Functional Analysis Of The Major (Hemi) Cellulase Transcription Factors In Penicillium Oxalicum And Strain Engineering

Filamentous fungi are the most efficient degraders of plant biomass and contribute significantly to the global carbon cycle.Degradation of lignocellulosic materials by filamentous fungi mainly depends on a diverse set of extracellular lignocellulolytic enzymes.The conversion of insoluble polysaccharides into fermentable sugars by cellulase and hemicellulase secreted by filamentous fungi is the key step in the production of biofuels and other bio-chemicals from lignocellulosic biomass.However the main obstacle in this routine is the low enzymatic production and efficiency.Despite considerable progress in improving the production level and performance of lignocellulolytic enzymes has been made through traditional mutagenesis techniques,the further improvement of mutants was limited by classical mutagenesis.Investigations of the synthesis and regulation of lignocellulolytic enzymes would provide theoretical support for strain rational engineering in cost-competitive biofuels production.The lignocellulolytic fungus Penicillium oxalicum was isolated from decayed straw-covered soil in 1979.P.oxalicum produces a more balanced lignocellulolytic enzyme system with more ?-glucosidase and hemicellulase composition compared with the wildly used cellulase producer Trichoderma reesei.The hyper-mutant after several rounds of mutagenesis has been used for industrial-scale cellulase production in China since 1996,and its enzymatic productivities are comparable with T.reesei industrial strains.Numerous genetic variations were found between the wild-type strain 114-2 and the hyper mutant JU-A10-T through comparative genomic analysis,while proteins with sequence mutations were significantly enriched for transcriptional regulators.Comparative transcriptomic analysis between strains 114-2 and JU-A10-T revealed that differential expression of several intracellular biological processes.However,the knowledge on the lignocellulolytic enzyme expression characteristics and the regulation of their production is still poor compared with Aspergilli and T.reesei,which obviously limits further strain improvements for cellulase hyper-production.In this study,we have studied the functions and regulatory mechanisms of conserved cellulase transcriptional factors in P.oxalicum.Comparative genomic and transcriptomic studies of different cellulolytic fungi have provided us with large lists of genes whose implications on the production of cellulases deserve further attention.Although a number of them lack any significant similarity to the well-characterized orthologs from public databases and their functions in cellulose degradation remain unclear,they could be applied in gene targeting for improved cellulase production through genetic engineering.1.Characterization and expression of?hemi?cellulase transcriptional factors CreA,X1nR and AceA in P.oxalicumThe compositions of extracellular enzyme system altered significantly in the cellulose hyper-producing mutants JU-A10 and JU-A10-T compared with the wild-type 114-2,with increasing proposition of lignocellulolytic enzymes and decreasing amylase production.This difference was apparently reflected in the transcriptional level of the major lignocellulase and amylase genes,which indicated that the transcriptional regulation system in hyper-mutants might be more concentrated on expression of cellulase and hemicellulase.Using the amino acid sequence of T.reesei CREI,ACE lor A.niger XlnR as the query,the respective ortholog CreA,AceA or XlnR was identified in the genome database of P.oxalicum 114-2 by BLASTp search.Alignment of the amino acid sequence of CreA,AceA or XlnR along with that of its relative othologs revealed that they contained the conserved Cys2His2 or Zn2Cys6 type zinc finger DNA binding domain.The expression of the major lignocellulolytic genes cel7A-2 and xyn10A in P.oxalicum was dedicatedly repressed or induced by the available carbon source.Similarly,the transcripts abundance of these three conserved transcriptional factors CreA,AceA or XlnR was regulated depending on the carbon source.While the expression of creA and xlnR was repressed by glucose but not induced by cellulose,the expression level of aceA was carbon source independent.Unexpectedly,the transcripts of these three transcriptional factors were highest under carbon starvation condition,implying their important roles in basal metabolism and carbon utilization in P.oxalicum.Moreover,the expression of xlnR,creA and aceA in hyper-mutants JU-A10 and JU-A10-T increased at variable extent compared with the wild-type and maintained high levels irrespective of carbon source available,indicating that they might paly crucial roles in the improved lignocellulase production of the mutants.2.The glucose repressor CreA modulates lignocellulolytic enzyme synthesis and fugal development in P.oxacliumUsing gene targeting manipulation,we have found that CreA mediated carbon catabolite repression of cellulase and hemicellulase.Deletion of creA in wild strain 114-2 caused derepression of lignocellulolytic enzyme synthesis in the presence of glucose.The ?creA mutant also showed elevated lignocellulolytic enzyme production under inducing condition.In accordance with this,the expression of the major cellulolytic and xylanolytic genes cel7A-2,cel7B and xyn10A exhibited 12.5-,3.5-and 1.2-fold increase respectively than the parental strain.Moreover,transcriptomic analysis revealed that a set of cellulase,hemicellulase and sugat transpoters was regulated by CreA mediated glucose repression and CreA negatively influences the transcription of most of the lignocellulose degrading enzymes in cellulose.The repressor function of CreA under inducing condition might be correlated with its higher expression on cellulose than glucose.While promoter regions of genes regulated by CreA contained several adjacent 5'-SYGGRG-3'motifs,the ability of CreA to bind to the cel7A-2 promoter was demonstrated in vitro,subsequently indicating CreA could directly repress transcription of lignocellulolytic enzymes by binding to their promoter.In addition,the absence of CreA resulted in 5-and 2-fold increase in xlnR transcripts on glucose and cellulose respectively,which suggested that CreA might indirectly repress lignocellulase synthesis through inhibition of xlnR expression.As XlnR was shown to activate the transcription of cellulase and hemicellulase in the following study,such regulatory mechanism of CreA might be important for its repressor role in carbon catabolite repression.Furthermore,our data suggested that the frameshift mutation in CreA?CreA-1?led to partial carbon catabolite derepression of JU-A10.Deletion of creA-1 from JU-A10 further improved cellulase production in the presence of glucose,whereas retransformation of the wild-type creA gene restored glucose repression in JU-A10.Partial loss of repressor function in CreA-1 was also observed under inducing condition,in which the ?creA-1 mutant exhibited further increase in lignocellulolytic enzyme secretion while the wild-type creA retransformation strain showed decreased enzyme production compared with the parental strain JU-A10.Meanwhlie,we found that CreA was also involved in hyphal morphogenesis in P.oxalicum.Deletion of creA from the wild strain 114-2 resulted in enhanced hyphal branches and slower polarized apical growth.Accordingly AcreA exhibited reduced colonies and delayed sporulation.In addition,the genome-wide transcriptional analysis showed that CreA influences the basal biological process including DNA recombination and repairation,amino acid/protein synthesis and energy metabolism,subsequently modulates mycelium development of P.oxaclium.,3.XlnR positively regulates hemicellulase and cellulase expression in P.oxalicumP.oxalicum XlnR was found to be involved in the regulation of xylan metabolism and utilization through gene targeting and overexpression-based manipulations.In contrast to the wild-type,the ?xlnR mutant showed strongly reduced growth with fewer aerial hyphae on xylan,but it grew as well as the parental strain 114-2 on xylose and glucose.The reduced ability of AxlnR to utilize xylan mainly resulted from decreased secretion of ?-xylosidase and endoxylanase,whereas xlnR overexpression led to increased xylanolytic enzyme production.Similar to other cellulolytic fungi,XlnR positively regulates the expression oflignocellulose degrading enzymes in P.oxalicum.However,the extent and stringency of regulation was somewhat different from the characterized othologs.XlnR in P.oxalicum apparently regulates hemicellulase expression while moderately regulates cellulase expression.Specifically,in comparison with the wild strain 114-2,the transcript level of the major lignocellulolytic genes cel7A-2,cel5B and xyn10A decreased by 79%,62%and 93%respectively in the xlnR deletion mutant upon growth on cellulose,while overexpression of xlnR significantly increased these lignocellulase gene expression.In vitro binding and gel mobility shift assays revealed that XlnR was able to bind to the promoter region of cel7A-2.Furthermore,the expression of creA and aceA was not influenced by XlnR.These results together indicated that XlnR might act as an activator through directly binding to the promoter sequence of target genes.Phylogenetic analysis showed that X1nR-like proteins were more inclined to regulate hemicellulase or xylose metabolism during the evolutionary process.As the xlnR expression was under the control of CreA mediated glucose repression,the up-regulation of xlnR in JU-A10 and JU-A10-T might result from partial loss of repressor function of CreA-1,which then might contribute to the hyper-producing of cellulase and hemicellulase in mutants.4.AceA modulates fungal development,carbon utilization and extracellular polymer degrading enzyme expression in P.oxalicumSpecially,we found that AceA was involved in hyphal morphogenesis and asexual development in P.oxalicum.In details,deletion of aceA resulted in delayed spore germination,reduced polarized apical growth and more hyphal braches.Thus the ?aceA mutant formed irregular colonies with strikingly reduced aerial hyphae and impaired sporulation.The regulatory roles in hyphal and colonial morphology were carbon source independent,correlating well with the constitutive expression of aceA.Despite the AaceA mutant showed limited growth on cellulose,clear cellulolytic zone was still existed around its colonies.Further investigation gave evidence that AceA modulates expression of extracellular polysaccharide degrading enzymes.The growth of AaceA was improved in liquid cellulose cultivation with altered extracellular enzymatic composition.Specifically,the proportion of cellulase and hemicellulase increased,while that of amylase decreased significantly in AaceA compared with the wild strain 114-2.Consistently,the expression level of the major lignocellulolytic genes cel7A-2,cel7B and xyn10A in the AaceA mutant increased at different extent in comparison with 114-2,whereas the major amylase gene amy15A decreased stringkly by 85-98%.In addition,we found that AceA might influence amylase expression through the regulation of transcript abundance of amylase regulator amyR.The T.reesei ace1 gene was introduced into the P.oxalicum ?aceA mutant to determine whether ACEI can functionally complement the aceA null mutation.The abnormal hyphal and colonial morphology as well as the growth defect on certain carbon source of AaceA could be rescued by Tracel complementation with exception of growth on glycerol.However,ACE1 could not restore the altered expression of extracellular polymer degrading enzymes caused by aceA deletion.These above results indicated the overlapping as well as distinct regulatory roles between P.oxalicum AceA and T.reesei ACEI.5.Improved cellulase production via disruption of PDE₀1641 in P.oxalicumPDE₀1641 was identified as a single orthologous gene in the genome sequence of P.oxalicum 114-2 using the amino acid sequence of N.crassa NCU05137 as the query.Comparison of the deduced amino acid sequence of PDE 01641 with available database from NCBI revealed that it was highly conserved in a number of filamentous ascomycete fungi.Although the expression of PDE 01641 was decreased in JU-A10-T,no mutation was found in this protein compared with that of 114-2.Deletion of PDE₀1641 from the wild type 114-2 resulted in improved cellulase production.The transcription levels of cellobiohydrolase gene cel7A-2,xylanase gene xyn10A and ?-glucosidase gene bgll in PDE₀1641 deletion mutant increased more than 1.01-,0.48-and 1.86-fold respectively as compared to that of the parental strain 114-2,which suggested that we supposed that PDE₀1641 might act in the process that eventually influences expression of cellulolytic and xylanolytic enzyme system.With respect to the influence of the PDE01641 deletion on cellulase production in 114-2,we asked whether enzyme production could be further improved by deleting the PDE₀1641 gene in the industrial strain JU-A10-T.The cellulase and xylanase activity of the PDE₀1641 deletion transformant were 36%and 80%higher than those of the parental strain JU-A10-T,respectively.Our work indicated that PDE₀1641 could be exploited as a valid target for industrial strain improvement through genetic engineering.