Functional characterization of lichen fungal (Cladonia uncialis) genes and exploration of its secondary metabolome
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Lichens are traditionally described as symbionts of fungi and algae and are renowned for their diverse secondary metabolites. Our group identified and annotated 48 secondary metabolite biosynthetic gene clusters of the fungal partner of the lichen Cladonia uncialis using de novo whole genome sequencing. Out of these, putative assignment of biosynthetic functions of ‘ten’ gene clusters in C. uncialis was done by Bertrand (2018) including the usnic acid (UA) gene cluster (the most extensively studied lichen secondary metabolite). This research work deduced biosynthetic pathways and proposed biosynthetic function of seven more gene clusters using a 'homology mapping’ approach in combination with phylogenetics. The UA gene cluster contains two genes, one encodes for PKS enzyme and other for a post PKS tailoring enzyme, that have been respectively named as methylphloracetophenone synthase (MPAS) and methylphloroacetophenone oxidase (MPAO, a cytochrome p450). MPAO is believed to catalyze the oxidative dimerization of methylphloroacetophenone (MPA) to usnic acid (UA). Electron transfer reactions from NAD(P)H to a cytochrome p450 are supported by redox partner, cytochrome P450 oxidoreductase (CPR). We have identified a gene, cu-cpr in the genome of C. uncialis that encodes for a similar redox partner. We have successfully developed heterologous expression protocols for first lichen cytochrome p450 (mpao) and CPR (cu-cpr) in E. coli and, the purification protocols for these lichen proteins. We have also established a protocol for heterologous co-expression system for mpao and cpr in E. coli. This study demonstrates the first successful biosynthesis of UA (a lichen polyketide) with the development of bioconversion protocol of MPA to UA catalyzed by both MPAO and CPR. Biofilm-disruption and antibacterial assays were carried out to compare the bioactivity of MPA and UA, where UA was found to be more bioactive. That could suggest a rationale for why lichen fungus exerts the effort required to produce UA. Phloroglucinol derivatives (including MPA derivatives) were chemically synthesized and tested for their antibiotic properties against S. aureus (an important CF pathogen). Out of which, diacylated derivatives displayed the strongest bactericidal activity against MRSA clinical isolates.