Studies Of The Transcription Regulatory Mechanism Of Lignocellulose-degrading Enzyme Synthesis And Construction Of Cellulase High-producer In Penicillium Oxalicum

Plant biomass from photosynthesis is the richest and renewable resource on earth. Development and utilization of the bulky substrate to produce biofuels and chemicals provides a promising way to solve the energy and environmental crisis in China. By far, high-cost, low-efficient cellulase is a major bottleneck for lignocellulose-ethanol conversion. Filamentous fungi have been the main producer of commercial cellulase preparations, because that they could secrete a large amount of cellulolytic enzymes in nature. In filamentous fungi, the expression of lignocellulose-degrading enzyme is controlled strictly on the transcriptional level to save the unnecessary energy consumption. The cellulase high-producer Penicillium oxalicum had been isolated in our laboratory and investigated for more than 30 years, and its mutant had been developed to produce cellulase mixture in industrial scale in China. Previous studies demonstrated that the transcription regulation mechanism of cellulase expression in P. oxalicum is not fully known. Based on second-generation high-throughput sequencing, the genomes of both wild type 114-2 and cellulase high-producer JU-A10-T had been unveiled. Comparative genomics studies showed that the mutations of transcription factors were significantly enriched in all mutant proteins. Together, these results implied that many transcription factors and the regulatory mechanism involved exert an essential role in cellulase production and the expression network of cellulolytic genes.To mine the novel transcription factors potentially involved in the cellulase synthesis and further dissert the molecular mechanism for cellulase high-level secretion, and improve the regulatory network of cellulase expression, we completed a serious work toward disserting the regulatory mechanism of several core transcription factors. The observations obtained not only contributed to perfect the regulatory network model for cellulase expression and replenish the knowledge about fungal transcription regulation theory, but pave the way for strain genetic engineering for cellulase higher-production. We successfully constructed the cellulase hyper-producer RE-10 by redesigning the regulatory pathway. In addition, for the first time, we identified the upstream development regulator PoFlbC and one component of chromatin remodelling complex RSCA.Major advances in this thesis are as follows:1. Deciphering the regulatory mechanism of core cellulolytic transcription factors ClrB, CreA, AmyR and XlnR in expression of cellulolytic genesThese results showed that transcription factor ClrB is an ennsential activator of controlling cellulase gene expression. Lack of ClrB reduced fungal growth on cellulose plate and blocked the expression of almost all cellulolytic genes. Comparative transcriptome analyses found that polysaccharide degradation and metabolism, sugar transport processes are strictly dependent on functional ClrB. On the contrary, CreA functions as a major repressor. Gene knock-out studies found that deletion of creA significantly up-regulated the expression level of cellulases and ClrB. In addition, our data indicated that CreA play roles in fungal growth and asexual development. Transcription factor AmyR negatively regulates the expression of cellulase and positively regulates the expression of amylase. Therefore, deletion of AmyR reduced the amylase secretion, but enhanced the cellulase production. In P. oxalicum, XlnR mainly involved in hemicellulase expression, and moderately affect the expression level of cellulases. To sum up, transcription factors ClrB, CreA, AmyR and XlnR constitute the core transcription regulatory components determining the cellulase yields in P. oxalicum.1. Transcription factor-mediated synergistic and dose-controlled regulation of cellulase gene expressionOver-expression of transcription activator ClrB or XlnR significantly improved the expression of cellulases and further protein production. On the contrary, over-expression of repressor CreA or AmyR drastically down-regulated cellulase expression and further reduced the enzyme secretion. These data demonstrated that the expression level positively correlated with the level of activators ClrB and XlnR; negatively correlated with the level of repressor CreA and AmyR. More importantly, double-overexpression of ClrB and XlnR significantly enhanced cellulase expression synergistically. Double deletion of CreA or AmyR significantly further improved the expression level and yield of cellulase compared to ΔcreA or AamyR. Interestingly, mutant gpd（P）::clrB-ΔcreA has an ability to secrete cellulase without inducer to a level of the wild type strain induced by cellulose. Further measurement of the cellulase expression of many mutants gpd（P）::clrB-gpd（P）::xlnR, gpd（P）::clrB-gpd（P）::creA, gpd（P）::clrB-AamyR, AamyR-AcreA and gpd（P）::clrB-ΔcreA, and comparing with their individual single-gene mutant, further signify that the " seesaw" model balanced by the core transcription factors dose-dependent controlled the fungal cellulase output. Transciptional abundance of cellulolytic genes correlated positively with the level of transactivator ClrB or XlnR, and negatively with the level of repressor CreA or AmyR.Protein interaction assayed by combining the Y2H （in vitro） and BiFC （in vivo） demonstrated that ClrB interacted with AmyR and XlnR, and CreA interacted with XlnR. Presumably, the interactions among core cellulolytic regulators have roles in the regulation of cellulase gene expression. Electrophoretic mobility shift assay found that ClrB, XlnR and CreA associated directly with the promoters of cellulolytic genes in vitro. Furthermore, proteomics studies revealed the differential response of different lignocellulose-degrading enzyme components to the transcription factors.3. Functional identification of nucleosome-remodeling complex protein RSCA and investigation of its role in regulation of vegetative growth, cell wall integrity （CWI） and cellulase expression in P. oxalicumThe biological roles of nucleosome-remodeling complex protein RSCA in P. oxalicum was preliminary explorated. Gene knockout study clearly showed that functional RSGA was-critical for maintaining the cell wall integrity. Of particular, the expression of major chitin synthase PDE₀6421 is RSCA strictly dependent. Our data demonstrated that RSCA involved in regulation of cellulase expression. Further, RSCA directly interacted with cellulolytic regulator CreA, their shared biological process involved fungal growth, CWI and cellulase expression. The details of the regulatory mechanism of RSCA and CreA remain further investigation.4. Functional identification and characterization of the developmental regulator FlbC and its role in cellulase synthesis in P. oxalicumThis study identified and characterized a C2H2-type transcription factor called PoFlbC, which is an Aspergillus FlbC ortholog, in cellulolytic fungus Penicillium oxalicum. Gene knockout study found that the lack of PoFlbC resulted in a reduction in expression level of major conidial gene brlA and in the conidiation. And, OEPoflbC strain also exhibited a similar defect in conidiation. These results demonstrated that the native level of PoFlbC was crucial for the normal growth and asexual development of P. oxalicum. Importantly, the deletion of PoflbC gene substantially reduced cellulase and hemicellulase production. Comparative transcriptome analysis by RNA sequencing revealed a global downregulation of genes encoding cellulases, hemicellulases, and other proteins with functions in lignocellulose degradation. A similar defect was also observed in the OEPoflbC strain, suggesting that the production of cellulolytic enzymes was maintained by native expression of the PoflbC. In this study, an essential regulator for both fungal asexual development and cellulase production was established in P. oxalicum.5. Redesigning the regulatory pathway of cellulase expression to construct cellulase high-producersA systematic strategy was developed 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, higher than those of the wild-type （WT） strain 114-2 both on pure cellulose and complex wheat bran media, respectively. Most strikingly, the cellulolytic ability of RE-10 was higher than that of the industrial P. oxalicum strain JU-A10-T obtained by long-term random mutagenesis and screening. 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, however, the β-glucosidase did not.Two strategies were utilised to over-express P-glucosidase in the strain RE-10. The constitutive promoter of gene PDE₀2864 encoding 40S ribosomal protein S8, was used to over-express three P-glucosidases:BGL1, BGL4, BGL5. It was found that all mutants show significantly enhanced levels of β-glucosidase at transcriptional, protein, and activity levels. Furthermore, the inducible promoter from BGL2 was also used to conditionally over-express the β-glucosidases BGL1 and BGL4. Surprisingly, this induced expression strategy enabled significantly improved expression efficiency. The BGL1 over-expressing mutant I1-13 particularly improved the β-glucosidase activity at a factor of 65 folds, resulting in levels of up to 150 U/ml. This is the highest reported P-glucosidase activity in fungal crude cellulases. All our BGL over-expression mutants displayed significant enhancement of cellulolytic ability on both microcrystalline cellulose and filter paper. In addition, they substantially reduced the enzyme loads in the saccharification of a natural lignocellulose material delignified corncob residue （DCCR） and enhanced the hydrolysis efficiency. The mutant I4-32 with over-expression of BGL4 achieved the highest glucose yield in the saccharification of DCCR to a level of commercial preparation. Rational strain engineering by redesigning the regulatory mechanism avoided a serious of time-consuming process, such as concentration and formulating, which is a promising strategy in cellulosic ethnol commercializaiton.