Combinatorial Biosynthesis And Gene Clusters Function Study Of Terpenoids

Terpenoids are a large class of natural products,which have broad applications in food,medical and cosmetic industries because of their diverse structures and biological activities.In nature,terpenoids are synthesized as follows:the precursor compounds pentene pyrophosphate(isopentenyl diphosphate,IPP)and allyl pyrophosphate(dimethylallyl diphosphate,DMAPP)that are produced by the MVA and MEP pathways,respectively,condense to form isoprene precursors with different chain lengths:geranyl pyrophosphate(GPP),farnesyl pyrophosphate(FPP),geranylgeranyl diphosphate(GGPP),and geranylfarnesyl pyrophosphate(GFPP),catalyzed by isoprene transferase.Thereafter,terpene synthases catalyze the substrates with different chain lengths to synthesize core skeletons of terpenoids.Eventually,a variety of terpenoids with different biological activities,under the action of a series of post-modification enzymes(cytochrome P450s,glycosyl transferase,and acyltransferase),are formed from these core skeletons.So far,researchers have elucidated the structures of more than 80,000 terpenoids,of which the vast majority of compounds existed in the form of skeletons;however,the study of their biological activities are far from sufficient.Cytochromes P450(CYPs)are major players in generating the structural and biological activity diversity of terpenoids,and more than 97%of the terpenoids are oxygenated via the biological activity of CYPs.Therefore,how to discover P450s with biological activity efficiently and then obtain the terpenoids with diverse structures and biological activities,is a major problem in this research field.To overcome the above problem,in this study,we combined the advantages of traditional genomic mining,heterologous expression,and emerging combinatorial biosynthesis strategies,and discovered and identified the functions of P450s and obtained a series of new post-modified sesquiterpenoids and sesterterpenoids,which enrich the diversity of terpenoids.In our previous work,we sequenced the genome of the filamentous fungus Fusarium graminearum J1-012,and found that it contained 9 type I terpene synthases.Based on the platform for overproduction of terpenoids established in our group,we verified four of the terpene synthases,Fg J01056,Fg J02895,Fg J09920 and Fg MS,and obtained three sesquiterpene skeletons longiborneol,guaiadiene,koraiol,and sesterterpene skeleton mangicdiene.To further mine terpenoids with diverse structures and good biological activities,we used the strategies of combinatorial biosynthesis of non-cognate P450s and mining the P450s in terpenoid clusters to study the post-modified products of the four terpene skeletons.In this study,we used a combinatorial biosynthesis strategy to combine P450enzyme CYP260B1 with the sesquiterpene synthases Fg J01056 and Fg J09920,and synthesized two oxygenated products culmorin and culmorone and one new compound koraidiol.This combinatorial biosynthesis approach suggests the potential use of non-cognate P450s in producing novel oxygenated terpenoids,or generating a novel biosynthetic route for known terpenoids.We found that sesterterpene mangicdiene was the core skeleton of mangicol compounds.These are an important type of sesterterpenoids,isolated from Fusarium heterosporum CNC-477 in mangroves,which have attracted the attention of a large number of researchers for their complex structures,anti-inflammatory activity and low cytotoxicity.However,at present,these compounds are mainly obtained by traditional separation method with low efficiency and high cost.To date,the biosynthetic pathways of these compounds have not been elucidated.To this end,we utilized Aspergillus oryzae platform to mine the biosynthetic gene cluster of Fg MS and deciphered the biosynthetic pathway of mangicol compounds in this study.We expressed the genes of Fg MS gene cluster in A.oryzae and constructed a series of mutant strains and detected6 post-modified products in three mutant strains,AO-mgc DE,AO-mgc DEF and AO-mgc CDE,of which compound 1-5 were new magicol compounds and intermidate compound 3 showed good anti-inflammatory activity.Based on the above results,we first deciphered the biosynthetic pathway of mangicol compounds,of which 4 enzymes were involved in the biosynthesis of these compounds,namely,epoxide hydrolase mgc C,sesterterpene synthase mgc D,two P450 enzymes-mgc E and mgc F.It was found that mgc E was a multi-function P450 enzyme,which could catalyze the multi-step reactions continuously.Thus,the findings of this study fill the gap in the field of biosynthesis of mangicol compounds.In this study,we also found that guaiadiene produced by Fg J02895 had the same core skeleton as sesquiterpenoid englerin A,which is a novel guaiane type sesquiterpene diester isolated from bark of Phyllanthus engleri,Euphorbiaceae and has good anti-renal cancer activity(growth inhibitory GI₅₀<20 n M).At present,it is mainly extracted from plants or chemically synthesized,however,the yield is low and cannot meet the demand.Moreover,the biosynthesis pathway of englerin A is still unknown.To this end,we used A.oryzae as heterologous expression host and constructed a series of mutant strains to study the function of Fg J02895 sesquiterpenoid biosynthesis gene cluster systematically.The results showed that three post-modified products were produced in the AO-Y2 mutant strain(containing three P450s and one acetyl transferase),and NMR and HR-LCMS detection,which indicated that they were oxygenated and acetylated guaiane type sesquiterpenoids,respectively,elucidated their structures.The discovery of these metabolic intermediates is expected to enable us to synthesize englerin A in fewer steps.Through this study,we found that in addition to sesquiterpene synthase Fg J02895,P450 enzymes and acetyltransferase also play a role in the biosynthesis pathway.The experimental results of this study show that the combination of genome mining with combinatorial biosynthesis and heterologous expression strategies could accelerate the mining process of biosynthetic gene clusters of terpenoids,lead to discovery of novel,diverse terpenoids with biological activities,and help decipher the biosynthesis pathways and mechanisms of these terpenoids,thus building an important foundation for the subsequent efficient synthesis of these terpenoids.