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. 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.

Many Penicillium species could produce extracellular enzyme systems with good lignocellulose hydrolysis performance. However, these species and their enzyme systems are still poorly understood and explored due to the lacking of genetic information. Here, we present the genomic and secretomic analyses of Penicillium decumbens that has been used in industrial production of lignocellulolytic enzymes in China for more than fifteen years. Comparative genomics analysis with the phylogenetically most similar species Penicillium chrysogenum revealed that P. decumbens has evolved with more genes involved in plant cell wall degradation, but fewer genes in cellular metabolism and regulation. Compared with the widely used cellulase producer Trichoderma reesei, P. decumbens has a lignocellulolytic enzyme system with more diverse components, particularly for cellulose binding domain-containing proteins and hemicellulases. Further, proteomic analysis of secretomes revealed that P. decumbens produced significantly more lignocellulolytic enzymes in the medium with cellulose-wheat bran as the carbon source than with glucose. The results expand our knowledge on the genetic information of lignocellulolytic enzyme systems in Penicillium species, and will facilitate rational strain improvement for the production of highly efficient enzyme systems used in lignocellulose utilization from Penicillium species.

Advances in synthetic biology and our understanding of the rules of promoter architecture have led to the development of diverse synthetic constitutive and inducible promoters in eukaryotes and prokaryotes. However, the design of promoters inducible by specific endogenous or environmental conditions is still rarely undertaken. In this study, we engineered and characterized a set of strong, synthetic promoters for budding yeast Saccharomyces cerevisiae that are inducible under acidic conditions (pH ≤ 3). Using available expression and transcription factor binding data, literature on transcriptional regulation, and known rules of promoter architecture we improved the low-pH performance of the YGP1 promoter by modifying transcription factor binding sites in its upstream activation sequence. The engineering strategy outlined for the YGP1 promoter was subsequently applied to create a response to low pH in the unrelated CCW14 promoter. We applied our best promoter variants to low-pH fermentations, enabling ten-fold increased production of lactic acid compared to titres obtained with the commonly used, native TEF1 promoter. Our findings outline and validate a general strategy to iteratively design and engineer synthetic yeast promoters inducible to environmental conditions or stresses of interest.

Long-term strain improvements through repeated mutagenesis and screening have generated a hyper-producer of cellulases and hemicellulases from Penicillium decumbens 114 which was isolated 30 years ago. Here, the genome of the hyper-producer P. decumbens JU-A10-T was sequenced and compared with that of the wild-type strain 114-2. Further, the transcriptomes and secretomes were compared between the strains. Selective hyper-production of cellulases and hemicellulases but not all the secreted proteins was observed in the mutant, making it a more specific producer of lignocellulolytic enzymes. Functional analysis identified that changes in several transcriptional regulatory elements played crucial roles in the cellulase hyper-producing characteristics of the mutant. Additionally, the mutant showed enhanced supply of amino acids and decreased synthesis of secondary metabolites compared with the wild-type. The results clearly point out that we can target gene regulators and promoters with minimal alterations of the genetic content but maximal effects in genetic engineering.

In cellulolytic fungi, induction and repression mechanisms synchronously regulate the synthesis of cellulolytic enzymes for accurate responses to carbon sources in the environment. Many proteins, particularly transcription regulatory factors involved in these processes, were identified and genetically engineered in Penicillium oxalicum and other cellulolytic fungi. Despite such great efforts, its effect of modifying a single target to improve the production of cellulase is highly limited. In this study, we developed a systematic strategy for the genetic engineering of P. oxalicum to enhance cellulase yields, by enhancing induction (by blocking intracellular inducer hydrolysis and increasing the activator level) and relieving the repression. We obtained a trigenic recombinant strain named ‘RE-10’ by deleting bgl2 and creA, along with over-expressing the gene clrB. The cellulolytic ability of RE-10 was significantly improved; the filter paper activity and extracellular protein concentration increased by up to over 20- and 10-fold, respectively, higher than those of the wild-type (WT) strain 114-2 both on pure cellulose and complex wheat bran media. Most strikingly, the cellulolytic ability of RE-10 was comparable with that of the industrial P. oxalicum strain JU-A10-T obtained by random mutagenesis. Comparative proteomics analysis provided further insights into the differential secretomes between RE-10 and WT strains. In particular, the enzymes and accessory proteins involved in lignocellulose degradation were elevated specifically and dramatically in the recombinant, thereby confirming the importance of them in biomass deconstruction and implying a possible co-regulatory mechanism. We established a novel route to substantially improve cellulolytic enzyme production up to the industrial level in P. oxalicum by combinational manipulation of three key genes to amplify the induction along with derepression, representing a milestone in strain engineering of filamentous fungi. Given the conservation in the mode of cellulose expression regulation among filamentous fungi, this strategy could be compatible with other cellulase-producing fungi.