Yudai Matsuda

Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.

Complete biosynthetic pathway of anditomin: nature's sophisticated synthetic route to a complex fungal meroterpenoid.

Anditomin and its precursors, andilesins, are fungal meroterpenoids isolated from Aspergillus variecolor and have unique, highly oxygenated chemical structures with a complex bridged-ring system. Previous isotope-feeding studies revealed their origins as 3,5-dimethylorsellinic acid and farnesyl pyrophosphate and suggested the possible involvement of a Diels-Alder reaction to afford the congested bicyclo[2.2.2]octane core structure of andilesins. Here we report the first identification of the biosynthetic gene cluster of anditomin and the determination of the complete biosynthetic pathway by characterizing the functions of 12 dedicated enzymes. The anditomin pathway actually does not employ a Diels-Alder reaction, but involves the nonheme iron-dependent dioxygenase AndA to synthesize the bridged-ring by an unprecedented skeletal reconstruction. Another dioxygenase, AndF, is also responsible for the structural complexification, generating the end product anditomin by an oxidative rearrangement.

Astellifadiene: Structure Determination by NMR Spectroscopy and Crystalline Sponge Method, and Elucidation of its Biosynthesis

Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, astellifadiene (1), having a 6-8-6-5-fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope-incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation-initiated second cyclization sequence, 1,2-alkyl migration, and 1,5-hydride shift to generate the novel scaffold of 1.

An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping

Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.

Uncovering the unusual D-ring construction in terretonin biosynthesis by collaboration of a multifunctional cytochrome P450 and a unique isomerase.

Terretonin (1) is a fungal meroterpenoid isolated from Aspergillus terreus, and possesses a highly oxygenated and unique tetracyclic structure. Although the biosynthetic gene cluster for 1 has been identified and the biosynthesis has recently been studied by heterologous reconstitution and targeted-gene deletion experiments, the last few steps of the terretonin pathway after terrenoid (6) have yet to be elucidated. Notably, the mechanism for the D-ring expansion to afford the terretonin scaffold has been a long-standing mystery to solve. Here we report the characterization of three enzymes that convert 6 into 1, as well as the complete biosynthetic pathway of 1. In the proposed terretonin pathway, the cytochrome P450 Trt6 catalyzes three successive oxidations to transform 6 into an unstable intermediate, which then undergoes the D-ring expansion and unusual rearrangement of the methoxy group to afford the core skeleton of 1. This unprecedented rearrangement is catalyzed by a novel isomerase Trt14. Finally, the nonheme iron-dependent dioxygenase Trt7 accomplishes the last two oxidation reactions steps to complete the biosynthesis.

Molecular Basis for Stellatic Acid Biosynthesis: A Genome Mining Approach for Discovery of Sesterterpene Synthases

The search for a new sesterterpene synthase in the genome of Emericella variecolor, which reportedly produces diverse sesterterpenoids, is described. One gene product (a chimeric protein with prenyltransferase and terpene cyclase domains) led to the synthesis of a novel tricyclic sesterterpene, stellata-2,6,19-triene (1), from DMAPP and IPP, and the hydrocarbon was further transformed into stellatic acid (2) by cytochrome P450 monooxygenase encoded by the gene adjacent to the sesterterpene synthase gene.

Terretonin Biosynthesis Requires Methylation as Essential Step for Cyclization

The fungal meroterpenoids have tremendous structural diversity. This group includes several medicinally important compounds, such as the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, pyripyropene A, the selective acetylcholinesterase inhibitor, arisugacin A, and the protein farnesyltransferase inhibitor, andrastin A (Scheme 1). Many of these compounds have polyketide and terpenoid origins. The diversities of these two groups contribute to the structural diversity of fungal meroterpenoids. Understanding the biosynthetic routes of meroterpenoids is important for utilizing the pathways for combinatorial biosynthesis. We have previously reported the functions of the enzymes catalyzing pyripyropene A biosynthesis, consisting of CoA-ligase, polyketide synthase (PKS), prenyltransferase (PT), flavin-dependent monooxygenase (FMO) and terpene cyclase (CYC). In the biosynthesis of pyripyropene A, the terpenoid moiety is cyclized by Pyr4, a novel transmembrane protein with very low similarity to the known terpene cyclases. The cyclization reaction in fungal meroterpenoid biosynthesis is one of the key steps that generate the structural diversity of this class of compounds. As exemplified by terretonin, austinol, andrastin A and anditomin, some compounds are derived from the same polyketide core and differently cyclized terpenoid moieties. These compounds are all derived from 3,5-dimethylorsellinic acid (DMOA, 1, Scheme 2), but they have various cyclic terpenoid moieties. These differences are due to the presence of CYCs with diverse cyclization activities. Terretonin is a toxic compound isolated from Aspergillus terreus. 8] The intriguing structure of terretonin has tempted many researchers to study its biosynthetic route. A previous isotope-feeding experiment revealed that terretonin is derived from 1 and farnesyl diphosphate (FPP). Recently, we identified the biosynthetic gene cluster of terretonin (8) in the A. terreus NIH2624 genome and revealed, using a fungal heterologous expression system, that the PKS (trt4), PT (trt2) and FMO (trt8) genes, which are homologous to the pyripyropene A biosynthetic genes, are responsible for the production of epoxyfarnesyl-DMOA (3), a precursor of terretonin (Scheme 2). However, the CYC gene responsible for the cyclization of the farnesyl moiety of 3 remained unknown. Therefore, we set out to clarify the cyclization step of terretonin biosynthesis. Since there are many DMOA-derived meroterpenoids, significant knowledge about a number of compounds should be obtained by such a study. We first focused on trt1, which is homologous to the pyr4 gene and encoded upstream of trt2, as the CYC gene responsible for the cyclization of 3. To characterize the function of Trt1, we expressed trt1 along with trt4, trt2 and trt8 in the heterologous fungal host A. oryzae NSAR1, a quadruple auxotrophic mutant strain (niaD , sC , DargB, adeA ). By using this host and four expression vectors—pTAex3 harboring a argB marker, pPTRI harboring a ptrA marker, pUSA harboring a sC marker and pAdeA harboring a adeA marker —the five genes were coexpressed under the control of amyB promoter. The transformant was cultured in Czapek–Dox (CD) medium, supplemented with starch to induce expression. After three days, the culture supernatant and the mycelial extract were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). 1 and dihydroxyfarnesyl-DMOA (4), a hydrolyzed compound derived from 3, were observed, but in contrast to our expectaScheme 1. Representative fungal meroterpenoids.

Genome-Based Discovery of an Unprecedented Cyclization Mode in Fungal Sesterterpenoid Biosynthesis

Sesterterpenoids are a group of terpenoid natural products that are primarily biosynthesized via cyclization of the C25 linear substrate geranylfarnesyl pyrophosphate (GFPP). Although the long carbon chain of GFPP in theory allows for many different cyclization patterns, sesterterpenoids are relatively rare species among terpenoids, suggesting that many intriguing sesterterpenoid scaffolds have been overlooked. Meanwhile, the recent identification of the first sesterterpene synthase has allowed the discovery of new sesterterpenoids by the genome mining approach. In this study, we characterized the unusual fungal sesterterpene synthase EvQS and successfully obtained the sesterterpene quiannulatene (1) with a novel and unique highly congested carbon skeleton, which is further oxidized to quiannulatic acid (2) by the cytochrome P450 Qnn-P450. A mechanistic study of its cyclization from GFPP indicated that the biosynthesis employs an unprecedented cyclization mode, which involves three rounds of hydride shifts and two successive C-C bond migrations to construct the 5-6-5-5-5 fused ring system of 1.

Biosynthesis of LL-Z1272β: Discovery of a New Member of NRPS-like Enzymes for Aryl-Aldehyde Formation.

LL‐Z1272β (1) is a prenylated aryl‐aldehyde produced by several fungi; it also serves as a key pathway intermediate for many fungal meroterpenoids. Despite its importance in the biosynthesis of natural products, the molecular basis for the biosynthesis of 1 has yet to be elucidated. Here we identified the biosynthetic gene cluster for 1 from Stachybotrys bisbyi PYH05‐7, and elucidated the biosynthetic route to 1. The biosynthesis involves a polyketide synthase, a prenyltransferase, and a nonribosomal peptide synthetase (NRPS)‐like enzyme, which is responsible for the generation of the aldehyde functionality. Interestingly, the NRPS‐like enzyme only accepts the farnesylated substrate to catalyze the carboxylate reduction; this represents a new example of a substrate for adenylation domains.

Unusual chemistries in fungal meroterpenoid biosynthesis

Meroterpenoids are polyketide and terpenoid hybrid natural products with remarkable biological activities. Recent progress in fungal meroterpenoid biosynthesis has revealed several unusual enzyme reactions and novel enzymes, including unique terpene cyclization reactions by a novel family of membrane-bound terpene cyclases and post-cyclization modification reactions by oxygenases, such as non-heme iron-dependent dioxygenases, flavin adenine dinucleotide-dependent monooxygenases, and cytochrome P450 monooxygenases. They contribute to the structural diversification and increase in complexity of fungal meroterpenoids. Structure-function studies of these enzymes provide strategies for engineering the biosynthetic machinery to create novel molecular scaffolds for drug discovery.