Flow cytometric sorting of the filamentous fungus Trichoderma reesei for improved strains

Abstract

Metabolic measurements and screening of Trichoderma reesei have conventionally been performed during the hyphal stage of fungal development. To determine if flow cytometric measurements of protein expression could be made on germinating spores we created a gene construct, placing the Renilla reniformis green fluorescent protein gene under control of the cellobiohydrolase I ( cbh1) promoter and terminator of T. reesei. This vector was transformed into T. reesei and GFP expression was measured in germlings by flow cytometry. Fluorescence associated with GFP expression was observed in germlings grown under conditions known to induce cellulases in Trichoderma. Spores were mutated using UV light and germinating spores were screened for increased GFP expression using high-speed cell sorting, to select for strains with genetic changes associated with increased protein expression. Secondary screens for cellulase production were conducted in microtitre plates. Flow cytometric screening of germinating spores expressing GFP yielded a mutant with improved ability to hydrolyse biomass.