Evolution and screening of Trichoderma reesei mutants for secreted protein production at elevated temperature

The filamentous fungus Trichoderma reesei is a mesophilic ascomycete commercially used to produce industrial enzymes for a variety of applications. Strain improvement efforts over many years have resulted not only in more productive hosts, but also in undesirable traits such as the need for lower temperatures to achieve maximum protein secretion rates. Lower fermentation temperatures increase the need for cooling resulting in higher manufacturing costs. We used a droplet-based evolution strategy to increase the protein secretion temperature of a highly productive T. reesei whole cellulase strain from 25 degrees C to 28 degrees C by first isolating an improved mutant and subsequently tracing the causative high-temperature mutation to one gene designated gef1. An industrial host with a gef1 deletion was found to be capable of improved productivity at higher temperature under industrially relevant fermentation conditions. One-Sentence Summary High-temperature droplet-based evolution resulted in the identification of a mutation in Trichoderma reesei gef1 enabling high productivity at elevated temperatures.