title: Putative identification of the usnic acid biosynthetic gene cluster by de novo whole-genome sequencing of a lichen-forming fungus.
abstract: To identify the biosynthetic gene cluster responsible for the biosynthesis of the polyketide usnic acid we carried out the de novo genome sequencing of the fungal partner of Cladonia uncialis. This was followed by comprehensive in silico annotation of polyketide synthase (PKS) genes. The biosynthesis of usnic acid requires a non-reducing PKS possessing a carbon methylation (CMeT) domain, a terminal Claisen cyclase (CLC) domain, and an accompanying oxidative enzyme that dimerizes methylphloracetophenone to usnic acid. Of the 32 candidate PKS genes identified in the mycobiont genome, only one was identified as consistent with these biosynthetic requirements. This gene cluster contains two genes encoding a non-reducing PKS and a cytochrome p450, which have been respectively named methylphloracetophenone synthase (MPAS) and methylphloracetophenone oxidase (MPAO). Both mpas and mpao were demonstrated to be transcriptionally active by reverse transcriptase-PCR of the mRNA in a lichen sample that was observed by HPLC to produce usnic acid. Phylogenetic analysis of the bioinformatically identified ketosynthase (KS) and CLC domains of MPAS demonstrated that mpas grouped within a unique clade and that mpas could be used as a phylogenetic probe to identify other MPAS genes.
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada. Electronic address: umabdelm@cc.umanitoba.ca.
authors.forename: Mona
authors.initials: M
authors.lastname: Abdel-Hameed
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada. Electronic address: bertrarl@cc.umanitoba.ca.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Bertrand
authors.affiliation: Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada. Electronic address: Michele.Piercey-Normore@umanitoba.ca.
authors.forename: Michele D
authors.initials: MD
authors.lastname: Piercey-Normore
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada. Electronic address: John_Sorensen@umanitoba.ca.
authors.forename: John L
authors.initials: JL
authors.lastname: Sorensen
mesh_terms: D001203:Ascomycota / Q000235:genetics* / Q000378:metabolism*; D001572:Benzofurans / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D020869:Gene Expression Profiling; D016681:Genome, Fungal; D008025:Ligases / Q000235:genetics; D005810:Multigene Family; D010088:Oxidoreductases / Q000235:genetics; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D001572:Benzofurans; C073339:usnic acid; D010088:Oxidoreductases; D008025:Ligases
keywords: Biosynthesis; Cladonia uncialis; Lichen fungi; Phylogenetics; Polyketide synthase; Secondary metabolite