Xinxing Lv

A copper-responsive promoter replacement system to investigate gene functions in Trichoderma reesei : a case study in characterizing SAGA genes

Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The molecular mechanism underlying the regulation of cellulase production as well as other physiological processes in T. reesei is still insufficiently understood. We constructed a Ptcu1-based promoter substitution cassette that allowed one-step replacement of the endogenous promoter for controlling the target gene expression with copper. We then showed that copper repression of the histone acetyltransferase gene gcn5 phenocopied the gcn5 deletion strain. Using the same strategy, we further characterized the function of another putative Spt-Ada-Gcn5 acetyltransferase (SAGA) complex subunit encoding gene, ada2, in T. reesei. Similar to the repression of gcn5, the addition of copper to the Ptcu1-ada2 strain not only drastically reduced the vegetative growth and conidiation in T. reesei but also severely compromised the induced cellulase gene expression. The developed strategy will thus be potentially useful to probe the biological function of the large fraction of T. reesei genes with unknown functions including those essential genes in the genome to expand its extraordinary biotechnological potential.

The mating type locus protein MAT1-2-1 of Trichoderma reesei interacts with Xyr1 and regulates cellulase gene expression in response to light

Cellulase production in the model cellulolytic fungus Trichoderma reesei is subject to a variety of environmental and physiological conditions involving an intricate regulatory network with multiple transcription factors. Here, we identified the mating type locus protein MAT1-2-1 as an interacting partner for the key transcriptional activator Xyr1 of T. reesei cellulase genes. Yeast two-hybrid and GST pulldown analyses revealed that MAT1-2-1 directly interacted with the putative transcription activation domain (AD, 767~940 aa) and the middle homology region (MHR2, 314~632 aa) of Xyr1. Disruption of the mat1-2-1 gene compromised the induced expression of cellulase genes with Avicel in response to light or with lactose. Chromatin immunoprecipitation (ChIP) demonstrated that MAT1-2-1 was recruited to the cbh1 (cellobiohydrolase 1-encoding) gene promoter in a Xyr1-dependent manner. These results strongly support an important role of MAT1-2-1 as a physiological cofactor of Xyr1, and suggest that MAT1-2-1 represents another regulatory node that integrates the light response with carbon source signaling to fine tune cellulase gene transcription.

Gluconolactone induces cellulase gene expression in cellulolytic filamentous fungus Trichoderma reesei

Filamentous fungus Trichoderma reesei is well known for its high capacity of producing cellulases for industrial application. Besides crystalline cellulose, several soluble sugars including cellobiose can effectively induce cellulase formation. In this study, gluconolactone, previously reported as a β-glucosidase inhibitor, was demonstrated to be capable of inducing cellulase gene expression at a level equivalent to that induced by cellobiose. Gluconolactone-induced formation of cellulase was abolished in T. reesei strain lacking Xyr1 or Crt1, two key regulators for cellulase gene expression. The induced expression of cellulase gene cbh1 was eliminated in the absence of intracellular β-glucosidase Cel1a on gluconolactone while it was hardly affected by the absence of extracellular β-glucosidase Bgl1. We further found that the absence of a cellobiose/glucose transporter Stp1 compromised cellulase production and led to a lower consumption of extracellular gluconolactone. These results suggest that the gluconolactone-derived inducing signal involves both its sensing at the membrane and its intracellular delivery for further processing to initiate cellulase formation.