Demonstration of a single nucleotide substitution with significant impact on specific productivity in Trichoderma reesei

As part of our continued effort to maximize the amount of secreted protein produced by our strains, we chemically mutagenized Trichoderma reesei strain RL-P37 and searched for strains with improved specific productivity. One such strain, Strain A, was found and subsequently sequenced against its RL-P37 parent strain to determine which gene(s) were responsible for the improvement in specific productivity. As a result, numerous intragenic and intergenic mutations were identified, of which a subset were chosen to be candidates for introduction back into the RL-P37 strain. Those strains were assessed for changes in specific productivity. It was determined that the introduction of a single nucleotide substitution in a histidine kinase gene (hk^mutant) into strain RL-P37 brought specific productivity to the heights seen in Strain A. The mutation in hk^mutant was responsible for phenotypic changes as well, specifically a sensitivity to high osmolarity. This is in line with the function of histidine kinase homologs as osmosensors in other filamentous fungi. Deletion of the native hk^wt allele in strain RL-P37 significantly reduced specific productivity, indicating that the hk^mutant allele is a gain of function mutation. Replacing the mutant hk^mutant allele with the wild type hk^wt allele in Strain A returned Strain A to RL-P37 levels of specific productivity, demonstrating that the mutated hk gene is the main gene responsible for the specific productivity improvement in Strain A. To demonstrate that we can achieve a benefit in other host backgrounds as well as strains overexpressing proteins of interest, we replaced the native hk^wt allele with the hk^mutant allele in a T. reesei high volumetric productivity strain, called Strain B, over-expressing Cbh1. An increase in specific productivity was observed, confirming that we can combine the hk^mutant allele with other useful host modifications. The use of the discovered hk^mutant allele, alone or combined with other beneficial mutations, will significantly improve secreted protein production.