Improvement of β‐Xylosidase and Endoxylanase Activities in <i>Talaromyces amestolkiae</i> by Genetic Manipulation of the Transcriptional Activator <scp>XlnR</scp>

The ascomycete Talaromyces amestolkiae is a promising source of glycosyl hydrolases for hemicellulose degradation, as it contains a considerably higher number of genes encoding these enzymes than other fungi exploited for plant biomass valorisation. The development of genetic engineering tools could further improve its biotechnological potential. We report here a transformation system for T. amestolkiae based on pyrimidine auxotrophy complementation, which was used to successfully introduce both integrative and autonomously replicating plasmids. Then, we applied this tool to force the expression of the transcriptional activator XlnR, generating an engineered strain with enhanced β-xylosidase (1.4-fold) and endoxylanase (2.0-fold) activities compared to the wild-type cultured on xylan. Markedly larger improvements were obtained after introducing Ala788Val or Val785Phe substitutions in XlnR, achieving 3.3-fold and 3.9-fold increases in β-xylosidase and endoxylanase activities, respectively, in the case of XlnR^V785F. This recombinant strain also displays a partial deregulation of the hemicellulolytic system when cultivated on glucose and glycerol (a low-cost and renewable substrate), yielding notably higher production of β-xylosidases (16.9-fold and 13.8-fold) and endoxylanases (31.9-fold and 22.7-fold) than the wild-type. Increased efficiencies of XlnR^V785F enzymatic crudes in xylan saccharifications showed the potential of XlnR engineering to develop robust T. amestolkiae strains for the valorisation of hemicellulosic residues.