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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: A 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 ?6-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 ?6-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 ?6-protoilludene to produce ?6-protoilludene-8-ol and ?6-protoilludene-5-ol. Furthermore, structural isomers of ?7-protoilludene-6-ol were obtained from incubation of ?6-protoilludene-8-ol in acidic culture medium.

SUBMITTER: Ichinose H

PROVIDER: S-EPMC6116744 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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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.

Ichinose Hirofumi H Kitaoka Takuya T

Microbial biotechnology 20180813 5

A 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 sesquiterpen ...[more]

PMID: 30105900

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Project description:Wood-degrading brown rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and non-selective, these two steps are likely segregated. A common hypothesis has been that brown rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that lignocellulose-oxidation (LOX) components involved in ROS production are differentially expressed by brown rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole transcriptome sequencing (RNAseq) and assayed relevant enzyme activities. We found a marked pattern of LOX upregulation in a narrow (5-mm; 48-hr) zone at the hyphal front, which included many genes likely involved in ROS generation. Upregulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for brown rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization. We sequenced mRNA from 9 Postia placenta samples taken from 3 wood sections of wafer design, with 3 bioreplicates for each wood section, to compare the gene expression during brown rot processes. Three wood sections of the wafer are representing early to late decay stages.

Dataset Information

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.

Publications

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.

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