Combinatorial Biosynthesis Of Highly Oxidized Tricyclic Sesquiterpenes From Fungi

Sesquiterpenoids are structurally diverse with C₁₅ compounds based on three isoprene units.Sesquiterpenes are important for drug development and display a vast array of biological and pharmacological activities.In nature,all sesquiterpenoids are derived from the precursor（FPP）.Typically,FPP is cyclized by sesquiterpene cyclase（STC）to generate sesquiterpene core-skeletons.The core sesquiterpene skeleton can be decorated by a series of post-modifying enzymes such as oxidases,acyltransferases and glycosyltransferases to form structurally and functionally diverse sesquiterpenoids.Among these modifying enzymes,oxidases play an important role in the formation of the structural and bioactive diversity of sesquiterpenoids.Cytochrome P450 oxidases（CYP450s）can selectivity catalyze different types of oxidation reactions on the sesquiterpene skeleton,which greatly broaden the structural complexity of terpenes and provide modifiable groups for other post-modifying enzymes.Due to the advantages of mild reaction conditions and high stereoselectivity,CYP450s have great potential as biocatalysts for the oxidation of inert carbon skeletons.However,most CYP450s are substrate specific,which limit its application in biolistic enzyme catalysis.Therefore,how to discover promiscuous CYP450s efficiently is a major problem in this research field.In this study,we use combinatorial biosynthesis strategy,genome mining,heterologous expression in Saccharomyces cerevisiae RC01 to find the promiscuous CYP450s and obtain a series of highly oxidized"unnatural"sesquiterpenoids.Our group has previously reported the synthesis of Peni A,a polyquatane silphinene（74）of fungal origin,and a multifunctional CYP450-PeniB that catalyzes a series of sequential oxidation reactions on the silphinene backbone to construct theγ-lactone-[5.5.5.5]oxidized windowane ring,a step that is essential for the synthesis of the significantly insecticidal activity of This step is a key step in the synthesis of penifulvin A,which has significant insecticidal activity.In addition,Bc BOT2 of fungal origin was reported in the literature as a synthase for poly（quinine）-presilphiperfolan-8β-ol（67）and CYP450-Bc BOT4 as its specific hydroxylase,a key step in the synthesis of the phytotoxin Botrydial.Furthermore,it was shown that tricyclic skeleton 67 is converted to tricyclic skeletons 103-105 under non-enzymatic catalytic conditions.therefore,we speculate that PeniB and Bc BOT4may be substrate broad（PeniB recognises 67 and non-natural substrates 103-105 and Bc BOT4 recognises non-natural substrates 74）.Based on this hypothesis,in order to discover the multifunctional abilities of promiscuous CYP450s and obtain highly oxidized tricyclic sesquiterpene products,we investigated the oxidizing capabilities of PeniB and Bc BOT4 on tricyclic sesquiterpenes using combinatorial biosynthesis and genome mining strategy.The main work completed as follows:（1）Combinatorial biosynthesis of bcbot4 and peni A,peniB and bcbot2,peniB and bcbot2,bcbot4 and peni A were paired and heterologously expressed in an engineered yeast platform（Saccharomyces cerevisiae RC01）,respectively.Metabolite analysis showed that Bc BOT4 could only oxidize the natural substrate 67;peniB could not only accept the natural substrate 74,but also catalyze the oxidation of substrate 103-105,showing a broad tolerance for substrate.Seven new sesquiterpenes（106-112）were isolated from the large-scale cultivation of the strains S.c-bcbot2-peniB,and their structures were subsequently confirmed by HR-MS and NMR analyses.（2）Research on the biosynthesis of clade1TC combined with peniB or bcbot4.The Clade1TC is the homolog of linear tricyclic sesquiterpene koraiol synthase Ffsc4.The gene clade1TC was combined with peniB or bcbot4 in yeast platform S.cerevisiae RC01,respectively.Analysis of the metabolites showed that Bc BOT4 did not accept the linear tricyclic sesquiterpene skeleton koraiol.However,PeniB could catalyze the oxidation of the substrate koraiol.A total of three products 114-116 were identified from recombinant strains S.c-clade1TC-peniB by large-scale cultivation.The results show that PeniB has a large substrate breadth and is able to recognize the linear tricyclic sesquiterpene backbone.（3）Research on the biosynthesis of the genes coll34TC combined with peniB or bcbot4.A sesquiterpene synthase Coll34TC derived from fungus Colletotrichum fructicola was identified and validated by sequence network similarity analysis combined with a chemical structure-guided genome mining approach.The heterologous expression experiments confirmed that the high expression stain of gene coll34TC could efficiently produce 117 and 118 which were characterized as tricyclic sesquiterpene skeletons presilphiperfol-1-ene and（-）-presilphiperfolan-1β-ol,respectively.Therefore,Coll34TC is a presilphiperfol-1-ene and（-）-presilphiperfolan-1β-ol cyclase which was not previously been reported.We deduced the cyclization mechanism of Coll34TC from FPP to（-）-presilphiperfolan-1β-ol and presilphiperfol-1-ene.Using combinatorial biosynthesis,coll34TC was paired with peniB or bcbot4,and coexpressed in an engineered yeast platform（Saccharomyces cerevisiae RC01）.Metabolite analysis showed that Bc BOT4 did not accept the tricyclic sesquiterpene scaffolds 117 and 118,while PeniB could selectively catalyse the oxidation of 117 and 118.A total of three oxidation products 119-121 were identified by large-scale cultivation.In conclusion,we developed a yeast-based platform for the oxidative derivatization of tricyclic sesquiterpene scaffolds using combination biosynthesis and genome mining.We mainly identified a promiscuous CYP450 PeniB which can catalyze a wide range of oxidation reactions on tricyclic sesquiterpene skeletons resulting in a total of 13 products.Our work expands the diversity of tricyclic sesquiterpene and provides an important material foundation for further activity screening.The discovery of multifunctional oxidase PeniB provides important biocatalysts for the selective oxidation of inert carbons in terpenoids in the future.