Genome Mining And High Efficiency Production Of Terpenoids In Microbes

Terpenoids comprise the largest family of natural products with high levels of structural diversity and play an important role in our daily life.Commercialized production of many terpenoids in microorganism was realized by the metabolic engineering and combinatorial biosynthesis strategy.Some of these compounds are widely used in perfume,health care products,agricultural production and medical industry.The mevalonate(MVA)pathway,the 2-C-methyl-D-erythritol-4-phosphate(MEP)pathway and the isopentenyl diphosphate isomerase(Idi)were used to produce the C5 precursors isopentenyl diphosphate(IPP)and dimethylallyl diphosphate(DMAPP).Followed,prenyltransferases assemble IPP and DMAPP into polyisoprenoid diphosphates with various lengths(GPP,FPP,GGPP and GFPP).Then terpene synthases cyclise these polyisoprenoid diphosphates to generate terpene products with structural diversity.Therefore,efficient IPP and DMAPP production platform are the committed step to produce terpenoids with high efficiency.Base on well established "Targeted Anabolism" strategy,the MVA pathway was engineered in E.coli,and the efficient IPP and DMAPP production platform was constructed.And as a proof of concept,taxadiene was successfully produced with limited steps of engineering in engineered E.coli,which laid a foundation for other diterpene production in these engineered IPP and DMAPP overproduction platform.Next,we transferred this platform into filamentous fungi Alternaria alternata and realized the stable production of taxadiene in this fungus.Before the engineering study of A.alternata TPF6,we first developed an Agrobacterium tumefaciens-mediated transformation(ATMT)method and verified the strength of heterologous promoters in Alternaria alternata TPF6.We next transformed the taxadiene-producing platform into A.alternata TPF6,and the MVA pathway was engineered,with introduction of the plant taxadiene-forming gene.Notably,by co-overexpression of isopentenyl diphosphate isomerase(Idi),a truncated version of 3-hydroxy-3-methylglutaryl-CoA reductase(tHMG1),and taxadiene synthase(TS),we could detect 61.9 ± 6.3 μg/L taxadiene in the engineered strain GB127.This is the first demonstration of taxadiene production in filamentous fungi,and the approach presented in this study provides a new method for microbial production of Taxol.The well-established ATMT method and the known promoter strengths facilitated further engineering of taxaenes in this fungus.In addition,we have also constructed an efficient precursor supply platform for terpenoids in S.cerevisiae,which laid the foundation for the efficient synthesis of terpenoids in S.cerevisiae.Terpene cyclase can catalyze polyisoprenoid diphosphates of different lengths to generate terpene products with a single ring or intricate multiple rings.Then,tailoring enzymes,including oxygenases,methyltransferases,acetyltransferases,and glycosyltransferases,add functional groups at different positions,further enhancing structural diversity to produce thousands of terpenoids.Consequently,digging new terpene cyclase is the committed step for us to expand the structural diversity of terpenoids.However,the discovery of terpene cyclases and relevant enzymes was far behind the process of terpenoids.The biosynthetic pathway of many high value added-terpenoids was still unknown,which hindered the metabolic engineering and commercialized production of these compounds.Genome sequencing and annotation provide a large number of terpenoids biosynthetic components with unknown function.And some of the terpene cyclases with inherent promiscuous property,can convert precursor polyisoprenoid diphosphates of different lengths to produce a variety of terpenoids.However,restricted by the low efficiency of heterologous overproduction platform,it is only possible for us to obtain the main products produced by these promiscuous terpene cyclise,which may covers the biosynthesis potential of terpenoids cyclase to a great extent.Therefore,develop an efficient terpenoids overproduction platform,and pay attention to those potential new terpene cyclases to discover new terpenoids is the key to accelerate the process of terpenoids discovery.Filamentous fungus Fusarium graryminearum J1-012 was genome sequenced and pomiscuous terpene cyclases of class I were predicted by the bioinformatic analysis.The terpene cyclase were predicted and verified by the in vitro assay.In this study,we first demonstrated that the promiscuous terpene synthases FgMS and FgGS can produce more variable terpenoids in vivo by converting precursor polyisoprenoid diphosphates of different lengths(C10,C15,C20,C25).To realise the full potential of these promiscuous terpene synthases,we developed a platform consisting of three parts:a terpene-overproducing chassis,which can provide sufficient IPP and DMAPP for terpenoids production;a prenyltransferases library,which can provide FPP,GGPP and GFPP for terpene cyclise;and the promiscuous terpene synthases of interest.Directed by this strategy,six strains was constructed to mining the terpenoids production ability of FgGS and FgMS.In total,52 terpenoids were generated and 12 of them were structurally characterized by NMR.Seven new terpenoids(1,2,5 and 7-10)were characterised by NMR and three new skeletons were revealed.Which indicate the powerful terpenoids production ability of FgMS and FgGS,and the high efficiency of the combinatorial biosynthesis platform.To further enhance product diversity,the three-dimensional model of the terpene cyclase domain of FgMS was built by homologous modelling.And the site-directed mutagenesis was performed by selecting the potentially noncatalytic sites within the pocket.As a result,6 potentially new sesterterpene(53-58)emerged from mutants F65L and F159G,and compound 56 was isolated and identified as a new sesterterpene.The rational combinatorial biosynthesis and protein engineering strategy enable us to fully exploit the biosynthetic potential of FgMS and FgGS,and accelerate the process of new terpenoids mining.At the same time,we also introduced the metabolic engineering strategy to facilitate the function verification of terpene cyclase and structural characterization of terpenoids.Promiscuous sesquiterpene cyclase FgFS from F.graminearum J1-012 was characterized by the in vitro assay.And the data showed that FgFS can produce a series of sesquiterpene.Followed,FgFS and ERG20 were overexpressed in S.cerevisiae YZL141 to generate S.cerevisiae T16 to produce sesquiterpene compounds with high efficiency,and provide enough compounds for structural characterization.Ten sesquiterpene compounds were produced by FgFS and eight of them were characterized.And the results showed that compounds 1-3 were new sesquiterpene skeletons,compounds 2 and 3 are the cis-trans isomers.Compound 4(nerolidol)with important application value in perfume and spices production field.At the same time,the isotope labeling experiments was introduced to elucidate the chemical mechanism of the new skeleton compounds 1-3.Systematic metabolic engineering aided natural products discovery strategy provideS a mature solution for functional verification of terpene cyclase component and the mining of new terpene skeletons.The experimental results shown that the combinatorial biosynthesis,metabolic engineering and enzyme rational modification strategies used in the function verification of terpene cyclases and the mining of terpenoids is a very meaningful work,which may accelerate the mining of new terpenoids and strengthen understanding of terpenoids cyclase to a great extent.