Engineered Yeasts for Sustainable dsRNA Production: Molecular Tool Development and Fermentation Optimization

Viticulture faces major sustainability challenges due to resource-intensive practices and the large volumes of byproducts such as grape pomace. The widespread use of chemical pesticides also raises environmental and health concerns, highlighting the need for eco-friendly alternatives. This project investigates the valorization of grape pomace as a substrate for engineered yeasts producing double-stranded RNA (dsRNA), a biotechnological tool to combat grapevine diseases like downy mildew and grey mold. To address the high cost of in vitro dsRNA synthesis, the study focuses on optimizing in vivo production and delivery. Genome editing strategies are used to express dsRNA molecules targeting plant susceptibility genes, promoting sustainable crop protection. Chardonnay pomace from the Franciacorta region was separated from stalks, thermally treated (50, 65, 75 °C), dehydrated, and milled. Microbiological analyses showed reduced contamination at 65 °C and 75 °C. Enzymatic hydrolysis with cellulase increased glucose content by ~25%. Growth trials with Saccharomyces cerevisiae EC1118 and Pichia pastoris GS115 showed superior performance of S. cerevisiae. A CRISPR/Cas9 strategy was used to generate an EC1118 mutant lacking the RNT1 gene, enhancing dsRNA accumulation. Two delivery approaches were developed targeting VviLBDIf7, a susceptibility gene in Vitis vinifera: (1) expression of hairpin RNA from the p426-Nat plasmid for in vivo self-annealing, and (2) encapsidation via MS2 bacteriophage coat proteins, using the pESC-URA plasmid co-expressing dsRNA fused to a 19-nt MS2 operator. In both cases, expression was confirmed within 72 hours, and dsRNA was purified by DNase/RNase treatment. Yield optimization and in vivo assays are ongoing. These efforts contribute to more sustainable viticulture, aligned with circular economy principles.