Insight into metabolic diversity of the brown‐rot basidiomycete Postia placenta responsible for sesquiterpene biosynthesis: semi‐comprehensive screening of cytochrome P450 monooxygenase involved in protoilludene metabolism

Abstract

SummaryA wide variety of sesquiterpenoids have been isolated from basidiomycetes, and their bioactive properties have attracted significant attention in an effort to understand biosynthetic machineries. As both sesquiterpene synthases and cytochrome P450 monooxygenases play key roles in the diversification of sesquiterpenoids, it is important to widely and mutually understand their biochemical properties. In this study, we performed genome‐wide annotation and functional characterization of sesquiterpene synthases from the brown‐rot basidiomycete Postia placenta. Using RT‐PCR, we isolated 16 sesquiterpene synthases genes as full‐length cDNAs. Heterologous expression revealed that the sesquiterpene synthases could produce a series of sesquiterpene scaffolds with distinct metabolic profiles. Based on metabolic studies, we identified 25 sesquiterpene scaffolds including Δ6‐protoilludene produced by the sesquiterpene synthases. In particular, a protoilludene synthase from the brown‐rot basidiomycete was characterized for the first time. Furthermore, we conducted a semi‐comprehensive functional screening of cytochrome P450 monooxygenases from P. placenta to elucidate biosynthetic machineries involved in metabolisms of Δ6‐protoilludene. Coexpression of protoilludene synthase and 184 isoforms of cytochrome P450 monooxygenases enabled the identification of CYP5344B1, CYP5348E1 and CYP5348J3, which catalysed the hydroxylation reaction of Δ6‐protoilludene to produce Δ6‐protoilludene‐8‐ol and Δ6‐protoilludene‐5‐ol. Furthermore, structural isomers of Δ7‐protoilludene‐6‐ol were obtained from incubation of Δ6‐protoilludene‐8‐ol in acidic culture medium.

Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences

“…Even if a gene is expressed, alternative splice variants may be produced that are not functional (2). All pioneering work to access the large diversity of terpenoid NPs made by basidiomycetes has therefore relied on the amplification of functional terpene synthase (TS) genes from cDNA for expression and characterization in Escherichia coli or yeasts, limiting the complete functional analysis of all computationally identified TSs in basidiomycete genomes (9)(10)(11)(12)(13). Being able to directly express basidiomycete biosynthetic genes from genomic DNA in a suitable fungal surrogate host would therefore greatly accelerate the functional characterization of NP pathways.…”

Bioinformatics-Aided Identification, Characterization of Fungal Linalool Synthases and Applications in Linalool biosynthesis

“…b-caryophyllene and cubenol 18 ) and monoterpenes (linalool 14 , limonene 14 and 1,8-cineole 19 ). To our knowledge, to date, there are only a couple of fungal monoterpene synthases reported in literature (Hyp3, a 1,8-cineole synthase 19 and the very recently identified PpSTS25, a bifunctional myrcene/linalool synthase 20 ). Here, we addressed this gap and specifically explored fungal linalool synthases.…”

“…(R)-linalool synthases were marked with "▲", (S)-linalool synthases with "△", LNSs with "=" and nerolidol synthases with "■". PpSTS25 is a myrcene/linalool synthase (MLS) from Postia placenta 20 . For plant and bacterial enzymes, UniProt accession number were used.…”

RNA-Seq de Novo Assembly and Differential Transcriptome Analysis of Chaga (Inonotus obliquus) Cultured with Different Betulin Sources and the Regulation of Genes Involved in Terpenoid Biosynthesis

“…Sesquiterpene synthases play an essential role in diversifying the skeletal structure of sesquiterpenoids by catalyzing the very complex cyclisation of the common precursor, FPP [38,70,71,72]. Sesquiterpenoids have been reported in Inonotus [43]; however, no biosynthetic genes have been identified.…”

“…The rich bouquet of several VOCs contribute to the aroma and flavor of mushrooms. Though the ecological function of fungal volatiles is unknown, studies showed their biological activities as antibiotics, antioxidants or inhibitor of germination [72,73,74].…”