FUNCTIONAL AND STRUCTURAL CHARACTERIZATION OF THE CELLOBIOSE TRANSPORTER CRT3 FROM TRICHODERMA REESEI

Trichoderma reesei is an industrially relevant filamentous fungus widely used in second-generation ethanol production due to its exceptional cellulase secretion capacity and its potential for genetic engineering to achieve higher enzyme yields. Although sugar transporters are critical for regulating these enzymes by sensing both inducing (e.g., cellobiose and sophorose) and repressing sugars (e.g., glucose), most remain functionally and structurally uncharacterized. In this work, we conducted a comprehensive functional and structural characterization of Cellulose Response Transporter 3 (CRT3), a cellobiose and sophorose transporter in T. reesei. To determine its functional role, the crt3 gene was deleted in T. reesei TU-6 using the CRISPR–Cas9 system, followed by gene expression analysis during growth on cellulose. Deletion of crt3 resulted in increased gene expression of major cellulase genes, including cellobiohydrolase cel7a, endoglucanase cel7b, and β-glucosidase cel3a, as well as the key transcriptional activator xyr1 and the essential cellobiose transporter crt1 after 24 hours of cultivation, suggesting a potential compensatory response. For structural characterization, recombinant CRT3 (54 kDa) was heterologously expressed in Saccharomyces cerevisiae DSY-5, purified, and subjected to cryogenic electron microscopy (cryo-EM) data collection. Initial purification tests using different detergent solubilization conditions (LMNG and DDM with soybean lipid) did not yield a 3D volume of the protein. Subsequently, reconstitution into peptidiscs promoted partial CRT3 dimerization, which effectively increased particle size, enhanced image alignment during processing, and resolved the contribution of detergent micelles (a common bottleneck for small membrane proteins), yielding a 3.1 Å density map. Altogether, these findings establish CRT3 as an important mediator of cellulase induction in T. reesei and demonstrate that peptidisc reconstitution is a good strategy for structural studies of small fungal transporters from the Major Facilitator Superfamily.

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2022/14399-1, FAPESP 2024/11106-9,   and 2021/01580-7).