Biosynthetic Research Of Fungal Insecticidal Fenestrane Sesquiterpene Penifulvin A

Penifulvin A,a sesquiterpene natural product contains unique fenstrane structure,has been isolated from cultures of fungus Penicillium griseofulvum NRRL 35584.It has a unique tetracyclic ring system in which four fused rings share a central quaternary carbon atom with a spiro fashion in its structure,forming a unprecedented dioxa-[5.5.5.6]-fenstrane ring,and the two lactone rings(?-and ?-lactone rings)share a chiral acetal center.Bioactivity research indicates that penifulvin A was considered to be one of the most valuable natural products for pharmaceutical insecticides development,due to its significantly insecticidal activity against the fall armyworm(Spodoptera frugiperda),whose larvae cause enormous damage on economic crops,especially corn.In view of its novel and unique chemical structure and good biological activity,penifulvin A has attracted great attention from chemists and biologists in recent years.At present,the chemists have achieved total chemical synthesis of penifulvin A by means of retrosynthetic analysis and meta photocycloaddition reaction,but there are still some difficulties in stereoselective construction of its central dioxa-[5.5.5.6]-fenstrane skeleton.Relative to total chemical synthesis,the biosynthetic mechanism of penifulvin A has not been reported.Therefore,the study of penifulvin A biosynthesis pathway is expected to guide the chemical biomimetic synthesis,while its special chemical structure must also bears a unique enzymatic machinery.Based on this objective,this paper identified the biosynthetic gene cluster(peni)of penifulvin A from P.griseofulvum NRRL 35584,systematically clarified the biosynthetic mechanism of penifulvin A through the method of bioinformatics analysis,in vivo gene knockout,compound feeding,in vitro enzymatic assay,and heterologous biosynthesis.Thus,this research deeply revealed the biosynthesis processes of unique fenstrane structure in nature,and laid a solid theoretical and material foundation for subsequent transformation of the different types of fenstrane-based natural products to obtain more derivatives with novel structure and strong insecticidal activity through combinatorial biosynthesis technology.The main work completed in this study is as follows:(1)Determination of the fermentation condition and the method for its detection and isolation of penifulvin A.External environment are important factors that affect the microbial metabolic profiles.In order to determine the material basis for studying penifulvin A biosynthesis,we first determined the conditions for producing penifulvin A from P.griseofulvum NRRL 35584 under laboratory culture conditions.Small-scale fermentation experiments showed that P.griseofulvum NRRL35584 could stably produce penifulvin A on the rice medium for seven days at 25 ?,which was confirmed by subsequent separation and structural identification.The determination of penifulvin A production and its detection conditions laid a good foundation for the subsequent culture conditions of other mutant strains and the detection and separation of intermediates.(2)Identification of the biosynthetic gene cluster of penifulvin A(peni).The whole genome of P.griseofulvum NRRL35584 contained six terpene biosynthetic gene clusters analyzed by fungal secondary metabolite gene clusters analysis website(anti-SMASH fungal version).Based on the structurally oriented phylogenetic tree analysis of the terpene cyclases,the biosynthetic gene cluster in the sacffold 14(peni)was considered to be related to the production of penifulvin A.Bioinformatics analysis revealed that the peni gene cluster contained six genes,in addition to the sesquiterpene cyclase gene(peniA),there were a P450 monooxygenase gene(peniB),a FAD-dependent monooxygenase gene(peniC),two dioxygenases genes(peniD and peniF)and a gene with unknown functional(peniE).The results of RT-PCR showed that the transcription of the peni gene cluster was associated with the production of penifulvin A.Further knockout of the sesquiterpene cyclase gene(peniA)revealed that ?peniA mutant strain completely lost the capacity to produce penifulvinA.This clearly clarified the correlation between the peni gene cluster and the production of penifulvinA.The identification of the peni gene cluster is an important prerequisite for elucidating the biosynthesis mechanism of penifulvin A.(3)Determination of the genes involved in the biosynthesis of penifulvin A.The five genes of peniB~peniF in the peni cluster were individually inactivated,while the three genes of peniD~peniF were inactivated simultaneously.The results showed that the ?peniB and ?peniC mutants lost the capacity to produce penifulvinA,while the single deletion or simultaneous deletion of the three genes peniD~peniF did not affect the production of penifulvin A,which indicated that only three genes of peniA~peniC in the peni cluster may be related to the biosynthesis of penifulvin A.Further study showed that penifulvin A could be efficiently produced by co-expressing three genes of peniA~peniC in the heterologous host A.nidulans A1145,demonstrating that only peniA,peniB and peniC three genes were required for the biosynthesis of penifulvin A.The results of gene knockout and heterologous production revealed that only three genes were involved in the construction of a unique dioxa-[5.5.5.6]-fenstrane heterocycle structure in nature.(4)Research on the catalytic function of sesquiterpene cyclase PeniA.The soluble recombinant protein of PeniA was purified by heterologously expressed in E.coli,and in vitro enzyme activity experiments showed that,1)PeniA could catalyze the cyclization of farnesyl pyrophosphate(FPP)to 209,the MS fragments of which are consistent with the standard of triquinane sesquiterpene silphinene in the database;2)PeniA exhibits strict substrate specificity toward FPP,and could not catalyze the cyclization of GPP and GGPP to produce the corresponding products.Yeast heterologous expression experiments confirmed that the high expression strain of the gene peniA could efficiently produce 209 in yeast,and it was fully characterized as triquinane sesquiterpene silphinene with further separation and nuclear magnetic resonance(NMR)analyses.Therefore,PeniA is a silphinene cyclase which has not previously been reported.Through the study of the in vitro catalytic function of PeniA and the structure confirmation of silphinene,we deduced the whole cyclization mechanism from FPP to silphinene catalyzed by PeniA.The chemical feeding of 209 into ?peniA mutant strain restored penifulvin A production,confirming that 209 is an important precursor in penifulvin A biosynthesis,and would provide important material support for the catalytic function study of subsequent oxidation modifying enzymes(PeniB and PeniC).(5)Research on the catalytic function of cytochrome oxidase P450 PeniB.Bioinformatics analysis showed that PeniB is a cytochrome P450 monooxygenase.Co-expression of peniA and peniB genes in A.nidulans(AN-peniAB)could produce three products,namely silphinen-15-oic acid(203),?-lactone-2-hydroxy [5.5.5.5] fenestrane(210),?-lactone-2-keto[5.5.5.5] fenestrane(211).This result indicated that PeniB,a multifunctional P450 oxidase,could catalyze the multistep oxidation on silphinene.In vitro enzymatic assay of PeniB microsomal complexes indicated the oxidation process of PeniB,1)PeniB catalyzes three-step oxidation of C-15 methyl group of 209 to form carboxyl product 203;2)Compound 203 is further oxidized by PeniB to give 210;3)PeniB continues to catalyze the dehydrogenation of the hydroxy group at C2 of 210 to generate 211.Chemical feeding of 203,210 and 211 individually into ?peniA mutant strain all restored the production of penifulvin A,confirming that the three oxidation products are all important biosynthesis intermediates of penifulvin A,and the conversion of 203 to 210 is a key step in the construction of the first ?-lactone ring.Thus,we deduced that PeniB may catalyze the oxidation of 203 to 210 by two different mechanisms,consisting of the C-15 carboxyl radical of 203 addition to the C1-C2 double bond or the C-15 carboxyl group attacking the epoxy at the C1-C2.To validate the mechanism of PeniB,we chemically synthesized 214,the carboxylate of which was esterified with a methyl ester.In vitro enzymatic assay of PeniB with 214 showed that the catalytic activity of PeniB was completely inhibited,thereby indicating the formation of a carboxyl radical at the C-15 is essential for the formation of the ?-lactone ring in 210.(6)Research on the catalytic function of Baeyer-Villiger monooxygenase PeniC.From 211 to the final product penifulvin A,a position-selective Baeyer-Villiger oxidation reaction at the C1-C2 is required.Bioinformatics analysis showed that PeniC is indeed a flavin-dependent Baeyer-Villiger monooxygenase that may be responsible for the conversion of 211 to penifulvin A.Co-expression of peniA,peniB and peniC genes in A.nidulans(AN-peniABC)could detect the production of the final product penifulvin A.we attempted to express PeniC as a soluble protein from E.coli to verify its catalytic conversion of 211 to penifulvin A,but after various attempts,PeniC or its fusion protein with other tags were insoluble in E.coli.Chemical feeding of 211 into ?peniB or ?peniC mutants respectively showed that 211 could restore the penifulvin A production in ?peniB mutant,but it was not detected in the ?peniC mutant,which indirectly demonstrated that PeniC is indeed a key enzyme that catalyzes 211 to form penifulvin A,and could catalyze the Baeyer-Villiger oxidation reaction between the C1 and C2 to form the ?-lactone ring.(7)Deduction of the biosynthetic pathway of penifulvin A.Based on the study of the gene function of peniA,peniB and peniC,and the catalytic function of their corresponding proteins,we have deduced the possible biosynthetic pathway of penifulvin A,1)PeniA catalyzes the cyclization of a linear FPP to give an triquinane sesquiterpene silphinene(209);2)PeniB catalyzes multistep oxidation reaction of 209 to form 211,which completes the construction of the ?-lactone ring;3)PeniC catalyzes the Baeyer-Villiger oxidation reaction between the C1 and C2 of 211 to construct the ?-lactone ring,thereby obtaining the final product of penifulvin A.The clarification of the penifulvin A biosynthesis pathway lays a good foundation for the biosynthesis study of other fenstrane-based natural products and synthetic biotransformation of penifulvin derivatives.