Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum

Abstract

Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular β-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a “seesaw model” in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors.

Regulation of the lignocellulolytic response in filamentous fungi

“…Aberrant expression of N. crassa CLR-2 through a constitutive promoter drives a nearly complete cellulase transcriptional response, even during growth on a normally repressive carbon source, indicating that CLR-2 does not require post-translational activation (Coradetti et al, 2013). This is not the case for other CLR-2 orthologs, however, since constitutive expression of A. nidulans and P. oxalicum ClrB does not result in constitutive activation of cellulolytic genes (Coradetti et al, 2013;Li et al, 2015). In N. crassa, regulation of clr-2 transcription is governed by a second zinc binuclear cluster transcription factor, CLR-1 .…”

“…In A. nidulans, CreA regulates xylanase genes as well as expression of their transcriptional regulator, xlnR (Orejas et al, 1999(Orejas et al, , 2001Tamayo et al, 2008). In N. crassa and P. oxalicum, regulation of cellulolytic genes as well as the transcription factor clr-2/clrB is thought to be under CCR control Li et al, 2015;Sun and Glass, 2011;Xiong et al, 2014b). Like in N. crassa and A. nidulans, in T. reesei, CRE1 regulates genes necessary for utilization of plant biomass as well as the transcription factors which regulate their expression.…”

The Different Roles of Penicillium oxalicum LaeA in the Production of Extracellular Cellulase and β-xylosidase

“…reesei, Aspergillus nidulans, N . crassa , and Penicillium oxalicum , such as the repressor CRE1/CreA (Strauss et al, 1995) and ACE1 (Aro et al, 2003) and the positive regulators XYR1/XlnR and CLR2/ClrB (Coradetti et al, 2012; Li et al, 2015). …”

“…The mechanism behind the regulation of lignocellulosic enzyme genes in P. oxalicum is similar to that in Aspergillus and Neurospora . Several transcription factors such as CreA (Liu et al, 2013a), ClrB, and XlnR (Li et al, 2015) have been found to play important roles in regulating cellulolytic enzyme gene expression. For instance, the overexpression of clrB , combined with both deletion of creA and an intracellular β-glucanse gene bgl2 , remarkably activated the cellulase gene expression and improved filter paper activity (FPA) by over 20-fold (Li et al, 2015; Yao et al, 2015).…”

Effect of different terminators on transcription regulatory factor ClrB and XlnR in Penicillium oxalicum 114-2

“…It is reported that the degradation intensity of cellulase is depends on the transcription regulatory factor. ClrB is a global positive regulator,and ClrB/CLR -2, is essential for cellulase expression in P. oxalicum, N.crassa and Aspergillus nidulans [7,8] . It also belongs to the zinc finger protein transcription factor superfamily.…”