Expression of mannanase and glucanases in lettuce chloroplasts and functional evaluation of enzyme cocktail against <i>Candida albicans</i> in oral cancer patient samples

Candida albicans is a human pathogen responsible for several diseases. C. albicans cell wall contains chitin, glucan and mannan. Therefore, this study evaluated efficacy of enzyme cocktail containing lettuce-expressed chitinases, glucanases and mannanase against C. albicans in cell culture or cancer patient samples. Site-specific integration of the Man1, CelO, Cbh1, Cbh2 expression cassettes and removal of the selectable marker gene from lettuce transplastome was confirmed using three sets of PCR primers. Homoplasmy in transplastomic lines was confirmed in Southern blots by the absence of untransformed genomes. Unlike tobacco, lettuce transplastomic lines had no mosaic phenotype and yielded more biomass than untransformed plants. Maternal inheritance of transgenes was confirmed by lack of segregation when seedlings were germinated in the selection medium. Mannanase expressed in lettuce chloroplasts is thermostable, exhibiting the highest activity at 70 °C; expression increased up to 70 days of growth and is active in a broad range of pH (5-8). Endoglucanases and exoglucanases expressed in lettuce chloroplasts have a broad range of activities between 30 and 75 °C and pH 4-8. Based on observed biomass and mannanase expression level, up to 8640 million enzyme units could be harvested per acre/year. The enzyme cocktail effectively degraded the fungal cell wall, significantly reducing C. albicans viability in cultures and complete inhibition in oral cancer patient samples. Beyond biomedical applications of antifungal drugs, plant cell-based enzyme market is anticipated to double from $52 to >$100 billion in 2028. Edible leaf-based enzyme production offers a new platform that is different from seed-based current technologies for various biotechnology applications.