Fungal pathogens represent major problems for human health and agriculture. As eukaryotic organisms, fungi share some important features with mammalian cells. Therefore, current anti-fungal antibiotics often can not distinguish between fungi and mammalian cells, resulting in serious side effects in mammalian cells. Accordingly, there is strong impetus to develop antifungal alternatives that are both safe and effective. The E1 family of colicin are channel-forming bacteriocins produced by Escherichia coli, which are bactericidal only to E. coli and related species. To target the channel-forming domain of colicin to fungal cell membrane, we engineered a sexual mating pheromone of Candida albicans, α-factor pheromone to colicin Ia. A peptide was constructed consisting of an α mating pheromone of C. albicans fused to the channel-forming domain of colicin Ia to create a new fusion protein, pheromonicin-CA (PMC-CA). Indirect immunolabeling showed that the PMC-CA bound to fungal cells and inhibited growth in the laboratory and field. In the field, the protective activity of pheromonicin against rice blast disease was significantly greater, on a molar basis, than that of triazoles, tricyclazole or isoprothiolane. These results suggest that fusion peptides may be of value as fungicidal agents under agricultural conditions.
Candida albicans ; chemistry ; Colicins ; chemistry ; Fungicides, Industrial ; chemistry ; Mating Factor ; Peptides ; chemistry ; Protein Engineering

The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL.
Coprinus ; enzymology ; Culture Media ; Cytoplasm ; Fermentation ; Glycoproteins ; metabolism ; Mass Spectrometry ; Mating Factor ; Oxidoreductases Acting on Sulfur Group Donors ; metabolism ; Peptides ; Peroxidases ; biosynthesis ; Pichia ; metabolism ; Protein Disulfide-Isomerases ; metabolism ; Protein Folding ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae Proteins ; metabolism