco Ⅰ and EcoR Ⅰ restriction sites, pET-30a/4CL::RS, was constructed and transformed to E. coli BL21(DE3). Then, the recombinant bacteria were also identified. After inducing with 28℃ and 8 h, the fusion protein was isolated and purified using ProteinIsoTM Ni-NTA Resin, and then was analysed by Western blot. The results further verified that the prokaryotic expression vector, pET-30a/4CL::RS, expressed the target fusion protein 4CL::RS successfully.4. Study on the biosynthesis of the resveratrol: To detect the resveratrol derived from the recombinant bacteria, the HPLC method was established. After 48 h of growth in LB media at 28℃, 80.524 mg/L of the resveratrol was detected in the culture media with 1 mM p-coumaric acid, equal to a 35.28% conversion yield(mol/mol). In this study, the results showed the fusion protein expressed from the recombinant strains had the activities of both enzymes(4CL and RS).In the present study, the 4CL gene and RS gene were linked by restriction site for the first time. In vitro, the results of enzymatic reaction indicated that the fusion protein expressed from the recombinant strain possessed the bifunctional activities. Finally, the biosynthetic pathway of resveratrol was eventually established in E. coli.