First functional expression of lichen cytochrome P450 enzymes enabling In vitro biosynthesis of usnic acid

Abstract

Lichens are symbiotic associations between fungi and photosynthetic partners, typically algae or cyanobacteria (Ingólfsdóttir, 2002). These stable communities of microorganisms are well known for producing wide array of secondary metabolites with diverse biological activities. One such notable metabolite is usnic acid (UA), a dibenzofuran compound extracted from lichen Cladonia uncialis, exhibits antimicrobial, anti-inflammatory, anti-viral and anticancer properties (Azhamuthu et al., 2024; Ingólfsdóttir, 2002). Despite its therapeutic potential, large-scale production is limited due to the extremely slow growth of lichens. To address this challenge, this thesis aims to elucidate and reconstruct the biosynthetic pathway of UA using a heterologous expression. In this work, biosynthetic gene clusters associate with UA production in C. uncialis were identified, and a heterologous expression system was established in Escherichia coli for lichen-derived cytochrome P450 enzymes. The study focuses on methylphloroacetophenone oxidase (MPAO), a cytochrome P450 enzyme, and its redox partner, cytochrome P450 reductase (CPR357), both of which are derived from C. uncialis. MPAO is proposed to catalyze the oxidative dimerization of methylphloroacetophenone (MPA) to form UA, with electron transfer facilitated by CPR357 from NAD(P)H (Abdel-Hameed et al., 2016; Mittal N., 2023). Following heterologous expression, both enzymes were purified using His-Tag Affinity Chromatography to obtain pure catalytically active enzyme. In vitro bioconversion assays using purified MPAO and CPR357 successfully converted MPA to UA, as confirmed by liquid chromatography–mass spectrometry, demonstrating the feasibility of enzymatic UA biosynthesis. In parallel, a comprehensive phylogenetic analysis of a lichen-derived cytochrome P450 associated with depsidone biosynthesis was conducted to investigate evolutionary relationships and identification which allowed us to identify a putative enzyme that catalyzes in this transformation. This work represents the first functional characterization of lichen-derived cytochrome P450 enzymes involved in usnic acid biosynthesis using purified enzymes. Additionally, the phylogenetic investigation offers insights into depsidone biosynthetic pathway. Together, these complementary approaches contribute to advancing the biotechnological potential of lichen-derived natural products.