Type I NR-PKS Gene Characterization of the Cultured Lichen Mycobiont Xanthoparmelia Substrigosa (Ascomycota)

Abstract

Secondary metabolites play an important role in drug discovery. Lichens, in the first instance the fungal symbiotic partner (referred to as mycobiont), produce a multitude of metabolites with interesting biological functions. A large share of these organic compounds represents polyketides (e.g., anthraquinones, depsides, and depsidones) biosynthesized by typical polyketide synthase (PKS) enzymes. This study addresses the detection of PKS genes that control the formation of secondary metabolites (polyketide-type compounds), specifically in the lichen species Xanthoparmelia substrigosa (Parmeliaceae). As a first step, the isolated mycobiont of X. substrigosa was cultured in large scale. For the molecular detection and identification of the PKS gene from the cultured mycobiont, the highly conserved β-ketoacyl synthase (KS) domain was used as starting point. Then, the SMARTer RACE cDNA amplification was performed using extracted RNA of the fungal cultures by means of specific primers generated from the obtained sequence of the KS domain. Since the PCR products were over several 1,000 base pairs (bps) long, cloning of the products was the next step to attain sufficient material for the sequencing procedure. The located type I PKS gene of the mycobiont (Xsm PKSI) comprises 2,273 amino acids and is composed of one KS, one acyltransferase (AT), two acyl carrier proteins (ACPs), and one thioesterase (TE) domain. Similar domain formation of the PKS gene has been found in the original lichen thallus of X. substrigosa (XsTPKSI), but differs from the cultured mycobiont sequence with five inserted introns arranged along the first 2,120 bps. Additionally, HPLC analyses of lichen thallus and fungal culture confirmed usnic acid, norstictic acid, and salazinic acid as secondary metabolites, which could be biosynthesized by the detected PKS gene.