Cellulolytic enzyme hydrolysis of lignocellulose biomass to release fermentable sugars is one of the key steps in biofuel refining. Gene expression of fungal cellulolytic enzymes is tightly controlled at the transcriptional level. Key transcription factors such as activator ClrB/CLR2 and XlnR/XYR1, as well as repressor CreA/CRE1 play crucial roles in this process. The putative protein methyltransferase LaeA/LAE1 has also been reported to regulate the gene expression of the cellulolytic enzyme. The formation and gene expression of the cellulolytic enzyme was compared among Penicillium oxalicum wild type (WT) and seven mutants, including ΔlaeA (deletion of laeA), OEclrB (clrB overexpression), OEclrBΔlaeA (clrB overexpression with deletion of laeA), OExlnR (xlnR overexpression), OExlnRΔlaeA (xlnR overexpression with deletion of laeA), ΔcreA (deletion of creA), and ΔcreAΔlaeA (double deletion of creA and laeA). Results revealed that LaeA extensively affected the expression of glycoside hydrolase genes. The expression of genes that encoded the top 10 glycoside hydrolases assayed in secretome was remarkably downregulated especially in later phases of prolonged batch cultures by the deletion of laeA. Cellulase synthesis of four mutants ΔlaeA, OEclrBΔlaeA, OExlnRΔlaeA, and ΔcreAΔlaeA was repressed remarkably compared with their parent strains WT, OEclrB, OExlnR, and ΔcreA, respectively. The overexpression of clrB or xlnR could not rescue the impairment of cellulolytic enzyme gene expression and cellulase synthesis when LaeA was absent, suggesting that LaeA was necessary for the expression of cellulolytic enzyme gene activated by ClrB or XlnR. In contrast to LaeA positive roles in regulating prominent cellulase and hemicellulase, the extracellular β-xylosidase formation was negatively regulated by LaeA. The extracellular β-xylosidase activities improved over 5-fold in the OExlnRΔlaeA mutant compared with that of WT, and the expression of prominent β-xylosidase gene xyl3A was activated remarkably. The cumulative effect of LaeA and transcription factor XlnR has potential applications in the production of more β-xylosidase.

There is a rising interest in bioethanol production from lignocellulose such as corn stover to decrease the need for fossil fuels, but most research mainly focuses on how to improve ethanol yield and pays less attention to the biorefinery of corn stover. To realize the utilization of different components of corn stover in this study, different pretreatment strategies were used to fractionate corn stover while enhancing enzymatic digestibility and cellulosic ethanol production. It was found that the pretreatment process combining dilute acid (DA) and alkaline sodium sulfite (ASS) could effectively fractionate the three main components of corn stover, i.e., cellulose, hemicellulose, and lignin, that xylose recovery reached 93.0%, and that removal rate of lignin was 85.0%. After the joint pretreatment of DA and ASS, the conversion of cellulose at 72 h of enzymatic hydrolysis reached 85.4%, and ethanol concentration reached 48.5 g/L through fed-batch semi-simultaneous saccharification and fermentation (S-SSF) process when the final concentration of substrate was 18% (w/v). Pretreatment with ammonium sulfite resulted in 83.8% of lignin removal, and the conversion of cellulose and ethanol concentration reached 86.6% and 50 g/L after enzymatic hydrolysis of 72 h and fed-batch S-SSF, respectively. The results provided a reference for effectively separating hemicellulose and lignin from corn stover and producing cellulosic ethanol for the biorefinery of corn stover.

Heterochromatin protein 1 (HP1, homologue HepA in Penicillium oxalicum) binding is associated with a highly compact chromatin state accompanied by gene silencing or repression. HP1 loss leads to the derepression of gene expression. We investigated HepA roles in regulating cellulolytic enzyme gene expression, as an increasingly number of studies have suggested that cellulolytic enzyme gene expression is not only regulated by transcription factors, but is also affected by the chromatin status. Among the genes that exhibited significant differences between the hepA deletion strain (ΔhepA) and the wild type (WT), most (95.0 %) were upregulated in ΔhepA compared with WT. The expression of the key transcription factor for cellulolytic enzyme gene (e.g., repressor CreA and activator ClrB) increased significantly. However, the deletion of hepA led to downregulation of prominent extracellular cellulolytic enzyme genes. Among the top 10 extracellular glycoside hydrolases (Amy15A, Amy13A, Cel7A/CBHI, Cel61A, Chi18A, Cel3A/BGLI, Xyn10A, Cel7B/EGI, Cel5B/EGII, and Cel6A/CBHII), in which secretion amount is from the highest to the tenth in P. oxalicum secretome, eight genes, including two amylase genes (amy15A and amy13A), all five cellulase genes (cel7A/cbh1, cel6A/cbh2, cel7B/eg1, cel5B/eg2, and cel3A/bgl1), and the cellulose-active LPMO gene (cel61A) expression were downregulated. Results of chromatin accessibility real-time PCR (CHART-PCR) showed that the chromatin of all three tested upstream regions opened specifically because of the deletion of hepA in the case of two prominent cellulase genes cel7A/cbh1 and cel7B/eg1. However, the open chromatin status did not occur along with the activation of cellulolytic enzyme gene expression. The overexpression of hepA upregulated the cellulolytic enzyme gene expression without chromatin modification. The overexpression of hepA remarkably activated the cellulolytic enzyme synthesis, not only in WT (~150 % filter paper activity (FPA) increase), but also in the industry strain RE-10 (~20–30 % FPA increase). HepA is required for chromatin condensation of prominent cellulase genes. However, the opening of chromatin mediated by the deletion of hepA was not positively correlated with cellulolytic enzyme gene activation. HepA is actually a positive regulator for cellulolytic enzyme gene expression and could be a promising target for genetic modification to improve cellulolytic enzyme synthesis.