Study for the Mass Production of Fungal Metabolite, Phleichrome from a Phytopathogenic Fungus Cladosporium phlei

Hypomycetous fungus Cladosporium phlei (C.T.Gregory) de Vries, known as a causal agent of leaf spot disease of timothy(Phleumpratense), is characterized by the production of deep red pigmented mycelia due to the presence of a fungal pigment phleichrome. Pheichrome, the secondary metabolite from the fungus Cladosporium phlei, has been known as a key precursor of photosensitizer which produce a reactive oxygen species due to the activation of photoenergy, photosensitizer is a key component of photodynamic theraphy(PDT). In our previous studies, the culture condition of C. phlei was previously achieved at 20℃ of temperature and using V8 juice-based media. Simple solvent extracting procedures using fungal mycelia as well as culture filtrate were developed to isolate the pigment from C. phlei and the resulting deep red pigment was identified to be phleichrome by NMR analysis. In the study, the fermentational features forthe mass production of phleichrome have been determined and the genes related to the biosynthesis of phleichrome is defined. The improved strain to produce phleichrome using UV mutagenesis and molecular technique as a genetic manipulation is found. Mycellial mass and culture filtrates were extracted with ethyl acetate(EtOAc). The residues were analyzed via thin-layer chromatography(TLC) using a resolving solution(CH2Cl2-MeOH=95:5, v/v). TLC screening method was progressed in the rapid and easy ways of determining the phleichrome content using analytical method. Phleichrome was produced from mycellial which were harvested at 12 to 15 days. About 50 isolated strains using UV-mutagenesis have analyzed by a rapid TLC screening method to obtain the improved strain. Phelichrome is among the perylenequinone of secondary metabolites. Therefore, polyketide synthase(PKS) gene have isolated by a comparative genomic approach on the genes related to the genes involved in biopsynthesis of fungal polyketide.