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

Zhonghai Li, Guangshan Yao, Ruimei Wu, Liwei Gao, Qinbiao Kan, Meng Liu, Piao Yang, Guodong Liu, Yuqi Qin, Xin Song, Yaohua Zhong, Xu Fang (+2 others)
2015 PLoS Genetics  
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
more » ... gulators 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. Cellulolytic fungi have evolved into sophisticated lignocellulolytic systems to adapt to their natural habitat. This trait is important for filamentous fungi, which are the main source of cellulases utilized to degrade lignocellulose to fermentable sugars. Penicillium oxalicum, which produces lignocellulolytic enzymes with more diverse components than Trichoderma reesei, has the capacity to secrete large amounts of cellulases. Meanwhile, cellulase expression is regulated by a complex network involved in many transcription factors in this organism. To better understand how cellulase genes are systematically regulated in P. oxalicum, we employed molecular genetics to uncover the cellulolytic transcription factors on a genome-wide scale. We discovered the synergistic and tunable 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. 6 / 45 Fig 2. Regulation of cellulolytic gene expression by ClrB, XlnR and CreA. (A) Northern blot analysis of the transcription of cellulolytic genes in the mutants versus wild-type strain. The strains were pre-cultured on glucose for 22 hours and then were shifted to Vogel's medium without carbon source for starvation cultivation for 2 hours, and then transferred to the same medium containing 2% (w/v) cellulose. 2 μg of total RNA was electrophoresed and blotted onto Hybond N+ nylon membrane. cbh1, eg2 or xyn1 mRNA was probed at different time points after shift to cellulose. (B) Under the same culture condition, gene expression levels of 3 cellobiohydrolase and 15 endoglucanase genes were determined in the mutants versus wild-type strain by q-PCR. All values Regulation of Cellulase Gene Expression PLOS Genetics | DOI:10.1371/journal.pgen.1005509 September 11, 2015 7 / 45 were normalized using the actin gene value/10000. Values reported were means of three biological replicates of each strain. Error bars represent the standard deviation of 3 biological replicates. Fig 3. Defining the categories of genes potentially regulated by ClrB. The ΔclrB mutant and wild-type strains were pre-cultured on glucose for 22 hours and then shifted to Vogel's media with no carbon source for 2 hours, and then transferred to Vogel's medium containing 2% (w/v) cellulose for 4 hours. These differential expression genes were subjected to gene ontology enrichment analysis and percentages of genes varying significantly for ClrB regulon distributed within each functional category. The x-axis represents the percentage of genes in the corresponding GO class.
doi:10.1371/journal.pgen.1005509 pmid:26360497 pmcid:PMC4567317 fatcat:li4zeplr2ffexm6donjkdlhnl4