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.

A single copy of the Renilla reniformis green fluorescent protein (GFP) gene under control of the cellobiohydrolase I (cbh1) promoter was inserted into a strain of Trichoderma reesei at the pyr4 locus by homologous recombination. The parent strain contained two copies (a tandem repeat) of the native glucoamylase (GA) gene integrated at a different locus, and controlled by a separate copy of the cbh1 promoter. A large positive correlation (r = 0.54) was observed between GFP expression and GA activity in ∼1900 randomly mutagenised germlings cultured in microtiter plates. The GFP-expressing strain was randomly mutagenised and screened using fluorescence activated cell sorting. Single germinating spores expressing GFP under conditions of carbon catabolite repression were sorted into individual wells of ten 96-well microtiter plates, cultured for 6 days, and assayed for GA activity. The strain producing the highest titres of GA from each of three rounds was re-screened by FACS. A variant (R3-14) was isolated that produced about 35% more total protein at 70% greater specific productivity in 14L fed-batch fermentation. The genome of strain R3-14 was analysed using comparative genome hybridization and large duplications totalling more than 750 kbp were identified on scaffolds 13, 2, 33 and 38 comprising 189 open reading frames. These included several putative transcriptional regulators, carbohydrate hydrolysing enzymes, and sugar transporters.

We have increased the production of bovine chymosin in Aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. Analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.