Mining And Identification Of Triterpene Synthase And Sesquiterpene Synthase Parts

Terpenoids are a large class of natural products widely found in plants,which are complex in structure and diverse in function.They not only play an important role in the interaction between plants and the environment,but also are used in the fields of medicines,perfumes,seasonings and industrial raw materials.In recent years,with the development of disciplines such as synthetic biology and bioinformatics,the research on the synthesis pathways of terpenoids has become more and more in-depth,and the mining and identification of related enzymes has become a research hotspot.The common precursor of terpenoids,isopentenyl diphosphate?IPP?and its isomer,dimethyl propylene diphosphate?DMAPP?,generate different steroid precursors under the action of prenyltransferase.Further,the terpene synthase catalyzes them to form different terpenoids skeletons,such as monoterpenes(C₁₀),sesquiterpenes(C₁₅),diterpenes(C₂₀),sesquiterpenes(C₂₅),triterpenoids(C₃₀)and so on.The traditional way to discover new terpenoids depends on the separation and identification of compounds extracted from plants.However,accumulation of many terpenoids may take a long time and be greatly affected by plants physiology and their environmental factors.Therefore,it becomes more and more difficulty to discover novel terpenoids by this traditional method.In the bioparts involved in biosynthetic pathway of terpenoids,terpene synthases are responsible for catalyzing the cyclization of common precursors,which is the determining step to yield the diverse structures of skeletons of terpenoids.Hence,the functional characterization of the terpene synthase encoded by the plant genome could provide a new method and means for systematical survey of novel terpene skeleton from plants.Enhancement of the terpenoid-producing precursor pathway of yeast chassis cells by synthetic biology techniques can provide the required chassis for systemic mining and identification of terpene synthase.All the terpene synthase encoded by the plant could be annotated from genome data and then could be introduced into the yeast chassis cells which could provide enough precursors?squalene-2,3-epoxide?for functional characterization of triterpene synthases.This"plug and play"method for the identification of terpene synthase provides new opportunities for the discovery of novel terpenoid skeletons.In this paper,we collected the functionally identified triterpene synthase bioparts and analyzed the correlation between their sequences and functions.It was found that triterpene synthase was relatively conservative in evolutionary relationship.Taking grain crop Zea mays as an example,preliminary exploration of mining the triterpenoid skeletons was carried out and find two triterpene synthase candidate genes up to now.The retention times of products of these two triterpene synthase are different from the common triterpenoids existing in our research group,which suggested that further structural identification is required.The structure of the zea3-2 compound was identified as simiarenol by and nuclear magnetic and mass spectrometry analysis.For the first time,we identified simiarenol in Zea mays and mined the triterpene synthase,which provided the necessary biopart for the future synthesis of simiarenol using synthetic biology techniques,and confirmed the advantages of this mining method,which is helpful for mining triterpene synthases with new functions.By collecting functionally identified sesquiterpene synthase bioparts,it was found that their sequences and functions were not closely related.Therefore,monoterpene synthase and diterpene synthase bioparts were further collected,and phylogenetic trees were constructed together with triterpene synthase and sesquiterpene synthase bioparts to analyze the evolutionary relationship of sesquiterpene synthase in terpene synthase.It was found that it can be clustered with monoterpene synthase and diterpene synthase in the terpene synthase phylogenetic tree,although there are crosses between the clusters.Therefore sesquiterpene synthase may be classified into defined by functional clusters,and then the representative sequences were selected from each OTU for expression.Taking Panax notoginseng as an example,a representative sequence of 10 OTUs belonging to 5 functional clusters of sesquiterpene synthases were cloned,and the representative sequence of 7 OTUs were expressed in a yeast chassis which could supply enough FPP for characterization of sesquiterpene synthases,however most of them fail to express or poorly expressed in this yeast chassis.Therefore,the expression system for sesquiterpene synthase still need to be optimized in future study.In the above studies,by systematically collating and analyzing the relationship between the known functions of terpene synthase sequences and functions.A systematic method for the identification of plant triterpenoids and sesquiterpene skeleton compounds in combination with synthetic biology techniques was proposed,which are expected to facilitate the systematic mining of triterpene and sesquiterpene skeleton compounds in plants.