Constructed Engineering Bacterium To Express Resveratrol Synthase And Optimized The Fermentation Conditions

Resveratrol is one of phytoalexins, belong to the non-flavonoid polyphenolcompounds. It is widely exists in many kinds of plants especially in Hu Zhang andgrape. Biology and medical research show that resveratrol has many pharmacologicalactivities and the healthcare function are beneficial to human health, such asanti-tumor, antioxidant, antiviral, anti-inflammatory, antibacterial, protect liver, treatcardiovascular disease and regulate estrogen content. Existing resveratrol productionis mainly the natural plant extraction and chemical synthesis, but there have relativelylimited. Therefore, the method of using genetic engineering is one of the feasibleways to improve the Res content in plants.This study first searched the resveratrol synthase gene coding sequence in thegrapes by NCBI (ID:AB046375). According to the gene sequence designed specificprimers and using cDNA from grape as a template, specific amplificated theresveratrol synthase gene by PCR (polymerase chain reaction) technique, it’s about1179bp. Resveratrol synthase gene was cloned into the prokaryote expression vectorpET22b which not contain the signal peptide. Using the pET22b-RS transferred intoE.coli DH5a and got the cloned strain DH5a-pET22b-RS. Sequencing was right andpreserved strain. Then the expression vector pET22b-RS transferred into E.coli BL21and obtained the genetic engineering bacterium BL21-pET22b-RS. The expression ofRS was induced by IPTG and using SDS-PAGE electrophoresis detected about42.9KDa recombinant protein, the same as the report.In the second part, we discussed the fermentation conditions of recombinationstrain BL21-pET22b-RS. By HPLC (high performance liquid chromatography)detection methods of resveratrol, detected production of resveratrol after fermentationthrough added the substrate of p-Coumaroyl-CoA and Malonyl-CoA and studied theinfluence of some important factors on the fermentation to optimized of theproduction of resveratrol. The results showed that: culture medium used LB medium,with2%glycerin. When the seed age of12h, inoculation amount to5%,0.1mMIPTG, culture temperature of30℃, pH6.5, the fermentation time of24h, resveratrolhad the highest yield can be up to25.21mg/L.In our study, we successfully constructed with resveratrol synthase geneengineering bacterium BL21-pET22b-RS, and optimized the fermentation conditions.It will contribute to using microorganism to produce resveratrol industrialization.