Identification of Ophiobolin F Synthase by a Genome Mining Approach: A Sesterterpene Synthase from <i>Aspergillus clavatus</i>

Chiba, Ryota; Minami, Atsushi; Gomi, Katsuya; Oikawa, Hideaki

doi:10.1021/ol303408a

Cited by 162 publications

(160 citation statements)

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“…6 Following this pioneering work, a homology-based cloning allowed us to identify homologous enzymes such as PaPS, ophiobolin F synthase and sesterfisherol synthase. [7][8][9] The cyclization products of the growing family of terpene synthases are unique structures featuring polycyclic skeletons such as tricyclic fusicoccadiene/ophiobolin F, 6,8 tetracyclic phomopsene (1)/sesterfisherol 7,9 and pentacyclic quiannulatene (Scheme 1 and Supplementary Figure S1). 10 The cyclization can occur under specific control of the terpene synthase, thus it is of particular interest for the researchers in the fields of organic and natural product chemistry.…”

Section: Introductionmentioning

confidence: 99%

Cyclization mechanism of phomopsene synthase: mass spectrometry based analysis of various site-specifically labeled terpenes

et al. 2017

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Elucidation of the cyclization mechanism catalyzed by terpene synthases is important for the rational engineering of terpene cyclases. We developed a chemoenzymatic method for the synthesis of systematically deuterium-labeled geranylgeranyl diphosphate (GGPP), starting from site-specifically deuterium-labeled isopentenyl diphosphates (IPPs) using IPP isomerase and three prenyltransferases. We examined the cyclization mechanism of tetracyclic diterpene phomopsene with phomopsene synthase. A detailed EI-MS analysis of phomopsene labeled at various positions allowed us to propose the structures corresponding to the most intense peaks, and thus elucidate a cyclization mechanism involving double 1,2-alkyl shifts and a 1,2-hydride shift via a dolabelladien-15-yl cation. Our study demonstrated that this newly developed method is highly sensitive and provides sufficient information for a reliable assignment of the structures of fragmented ions.

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Section: Introductionmentioning

confidence: 99%

Cyclization mechanism of phomopsene synthase: mass spectrometry based analysis of various site-specifically labeled terpenes

et al. 2017

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“…In fungi, six STSs have recently been identified and shown to synthesize ophiobolin F (in Aspergillus clavatus) (11), sesterfisherol (in Neosartorya fischeri) (12), and stellata-2,6,19-triene, (2E)-α-cericerene, quiannulatene, and astellifadiene (in Emericella variecolor) (13)(14)(15)(16) (Fig. 1A).…”

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confidence: 99%

Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution

Huang

Kautsar

Hong

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

116

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Sesterterpenoids are a rare terpene class harboring untapped chemodiversity and bioactivities. Their structural diversity originates primarily from the scaffold-generating sesterterpene synthases (STSs). In fungi, all six known STSs are bifunctional, containing C-terminal trans-prenyltransferase (PT) and N-terminal terpene synthase (TPS) domains. In plants, two colocalized PT and TPS gene pairs from Arabidopsis thaliana were recently reported to synthesize sesterterpenes. However, the landscape of PT and TPS genes in plant genomes is unclear. Here, using a customized algorithm for systematically searching plant genomes, we reveal a suite of physically colocalized pairs of PT and TPS genes for the biosynthesis of a large sesterterpene repertoire in the wider Brassicaceae. Transient expression of seven TPSs from A. thaliana, Capsella rubella, and Brassica oleracea in Nicotiana benthamiana yielded fungal-type sesterterpenes with tri-, tetra-, and pentacyclic scaffolds, and notably (−)-ent-quiannulatene, an enantiomer of the fungal metabolite (+)-quiannulatene. Protein and structural modeling analysis identified an amino acid site implicated in structural diversification. Mutation of this site in one STS (AtTPS19) resulted in premature termination of carbocation intermediates and accumulation of bi-, tri-, and tetracyclic sesterterpenes, revealing the cyclization path for the pentacyclic sesterterpene (−)-retigeranin B. These structural and mechanistic insights, together with phylogenetic analysis, suggest convergent evolution of plant and fungal STSs, and also indicate that the colocalized PT-TPS gene pairs in the Brassicaceae may have originated from a common ancestral gene pair present before speciation. Our findings further provide opportunities for rapid discovery and production of sesterterpenes through metabolic and protein engineering.sesterterpene biosynthesis | plant natural products | cyclization mechanism | convergent evolution | Brassicaceae S esterterpenoids are a largely unexplored class of terpenes with only around 1,000 members isolated from nature so far, representing a mere <2% of the reported terpene family members (>70,000). These compounds are structurally diverse and have a wide spectrum of biological activities, ranging from antiinflammatory, anticancer, cytotoxic, and antimicrobial bioactivities to phytotoxicity and plant defense (1). Recent work on sesterterpenoids has suggested that this terpene class could be an important new source of anticancer drugs (2, 3). The majority of sesterterpenoids characterized to date are from marine sponges and terrestrial fungi, with only ∼60-70 being of plant origin. Many of these plant sesterterpenes were isolated from the mint family (Lamiaceae) and have been implicated in plant defense (4-7). Although large transcriptome datasets for the mint family have been generated (8), very limited genome sequences for the Lamiaceae are currently available (9, 10).Like other classes of terpenes, the structural diversity of sesterterpenes largely originates from the...

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“…1 We successfully applied Aspergillus oryzae expression system to total biosynthesis of diterpene, 2 indole diterpenes, 3 polyketide 4 and to genome mining of novel metabolite. 5 During the projects on fungal metabolites, we have found unexpected reactions catalyzed by host, resulting in formation of by-products derived from intermediates accumulated in the transformants harboring the biosynthetic genes of two polyketide metabolites cytochalasin K (1) and solanapyrone A (2a). Here, we describe structure determination of modified intermediates and a method for suppressing undesired products.…”

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Reconstitution of biosynthetic machinery of fungal polyketides: unexpected oxidations of biosynthetic intermediates by expression host

Fujii

Ugai

Ichinose

et al. 2016

Bioscience, Biotechnology, and Biochemistry

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AbstractsReconstitution of whole biosynthetic genes in Aspergillus oryzae has successfully applied for total biosynthesis of various fungal natural products. Heterologous production of fungal metabolites sometimes suffers unexpected side reactions by host enzymes. In the studies on fungal polyketides solanapyrone and cytochalasin, unexpected oxidations of terminal olefin of biosynthetic intermediates were found to give one and four by-products by host enzymes of the transformants harboring biosynthetic genes. In this paper, we reported structure determination of by-products and described a simple solution to avoid the undesired reaction by introducing the downstream gene in the heterologous production of solanapyrone C. Initial introduction using pTAex3 harboring sol1 in A. oryzae NSAR1 has successfully gave transformants AO-sol1. AO-sol1 produced desmethylprosolanapyrone I (5) in 120 mg/kg whose methylation product (Me 2 SO 4 , acetone) was identical to the synthetic prosolanapyrone I (6) (Figure 2). 14 To the AO-sol1, we introduced the second gene sol2 with a plasmid pPTRI-sol2. The resultant AO-sol1/2 produced 6 and significant amount of by-products 11a-11d (Figure 3). Based on their characteristic UV spectra, we speculated that all of them possessed 2-pyrone moiety derived from 6. Molecular formula C 18 H 26 O 5 of a major product 11a suggested that 11a Keywordshad the same carbon framework of 6. Compared its 1 H NMR spectrum with that of 6, up-field shifted doublet methyl signal (1.13 ppm), and two oxymethine signals (3.85, 4.01 ppm) were newly observed in place of a terminal propenyl moiety in 6, suggesting that the terminal olefin in 6 was oxidized to a vicinal diol in 11a. Structure of 11a was further confirmed by several 2D-NMR data as shown in Figure 3. Similarly, structures of three additional metabolites were determined in a similar manner as shown in the Figure 3. Length of the side chain on the pyrone ring in 11b (C7), 11c (C5) and 11d (C3) suggested that putative C9-product derived by the oxidative cleavage of vic-diol in 11a further degraded by β-oxidation to give 11b-11d. Thus we speculated that host monooxygenase oxidized terminal olefin in 6 to epoxide 10 in which hydrolysis underwent to give diol 11a as in the case of the total biosynthesis of paxilline. 3a Further oxidations were also likely catalyzed by oxidases in A. oryzae. Again, we faced a similar problem which occurred in the cytochalasin project.To avoid the side reaction, we decided to introduce the downstream gene sol6. Our initial attempt to introduce sol6 to the AO-sol1/2 failed to produce prosolanapyrone II (7). 14 When we reexamined DNA sequence of gene cluster with a program 2ndFind which has recently developed for rapid identification of natural product gene cluster, we found the previous prediction of sol6 intron was not correct. 13 Based on this obsevation, we prepared the plasmids pUSA-sol2 and pUSA2-sol2/6 which were subsequently introduced into AO-sol1 to yield AO-sol1/2 and AO-sol1/2/6. We gratifyingly found that the AO-...

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Identification of Ophiobolin F Synthase by a Genome Mining Approach: A Sesterterpene Synthase from Aspergillus clavatus

Cited by 162 publications

References 27 publications

Cyclization mechanism of phomopsene synthase: mass spectrometry based analysis of various site-specifically labeled terpenes

Cyclization mechanism of phomopsene synthase: mass spectrometry based analysis of various site-specifically labeled terpenes

Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution

Reconstitution of biosynthetic machinery of fungal polyketides: unexpected oxidations of biosynthetic intermediates by expression host

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