Cercosporamide, a polyketide-derived fungal metabolite, serves as an antifungal agent against phytopathogenic fungi.

An endophytic fungus, Phoma sp. NG-25, produces a set of structurally related polyketides including cercosporamide, phomodione, and usnic acid, among which, cercosporamide has been reported to have strong antifungal and anticancer activities. In this study, Phoma sp. NG-25 was grown in seven growth media to determine the optimal culture condition conducive for cercosporamide production. Cercosporamide production peaked on the eighteenth day of incubation in beef peptone dextrose (BPD) broth media. The cercosporamide titer reached to an average of 77.5 µg/mL in BPD. Paper disk diffusion assay revealed that culture filtrate containing cercosporamide as a major constituent inhibited the growth of taxonomically diverse plant pathogens, including ascomycetous, basidiomycetous, and oomycete fungi. Cercosporamide exhibited strong antifungal activities against two pepper anthracnose pathogens, Colletotrichum gloeosporioides and C. scovillei with EC50 values of 3.8 and 7.0 µg/mL, respectively. This study suggests the potential application of cercosporamide as an effective antifungal agent in controlling anthracnose in pepper.

Identification of a biosynthetic gene cluster for a red pigment cristazarin produced by a lichen-forming fungus Cladonia metacorallifera.

Lichens are known to produce many novel bioactive metabolites. To date, approximately 1,000 secondary metabolites have been discovered, which are predominantly produced by the lichen mycobionts. However, despite the extensive studies on production of lichen secondary metabolites, little is known about the responsible biosynthetic gene clusters (BGCs). Here, we identified a putative BGC that is implicated in production of a red pigment, cristazarin (a naphthazarin derivative), in Cladonia metacorallifera. Previously, cristazarin was shown to be specifically induced in growth media containing fructose as a sole carbon source. Thus, we performed transcriptome analysis of C. metacorallifera growing on different carbon sources including fructose to identify the BGC for cristazarin. Among 39 polyketide synthase (PKS) genes found in the genome of C. metacorallifera, a non-reducing PKS (coined crz7) was highly expressed in growth media containing either fructose or glucose. The borders of a cristazarin gene cluster were delimited by co-expression patterns of neighboring genes of the crz7. BGCs highly conserved to the cristazarin BGC were also found in C. borealis and C. macilenta, indicating that these related species also have metabolic potentials to produce cristazarin. Phylogenetic analysis revealed that the Crz7 is sister to fungal PKSs that biosynthesize an acetylated tetrahydoxynaphthalene as a precursor of melanin pigment. Based on the phylogenetic placement of the Crz7 and putative functions of its neighboring genes, we proposed a plausible biosynthetic route for cristazarin. In this study, we identified a lichen-specific BGC that is likely involved in the biosynthesis of a naphthazarin derivative, cristazarin, and confirmed that transcriptome profiling under inducing and non-inducing conditions is an effective strategy for linking metabolites of interest to biosynthetic genes.

Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus.

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.