Structure-guided engineering of transcriptional activator XYR1 for inducer-free production of lignocellulolytic...

The filamentous fungus Trichoderma reesei is widely used for the production of lignocellulolytic enzymes in industry. XYR1 is the major transcriptional activator of cellulases and hemicellulases in T. reesei. However, rational engineering of XYR1 for improved lignocellulolytic enzymes production has been limited by the lack of structure information. Here, alanine 873 was identified as a new potential target for the engineering of XYR1 based on its structure predicted by AlphaFold2. The mutation of this residue to tyrosine enabled significantly enhanced production of xylanolytic enzymes in the medium with cellulose as the carbon source. Moreover, xylanase and cellulase production increased by 56.7- and 3.3-fold, respectively, when glucose was used as the sole carbon source. Under both conditions, the improvements of lignocellulolytic enzyme production were higher than those in the previously reported V821F mutant. With the enriched hemicellulases and cellulases, the crude enzymes secreted by the A873Y mutant strain produced 51 % more glucose and 52 % more xylose from pretreated corn stover than those of the parent strain. The results provide a novel strategy for engineering the lignocellulolytic enzyme-producing capacity of T. reesei, and would be helpful for understanding the molecular mechanisms of XYR1 regulation.