Enhancement of Postharvest Biocontrol Activity of the Yeast Candida oleophila by Overexpression of Lytic Enzymes

Enhancing the activity of biocontrol agents could be the most important factor in their success in controlling fruit disease and their ultimate acceptance in commercial disease management. Direct manipulation of a biocontrol agent resulting in enhancement of diseases control could be achieved by using recent advances in molecular biology techniques. The objectives of this project were to isolate genes from yeast species that were used as postharvest biocontrol agents against postharvest diseases and to determine their role in biocontrol efficacy. The emphasis was to be placed on the yeast, Candida oleophila, which was jointly discovered and developed in our laboratories, and commercialized as the product, Aspire. The general plan was to develop a transformation system for C . oleophila and either knockout or overexpress particular genes of interest. Additionally, biochemical characterization of the lytic peptides was conducted in the wild-type and transgenic isolates. In addition to developing a better understanding of the mode of action of the yeast biocontrol agents, it was also our intent to demonstrate the feasibility of enhancing biocontrol activity via genetic enhancement of yeast with genes known to code for proteins with antimicrobial activity. Major achievements are: 1) Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila; 2) Development of a transformation system for Candida oleophila; 3) Cloning and analysis of C.oleophila glucanase gene; 4) Overexpression of and knockout of C. oleophila glucanase gene and evaluating its role in the biocontrol activity of C. oleophila; 5) Characterization of defensin gene and its expression in the yeast Pichiapastoris; 6) Cloning and Analysis of Chitinase and Adhesin Genes; 7) Characterization of the rnase secreted by C . oleophila and its inhibitory activity against P. digitatum. This project has resulted in information that enhanced our understanding of the mode of action of the yeast C . oleophila. This was important step towards enhancing the biocontrol activity of the yeast. Fungal cell wall enzymes produced by the yeast antagonist were characterized. Different substrates were identified to enhance there production in vitro. Exo-b-1, 3 glucanase, chitinase and protease production was stimulated by the presence of cell-wall fragments of Penicillium digitatum in the growing medium, in addition to glucose. A transformation system developed was used to study the role of lytic enzymes in the biocontrol activity of the yeast antagonist and was essential for genetic manipulation of C . oleqphila. After cloning and characterization of the exo-glucanase gene from the yeast, the transformation system was efficiently used to study the role of the enzyme in the biocontrol activity by over-expressing or knocking out the activity of the enzyme. At the last phase of the research (still ongoing) the transformation system is being used to study the role of chitinase gene in the mode of action. Knockout and over expression experiments are underway.