Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from Cladonia macilenta. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with Agrobacterium cells, and incubation temperature, were tested to identify an optimized ATMT condition for the C. macilenta mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in C. macilenta, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.

Summary: Depsides and depsidones are polyketide‐derived lichen acids widely distributed in lichen thalli, yet the biosynthetic gene clusters (BGCs) responsible for their production remain poorly understood. To address this gap, we investigated the diversity and evolutionary relationships of polyketide BGCs in lichens. We conducted genome mining and phylogenetic analyses of polyketide synthases (PKSs) across 100 lichen genomes, followed by BGC network analysis to assess BGC diversity. Functional validation was performed through heterologous expression of PKS and tailoring enzyme genes in heterologous hosts. Lecanorales species showed the highest diversity of PKSs. Phylogenetic analysis revealed that pks1 is closely related to pks23 responsible for atranoric acid biosynthesis. The pks1 BGCs exhibited gene content variation between species producing different depsides/depsidones. Heterologous expression of pks1 produced 4‐ O ‐demethylbarbatic acid, the same compound from pks23 . Co‐expression of pks1 with tailoring enzymes yielded virensic acid, a depsidone precursor. High pks1 expression in barbatic acid–containing thalli supported its role in medullary depside/depsidone biosynthesis. These findings establish pks1 and pks23 as paralogous PKSs and reveal how tailoring enzyme diversity contributes to chemical diversity of lichen acids. This study provides new insight into the biosynthetic and evolutionary mechanisms driving structural diversification of lichen acids.