Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases

Trichoderma reesei endoglucanase I (EGI) was used as a reporter enzyme for screening mutagenized yeast strains for increased ability to produce protein. Sixteen haploid Saccharomyces cerevisiae strains, transformed with a yeast multicopy vector pALK222, containing the EGI cDNA under the ADH1 promoter, produced EGI activity of 10(-5)-10(-4) g/l. On the average 93% of the total activity was secreted into the culture medium. Two strains with opposite mating types were mutagenized, and several mutants were isolated possessing up to 45-fold higher EGI activity. The best mutants were remutagenized and a second-generation mutant, strain 2804, with an additional twofold increase in EGI activity was selected. The mutant strain 2804 grew more slowly and reached a lower final cell density than the parental strain. In the selective minimal medium, the 2804 strain produced 40 mg/l immunoreactive EGI protein, but only 2% was active enzyme. In the rich medium the secreted EGI enzyme stayed active, but without selection pressure the EGI production ceased after 2 days of cultivation, when the strain 2804 had produced 10 mg/l of EGI. A sevenfold difference was found between the parental and the 2804 strain in their total EGI production relative to cell density. The difference in favour of the mutant strain was also detected on the mRNA level. The 2804 mutant was found to be more active than the parental strain also in the production of T. reesei cellulases, cellobiohydrolase I, and cellobiohydrolase II.