Regulation Of Cellulase And Xylanase Gene Expression Induced By Different Carbon Sources In Penicillium Oxalicum

Lignocellulose is the most aboundant renewable biomass resource.Enzymatic hydrolysis of cellulose and xylan by cellulase and xylanase into fermentable sugars is one of the important ways to promote value-added utilization of lignocellulose.However,the yields of cellulase and xylanase in filamentous fungi are low,which is the biggest obstacle for large-scale utilization of lignocellulose.Penicillium oxalicum,as an important fungus producing cellulase and xylanase,has attracted extensive attention and research on the mechanism of expressional regulation of cellulase and xylanase genes.Nevertheless,it is still unclear about efficient inducers of cellulase and xylanase gene expression in P.oxalicum and their coexpression mode of cellulase and xylanase genes under differently environmental conditions.In this thesis,we mainly screened for efficient inducers for the expression of cellulase and xylanase genes in P.oxalicum,and analyzed large-scalely transcriptomic data with bioinformatics to reveal the co-expression mode of cellulase and xylanase genes in P.oxalicum.By screening for 23 carbon sources,two new highly effective inducers,methylcellulose and 2-hydroxyethylcellulose,were identified for the synthesis of cellulase and xylanase in P.oxalicum.Enzymatic production tests revealed filter paper(FPase)and xylanase yields of P.oxalicum cultivated on methylcellulose as the sole carbon source showed 60.79-and44.19-fold increase compared with those on wheat bran plus Avicel,when cultivated for four days following a 24 h pre-culture on glucose.Furthermore,combination of methylcellulose with Avicel and/or wheat bran showed a decreased enzyme production compared with the sole methylcellulose.Therefore,the carbon source with the best induction effect is methylcellulose,followed by the combination of methylcellulose and Avicel.Transcriptomes of P.oxalicum △Pox Ku70 under cultivation conditions with five different carbon sources and without carbon source as a control at four time points were comparatively analyzed.It was found that major cellulase and xylanase genes in P.oxalicum,as well as the related genes involved in basic metabolism such as glycolysis pathway and tricarboxylic acid cycle,were specifically carbon source-dependent,and their expression varied on different carbon sources and at different induction time points.Weighted Gene Co-Expression Network Analysis(WGCNA)of 72 transcriptomes on carbon sources at different time points identified 17 coexpression modules.Among them,the MEivory module is most related to the production of FPase,carboxymethylcellulase(CMCase)and xylanase by P.oxalicum.There were 120 genes in MEivory,including major cellulase and xylanase genes,two important regulatory gene Pox Clr B and Pox Xln R,and a cellodextrin transporter-encoding gene POX06051/Cdt C.Further analysis of gene expression kinetics in MEivory showed that the level of gene expression in MEivory was related to the type of carbon sources and the duration of carbon source induction.Global analysis of 276 transcriptomes of P.oxalicum under different cultivation conditions further identified 125 genes related to cellulase synthesis.By comparison with genes in MEivory,59 core genes related to cellulase synthesis in P.oxalicum were identified,defined as cellulase synthesis gene group.Through Multi-level association analysis(Ml A),the transcription factors that regulated cellulase synthesis showed spatial position relationship with cellulase synthesis gene group.To further confirm whether the regulatory genes in MEivory and Ml A2 be involved in the production of cellulase,three candidate genes with unknown function in MEivory and six ones in Ml A2 were attempted to be knocked-out in P.oxalicum △Pox Ku70,respectively.As a result,seven genes were successfully knocked-out.Enzymatic acitivity tests revealed that all the mutants showed significant alterations in the production of cellulase and xylanase.For example,△POX01678 increased cellulase and xylanase production by 10%-30% in comparison with those of the parental strain △Pox Ku70.By contrast,the β-glucosidase production of△POX04567,△POX05623 and △POX08723 decreased by 50%-65% on the fourth day after the transfer,and the production of cellulase and xylanase of other mutants decreased significantly by 10–40%.The high efficient inducers obtained in this work are of great significance for industrial production of cellulase and xylanase,thereby leading to the reduction of costs.Cellulase synthesis gene cluster and coexpression module identified by bioinformatics analysis will help deeply understand the mechanism of fungal cellulase and xylanase synthesis and secretion.Those also provide a theoretical guidance for genetic engineering of P.oxalicum to increase enzyme production.