Characterization of a mutant strain of a filamentous fungus Cladosporium phlei for the mass production of the secondary metabolite phleichrome

Abstract

UV-mutagenesis was performed to obtain mutant strains that demonstrate altered production of phleichrome, a secondary metabolite of Cladosporium phlei. Among fifty mutants selected, based on the increased area and intensity of the purple pigment surrounding the colonies, the strain M0035 showed the highest production of phleichrome, more than seven fold over wild type. Plate cultures of the M0035 strain resulted in a total of 592 mg phleichrome consisting of 146 mg and 446 mg from the mycelia and agar media, respectively. The M0035 strain displayed a growth rate and a mycelial mass comparable to the parental strain but had significantly reduced asexual sporulation.

Improved production of phleichrome from the phytopathogenic fungus Cladosporium phlei using synthetic inducers and photodynamic ROS production by phleichrome

“…The C. phlei wildtype strain obtained from the American Type Culture Collection (ATCC 36193) and the bred strain M0035 obtained from the previous UV-mutagenesis study (10) were maintained on V8 juice agar plates under constant dark condition at 20 C, as previously described (9,10). The culture conditions and methods for the preparation of cultures on cellophane membrane overlaying agar plates have been previously described (9,10).…”

“…The culture conditions and methods for the preparation of cultures on cellophane membrane overlaying agar plates have been previously described (9,10). Briefly, experimental cultures were initiated by placing uniform sized agar blocks, excised from the margins of actively growing colonies, at the center of the plates containing appropriate media with or without cellophane overlay.…”

“…The mycelia were then harvested by scraping from the detached cellophane overlaying media, which was used to determine the yield of phleichrome. In order to determine the yield of secreted phleichrome, 20 uniform-size (0.5-cm-diameter) agar plugs of media were obtained from positions 2 cm away from the center of the plate (10).…”

“…To determine phleichrome content, phleichrome was extracted from mycelia and agar media using EtOAc, as described previously (10). To rapidly determine if phleichrome was present, the crude extract was analyzed using thin-layer chromatography (TLC) on silica gel with a resolving solution (CH 2 Cl 2 /MeOH ¼ 19:1, v/v) and purified phleichrome as a control, as described previously (10).…”

“…In our previous study, we described the culture characteristics of C. phlei, the extraction of phleichrome, and the UV-mutagenesis of C. phlei to breed a fungal strain for the stable overproduction of phleichrome, which yielded 146 mg and 446 mg of phleichrome in the mycelia and the agar media of the bred strain, respectively, from 25 plates produced by pouring 40 ml of melted V8 juice agar (9,10). Although these studies demonstrated that it was possible to prepare phleichrome from biological sources, a further increase in phleichrome production is required for the sustainable conversion of phleichrome to hypocrellins and the development of other effective derivatives.…”