Genome-Wide Analysis of Biosynthetic Gene Cluster Reveals Correlated Gene Loss with Absence of Usnic Acid in Lichen-Forming Fungi

Abstract

Lichen-forming fungi are known to produce a large number of secondary metabolites. Some metabolites are deposited in the cortical layer of the lichen thallus where they exert important ecological functions, such as UV filtering. The fact that closely related lineages of lichen-forming fungi can differ in cortical chemistry suggests that natural product biosynthesis in lichens can evolve independent from phylogenetic constraints. Usnic acid is one of the major cortical pigments in lichens. Here we used a comparative genomic approach on 46 lichen-forming fungal species of the Lecanoromycetes to elucidate the biosynthetic gene content and evolution of the gene cluster putatively responsible for the biosynthesis of usnic acid. Whole-genome sequences were gathered from taxa belonging to different orders and families of Lecanoromycetes, where Parmeliaceae is the most well-represented taxon, and analyzed with a variety of genomic tools. The highest number of biosynthetic gene clusters was found in Evernia prunastri, Pannoparmelia angustata, and Parmotrema austrosinense, respectively, and lowest in Canoparmelia nairobiensis, Bulbothrix sensibilis, and Hypotrachyna scytodes. We found that all studied species producing usnic acid contain the putative usnic acid biosynthetic gene cluster, whereas the cluster was absent in all genomes of species lacking usnic acid. The absence of the gene cluster was supported by an additional unsuccessful search for ß-ketoacylsynthase, the most conserved domain of the gene cluster, in the genomes of species lacking usnic acid. The domain architecture of this PKS cluster-homologous to the already known usnic acid PKS cluster (MPAS) and CYT450 (MPAO)-varies within the studied species, whereas the gene arrangement is highly similar in closely related taxa. We hypothesize that the ancestor of these lichen-forming fungi contained the putative usnic acid producing PKS cluster and that the gene cluster was lost repeatedly during the evolution of these groups. Our study provides insight into the genomic adaptations to the evolutionary success of these lichen-forming fungal species and sets a baseline for further exploration of biosynthetic gene content and its evolutionary significance.

Keywords: Parmeliaceae; ascomycota; comparative-genomics; evolution; metabolic gene cluster; usnic acid.

Fig. 1.

Genome completeness and BGC assessment of 46 lichen-forming fungi genomes used for this study. Bars represent the number of BGC (blue), number of non-reducing PKS (orange), and percentage of genome completeness (grey). The Y-axis represents the values of percentage and the number of BGCs.

Fig. 2.

Gene tree of NR-PKS dataset inferred by ML analysis in IQ-Tree using 6 Methylsalicylic PKS protein sequences as outgroup. The distinct colors represent the different groups corresponding to the domain arrangement. In each group the domain arrangement of NR-PKS is highlighted with distinct colors. SAT = starter unit-ACP transacylase, KS = ketosynthase, AT = acyl transferase, PT = product template, ACP = acyl carrier protein, TE = thioesterase, TE/CLC = thioesterase/Claisen cyclase, CMeT = C-methyltransferase, R = reductase.

Fig. 3.

Fragment of NR-PKS tree showing the relationship of putative PKS responsible for the biosynthesis of usnic acid.

Fig. 4.

Putative usnic acid compound cluster (UA) conservation and synteny. The UA cluster from other lichen-forming fungi resembled to the characterized usnic acid core genes PKS (MPAS) and Cytochrome P450 (MPAO) from C. uncialis (high percentage of identity in protein-by-protein comparisons of both genes in all species included).

Fig. 5.

Phylogenetic tree from the IQ-Tree analysis based on a concatenated dataset of 2556 BUSCO genes (Pizarro et al. 2018). Numbers at the nodes represent ML bootstrap support values based on 1,000 bootstrap pseudoreplicates. Species in orange and black indicate the usnic acid producer species and non-producer species, respectively.