Optimization of Growth Medium and Fermentation Conditions for the Production of Laccase3 from Cryphonectria parasitica Using Recombinant Saccharomyces cerevisiae

Statistical experimental methods were used to optimize the medium for mass production of a novel laccase3 (Lac3) by recombinant Saccharomyces cerevisiae TYEGLAC3-1. The basic medium was composed of glucose, casamino acids, yeast nitrogen base without amino acids (YNB w/o AA), tryptophan, and adenine. A one-factor-at-a-time approach followed by the fractional factorial design identified galactose, glutamic acid, and ammonium sulfate, as significant carbon, nitrogen, and mineral sources, respectively. The steepest ascent method and response surface methodology (RSM) determined that the optimal medium was (g/L): galactose, 19.16; glutamic acid, 5.0; and YNB w/o AA, 10.46. In this medium, the Lac3 activity (277.04 mU/mL) was 13.5 times higher than that of the basic medium (20.50 mU/mL). The effect of temperature, pH, agitation (rpm), and aeration (vvm) was further examined in a batch fermenter. The best Lac3 activity was 1176.04 mU/mL at 25 °C, pH 3.5, 100 rpm, and 1 vvm in batch culture.

Antimicrobial and Antitumor Photodynamic Effects of Phleichrome from the Phytopathogenic Fungus Cladosporium Phlei

Fungal perylenequinones have photodynamic activity and are promising photosensitizers for photodynamic therapy (PDT). Here, we investigated the bactericidal and antitumor activities of phleichrome from the fungal perylenequinone family in vitro. Photodynamic bactericidal activity of phleichrome was analyzed by agar-well diffusion method under dark and illuminated conditions. The photodynamic antitumor activity of phleichrome was analyzed in MCF-7, HeLa, SW480, and HepG2 human cancer cell lines using in vitro cytotoxicity assays. Photodynamic bactericidal activities against Gram-negative and Gram-positive bacteria were species-specific. Antitumor activity against all tumor cell lines increased under the illuminated condition. Depending on the results of the analyses, Phleichrome has potential for further drug development related to its antibacterial and antitumor activities.

Role of MAPK Signaling Pathways in Regulating the Hydrophobin Cryparin in the Chestnut Blight Fungus Cryphonectria parasitica

We assessed the regulation of cryparin, a class II hydrophobin, using three representative mitogen-activated protein kinase (MAPK) pathways in Cryphonectria parasitica. Mutation of the CpSlt2 gene, an ortholog of yeast SLT2 in the cell wall integrity (CWI) pathway, resulted in a dramatic decrease in cryparin production. Similarly, a mutant of the CpBck1 gene, a MAP kinase kinase kinase gene in the CWI pathway, showed decreased cryparin production. Additionally, mutation of the cpmk1 gene, an ortholog of yeast HOG1, showed decreased cryparin production. However, mutation of the cpmk2 gene, an ortholog of yeast Kss1/Fus3, showed increased cryparin production. The easy-wet phenotype and accumulation of the cryparin transcript in corresponding mutants were consistent with the cryparin production results. In silico analysis of the promoter region of the cryparin gene revealed the presence of binding motifs related to downstream transcription factors of CWI, HOG1, and pheromone responsive pathways including MADS-box- and Ste12-binding domains. Real-time reverse transcriptase PCR analyses indicated that both CpRlm1, an ortholog of yeast RLM1 in the CWI pathway, and cpst12, an ortholog of yeast STE12 in the mating pathway, showed significantly reduced transcription levels in the mutant strains showing lower cryparin production in C. prasitica. However, the transcription of CpMcm1, an ortholog of yeast MCM1, did not correlate with that of the mutant strains showing downregulation of cryparin. These results indicate that three representative MAPK pathways played a role in regulating cryparin production. However, regulation varied depending on the MAPK pathways: the CWI and HOG1 pathways were stimulatory, whereas the pheromone-responsive MAPK was repressive.

Mycoflora and Enzymatic Characterization of Fungal Isolates in Commercial Meju, Starter for a Korean Traditional Fermented Soybean Product

Mycoflora was assessed in the commercial meju from four well-separated geographic origins. A total of 112 fungal isolates were identified by phenotypic characteristics and molecular taxonomy using sequencing the internal transcribed spacer of the rDNA and revealed 19 species from 13 genera. Enzymatic characteristics of protease and amylase, and mycotoxin production were analyzed.