In Vitro Reconstitution Of Mevalonate Pathway Via Yeast Surface Display For Synthesis Of Geraniol

Medicinal plants produce a large number of secondary metabolites with rich species and specific structures,which are widely employed to preventing and treating various diseases.Terpenoids are so important sources of modern drugs that have great research value.The limited content of terpenoids in most plants severely limits its application.Although some terpenoids can be synthesized by chemical synthesis,which has some disadvantages such as high energy consumption,low yield and heavy environmental pollution,it is still difficult to synthesize terpenoids with complex structures.Synthetic biology provides effective means for the synthesis of terpenoids and other natural products.At present,it is the main research direction of synthetic biology to synthesize some important natural products by using microbial cell factories,some of which have reached the level of industrial application.Due to the influence and limitation of complex background metabolic background,the requirements of cell factory technology are extremely demanding,and a set of mature technologies often need to be accumulated for many years.As an extension of biological engineering,cell-free metabolic engineering has a broad application prospect.The immobilization of renewable biological enzymes is a mature and applied industrial technology,which can realize the repeated use of enzyme,and has the characteristics of cost saving and environmental pollution reduction.Yeast surface display(YSD)technology is a molecular biological tool to display exogenous molecules covalently on the surface of yeast.It can be used to display catalytic enzymes to form biological immobilized enzymes,which avoid complex and rigorous enzyme expression and purification process of enzymes,and improve the catalytic activity and stability of enzymes.In this study,using the surface display technology of Saccharomyces cerevisiae,the 8 enzymes of the mevalonate(MVA)biosynthesis pathway,acetyl-Co A synthase and geraniol synthase were innovatively expressed on the surface of yeast.And then a reusable in vitro multi-enzyme catalytic system was build to synthesize monoterpene geraniol via YSD immobilized enzymes,providing new ideas and technical means for the future biosynthesis of geraniol and other terpenoids.The main results are as follows:1.Enzymes in the MVA synthesis pathway,including Atob,Erg13,mva A,Erg12,Erg8,Erg19,Idi1 and Erg20,as well as truncated geraniol synthase Ges,were immobilized to the cell surface of Saccharomyces cerevisiae EBY100 applying a a-agglutinin surface display system,which produced biological immobilized enzymes.Geraniol was successfully synthesized using 1 mmol/L acetyl-co A as the substrate with the maximum yield of 0.32 ± 0.02 mg/L.It was proved that the bio-immobilized enzyme constructed in this study has the expected catalytic function2.By optimizing the proportion of 9 kinds of biological immobilized enzyme,fixing catalytic enzyme Erg20 catalytic activity,coupling NADPH and ATP regeneration module as well as pyrophosphoric acid removal module,effects of substrate channel and enzyme’s proximity from dual bio-immobilized enzymes,efficiency of geraniol synthesis module was improved by more than 6.8 times,the maximum yield of 2.17 ± 0.09 mg/L.The above experimental results indicate that the in vitro multi-enzyme catalytic system constructed in this study has a large space for optimization,which should be carried out based on the overall situation.3.Geraniol was successfully synthesized using acetyl-co A synthetase(Acs2)with 5 mmol/L sodium acetate as substrate in the multi-enzyme catalysis system.The maximum yield of geraniol reached 1.31 ± 0.08 mg/L after 24 h.Through module immobilization,geraniol synthesis system in vitro was reused,after 7 times of reuse,the highest yield of geraniol reached 7.55 ± 0.43 mg/L.The above experimental conclusions indicate that the biological immobilized enzyme constructed in this study can be reused and can maintain the biological activity of the enzyme at a higher level,which is beneficial to reduce the production cost of the purified enzyme.