Transcriptome Analysis Identifies a Gene Cluster for the Biosynthesis of Biruloquinone, a Rare Phenanthraquinone, in a Lichen-Forming Fungus Cladonia macilenta

Abstract

Lichens are prolific producers of natural products of polyketide origin. We previously described a culture of lichen-forming fungus (LFF) Cladonia macilenta that produces biruloquinone, a purple pigment that is a phenanthraquinone rarely found in nature. However, there was no genetic information on the biosynthesis of biruloquinone. To identify a biosynthetic gene cluster for biruloquinone, we mined polyketide synthase (PKS) genes from the genome sequence of a LFF isolated from thalli of C. macilenta. The 38 PKS in C. macilenta are highly diverse, many of which form phylogenetic clades with PKS previously characterized in non-lichenized fungi. We compared transcriptional profiles of the 38 PKS genes in two chemotypic variants, one producing biruloquinone and the other producing no appreciable metabolite in vitro. We identified a PKS gene (hereafter PKS21) that was highly upregulated in the LFF that produces biruloquinone. The boundaries of a putative biruloquinone gene cluster were demarcated by co-expression patterns of six clustered genes, including the PKS21. Biruloquinone gene clusters exhibited a high degree of synteny between related species. In this study we identified a novel PKS family responsible for the biosynthesis of biruloquinone through whole-transcriptome analysis.

Identification of a biosynthetic gene cluster for a red pigment cristazarin produced by a lichen-forming fungus Cladonia metacorallifera

“…Crz7 was highly upregulated in growth media containing fructose or glucose. The second most upregulated gene was PKS21, an ortholog of the brq5 involved in the biosynthesis of biruloquinone, a rare phenanthraquinone-class metabolite, which we previously identified in C. macilenta [21]. However, biruloquinone has never been detected in culture extracts of C. metacorallifera, indicating a high level of PKS gene expression is required to produce final polyketide products above the detection limit of analytical devices [69][70][71].…”

“…To date, only the two BGCs for atranorin [12] and lecanoric acid [9] were identified by heterologous expression. In addition, a few lichen natural products have been connected to BGCs including biruloquinone [21], grayanic acid [10], and usnic acid [35,36] with transcriptional evidence. The BGCs of gyrophoric acid [37], olivetoric acid/ physodic acid [38] were tentatively assigned via phylogenetic analysis.…”

“…Also, PKS13 and PKS15 were phylogenetically related to PKS genes involved in melanin production [12]. Notably, PKS8, PKS16, and PKS21 were previously connected to the biosynthesis of usnic acid [29,35], grayanic acid [10], gyrophoric acid [37], lecanoric acid [9], olivetoric acid, physodic acid [38], and biruloquinone [21]. However, there were still many undescribed PKS genes in the genome of the C. metacorallifera mycobiont.…”

“…Due to the increasing availability of genomic resources [17,18], and natural product databases in fungi [19], in silico approaches for linking secondary metabolites with their respective BGCs are becoming more common [20][21][22][23][24][25]. BGCs are a locally clustered group of genes that together encode a biosynthetic pathway for a compound [26,27].…”

“…Despite a great deal of natural products from fungal lineages [2,26,39,40], BGCs responsible for the biosynthesis of major lichen metabolites are still unknown [41] due to extremely slow growth rate of mycobionts in culture [8] and limited molecular tools for manipulating mycobionts recalcitrant to genetic transformation [12]. To overcome these hurdles in studying on biosynthetic genes in lichens, the expression levels of biosynthetic genes were correlated with the amount of produced metabolites of interest [10,21,42]. Genome-guided gene discovery and metatranscriptomic analysis have aided the identification of BGCs for metabolite of interest in lichens, indicating that the number of BGCs found in fungal genomes outnumber the known fungal products they produce [7,22,28,35,36].…”