Studies On The Mechanisms For The Transcriptional Activation Of Lignocellulolytic Enzyme Genes In Penicillium Oxalicum And The Construction Of Enzyme High-producing Strains

Bioconversion of lignocellulosic materials to fuels and chemicals is an important solution to the resource,energy and environmental problems of human society.The high cost of cellulase for the hydrolysis of cellulose to fermentable sugars is one of the most significant difficulties in this route.Lignocellulose has complex composition and structure,and thus its degradation needs the synergistic action of dozens of enzymes.Trichoderma,Penicillium and other filamentous fungi are usually used for the production of such complex enzyme mixtures,due to their natural abilities to secret cellulases at high levels.The construction of cellulase high-producing strains can reduce the cost of cellulase production,and is beneficial for the development of large-scale industry for the bioconversion of lignocellulosic materials.The production of lignocellulolytic enzymes by filamentous fungi is mainly regulated at the transcription level of enzyme-encoding genes.Specifically,the transcription of many cellulase and hemicellulase genes are repressed by easy-to-use carbon sources?e.g.glucose?,and induced by cellulosic molecules as well as their analogs.Several transcription factors have been reported to be involved in this process in a combinatorial manner,including transcription activators such as ClrB and XlnR,and repressors such as CreA and ACEI.The investigation of the mechanisms for the transcriptional regulation of fungal cellulases could deepenour understanding about the natural carbon cycle mediated by microbes,and provide efficient targets for the genetic engineering of cellulase-producing strains.The main results of the research are as follows:1.Transcriptional activation mechanisms of lignocellulolytic enzymes genes by ClrBThe transcriptional regulating function of ClrB in the expression of lignocellulolytic enzymes in Penicillium oxalicumwas investigated.Deletion of clrB resulted in the dramatic decrease of cellulase and hemicellulase production under cellulose-inducing condition,and enzyme production was restored in the clrB complementation strain.The regulation of cellulase expression by ClrB showed a dose effect.The production level of cellulase significantly increasedthrough constitutive expression of clrBunder inducing condition,which was further enhanced through the overexpression of clrB by the promoter of gene encoding ribosomal protein S8e.RE-27 strain was constructed by increasing the expression of clrB in a previously constructed cellulase high-producing mutant,and showed a further increase of cellulase production.Additionally,the functional domains in ClrBwere identified.Constitutive expression of the truncation mutants of ClrB lacking the 146-173 or 174-201 amino acids led to cellulase production under carbon source-free condition,suggesting the involvement of this region in the regulation of ClrB.The 685-780 amino acids of ClrB were able to activate the expression of reporter genes when fused with the DNA-binding domain of transcriptional activator Gal4 in yeast,whichsuggested this regionbeas a transcriptional activation domain.The genetic interaction between clrB and intracellular ?-glucosidase gene bgl2was also investigated.ClrB regulated the expression of bgl2,while the deletion of bgl2 did not affect the expression of clrB.However,the increase in cellulase production caused by bgl2 deletion was dependent on the existence of ClrB.Combination of bgl2 deletion and clrB overexpression had a cumulative effect on the increase of cellulase production.Interestingly,the mutant showed the expression of cellulase in the absence of cellulose,which was not observed in bgl2 or clrB single-manipulating strains.2.The effect of XlnR engineering on the expression of lignocellulolytic enzymesThe transcriptional activating function of XlnR in the expression of hemicellulases in P.oxalicumwas investigated.Hemicellulase productiondramatically decreased in xlnRdeletion mutant,and increased after theconstitutive expression of xlnR.The mutation of alanine at 871 site in XlnR to valine significantly increased the production of lignocellulolytic enzymes,especially hemicellulases,in the presence of cellulose or xylan.In addition,the mutant increased the basal expression of hemicellulases under non-inducing condition.A lignocellulolytic enzyme high-producing strain RE-8 was constructed from strain M12 through two steps of genetic manipulation.First,simultaneous deletion of creA and overexpression of clrB in strain RE-6 resulted insignificantly increased production of lignocellulolytic enzymes.Second,the expression of xlnRA871V and cellulase genes cbhl and egl in RE-6 to obtain strain RE-8 with further increased enzyme production.RE-8 showed a similar level of extracellular filter paper enzyme activity,and higher activities of cellobiohydrolase,endoglucanase and hemicellulases in comparison with a previously constructed enzyme high-producing mutant RE-10.3.Regulation of the expression of lignocellulolytic enzyme genes by chimeric transcription factors CXCThe possibility of celluloseproduction under non-inducing conditions is explored by overexpressing two chimeric transcription factors.The chimeric transcriptionfactors contain the DNA binding domain of ClrB linked to the C-terminal sequences of XlnR^A871V.The obtainedrecombinant mutants exhibited dramatically improved basal production ofcellulase,which was not observed with the overexpression of intact ClrB.The expression of cellulase was still repressed by glucose in the mutants.When cultivated in a complex cellulosic medium containing wheat bran and cellulose,one of these mutantsdisplayed a 7.3-fold increase in cellulase production?2.8U/mL?relative to the parent strain.The results demonstrate that the dependence ofcellulase synthesis on cellulose could be reduced by the overexpression ofartificially designed chimeric transcription factors,and offers a potentialstrategy to engineer fungal strains for improving cellulase production.4.Investigation of the biological function of AraR and PDE₀7172 as homologs of XlnRThe biological function of AraR.and PDE₀7172,two homologs of XlnR,was studied in P.oxalicum.AraR regulates the catabolism of L-arabinose and D-xylose,as well as the expression of arabinofuranosidase involved in the degradation of hemicellulose.In addition,AraR can compensate the regulatory function of XlnR.The expression level of AraR was upregulated in xlnR deletion mutant,which maintained the expression of xylose-catabolic enzymes.Analysis of the utilization of multiple carbon sources by PDE₀7172 deletion mutant did not reveal the target of this potential regulator.