Isolation of a strain of Trichoderma reesei with improved glucoamylase secretion by flow cytometric sorting

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.