Purification-free antifungal biocontrol platform using engineered Saccharomyces cerevisiae secreting Trichoderma atroviride chitinase.

Abstract

Chitinases from Trichoderma species exhibit strong antifungal activity and high biocontrol potential, yet their industrial utilization has been constrained by low heterologous secretion efficiency and costly purification processes. In this study, the Generally Recognized As Safe yeast Saccharomyces cerevisiae Y2805 was engineered to secrete chitinase Tch36 from a Korean isolate of T. atroviride, using a rice α-amylase signal peptide under a constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. When cultivated in glycerol–colloidal chitin medium, the recombinant yeast exhibited approximately a fivefold increase in measurable chitinase activity relative to the SC mock control, whereas the empty-vector control showed only a modest increase. The crude, non-concentrated culture filtrate (approximately 1000 U L⁻1) displayed statistically significant antifungal activity against 12 fungal species, including 9 phytopathogens and 3 opportunistic Aspergillus species. Among the phytopathogens, Fusarium graminearum was one of the most strongly inhibited species, showing approximately 40–50% suppression of colonial growth on solid medium. In liquid culture, microscopy-based germination assays revealed ≥ 63% inhibition of early hyphal elongation in Botrytis cinerea and A. niger. All antifungal effects were evaluated relative to filtrates from the empty-vector control prepared under equivalent dilution conditions. The culture filtrate also enhanced protoplast formation, providing direct evidence of chitinase-associated cell wall weakening. Collectively, these results establish a purification-free, yeast-based heterologous secretion platform capable of producing active Trichoderma chitinase with inhibitory effects on diverse plant- and animal-associated fungi. This strategy has practical potential for biocontrol applications and for sustainable bioprocessing technologies based on microbial chitinase production.