| Filamentous fungi are generally used for the production of industrial cellulase due to their abilities to secrete complete lignocellulose-degrading enzymes at high levels.The construction of cellulase high-producing strain is an important way to reduce the cost of cellulase production and promote the development of cellulosic ethanol industry.The synthesis of lignocellulose-degrading enzymes by filamentous fungi is mainly regulated by transcriptional factors,which are generally divided into two categories.The first type is transcriptional activators,which mainly include CLR-1,CLR-2/ClrB,Xyrl/XLR-1/XlnR,ACEII,ACEIII,AraR/ARA1,etc.;the second type is transcriptional repressors,mainly including CRE1/CreA,ACEI,BglR,etc.At present,the core transcription factors regulating the expression of lignocellulose-degrading enzymes that have been identified in Penicillium oxalicum are CreA,ClrB,XlnR and AmyR.In which,XlnR is the most important transcriptional activator regulating xylanase gene expression,and is involved in the regulation of parts of cellulase genes.XlnR homologues can be found in most lignocellulose-degrading filamentous fungi.The sequence and function of this protein are relatively conserved,and it plays a crucial role in hemicellulose degradation and xylose metabolism.In this study,we studied the structural domain characteristics of XlnR of P.oxalicum and explored the regulatory properties of heterologous XlnR homologues in P.oxalicum.The study of XlnR and its homologues will help us to understand the function of XlnR homologues among different species,and provide more effective targets for the rational engineering of strains and the construction of high cellulase-producing strains.The main results of the research are as follows:1.Analysis of functional domains of transactivator XlnR of P.oxalicumBy means of the yeast reporter gene system,the activation domain of P.oxalicum of XlnR was identified by constructing fusion proteins,which were composed of various regions of XlnR and yeast transcription factor Gal4 DNA binding domain.Amino acids 351-694 were identified as an activation domain.After deleting a polyglutamine sequence near to N-terminus of XlnR,the regulatory ability of XlnR on cellulase and hemicellulase genes was enhanced,indicating that the glutamine sequence inhibited the activity of XlnR at a certain degree.It was found that the sequence at the C-terminus of XlnR is critical for the activity of P.oxalicum XlnR,and its deletion led XlnR to lose its regulatory activity,which is different from the result of the deletion of C-terminus of XlnR in Aspergillus niger.By studying the conserved amino acids in XlnR that may be involved in the release of glucose inhibition(amino acids 868-871),we found that when the amino acids of the conserved site were deleted,the activity of XlnR was lost,which proved that the presence of these sites is critical to the activity of XlnR.When the alanine at position 871 was mutated to more hydrophobic amino acids,the activating activity of XlnR was enhanced significantly;when the site was mutated to hydrophilic amino acids,the activity of XlnR was reduced or lost.By means of yeast two-hybrid assay,we determined that there was an interaction between the activation domain and the amino acid sequence at the C-terminus of XlnR,indicating that XlnR activity may be regulated by changing the interaction between them.2.Investigation of the biological function of heterologous XlnR homologs in P.oxaalicumHeterologous expression of A.niger AXlnR,Trichoderma reesei Xyr1 and Neurospora crassa XLR-1 were performed in the PxlnR deletion mutant of P.oxalicum,respectively.These three heterologous XlnR homologues were found to activate the expression of the lignocellulose degrading enzyme genes in P.oxalicum Among the four XlnR homologues including P.oxalicum XlnR,T.reesei Xyrl had the strongest ability to activate the cellulase genes of P.oxalicum,which may be related to that Xyrl possesses a strong cellulase gene regulation ability in the native host T.reesei.XLR-1 of N.crassa showed the weakest ability to activate the cellulase genes of P.oxalicum.Previous studies have shown that XLR-1 is not involved in the regulation of cellulase genes in N.crassa,but in P.oxalicum,XLR-1 can participate in cellulase gene regulation and increase the cellulase activity of PxlnR deletion strain.In summary,we demonstrated that XlnR homologues can participate in the regulation of lignocellulose-degrading enzyme genes in a heterologous host.Point mutants Xyr1A824v,XLR-1A828V and AXlnRA805V showed stronger abilities to activate the lignocellulose degrading enzyme genes of P.oxalicum than Xyrl,XLR-1 and AXlnR,respectively.Xyr1A824V had the strongest activation ability in P.oxalicum among the four kinds of XlnR homologue mutants.In addition,it was for the first time found that the A805V mutation of AXlnR could increase its activation effect on the expression of lignocellulose degrading enzyme genes of P.oxalicum,and the activation effect of this mutant was similar to that of P.oxalicum XlnRA871V.3.Functional study of heterologous XlnR regulatory modules in P.oxalicumP.oxalicum M12 was selected as a parent strain to obtain strain DB2,which established the Rec/six screening marker recycling system successfully.It was found that the system had no effect on the cellulase production of P.oxalicum.The heterologous regulatory module which contained Txyr1A824V and its cellulase target genes Tcbhl-Teg1 was introduced to P.oxalicum.We found that cellulase genes Tcbhl and Tegl showed low levels of expression,and the Xyr1A824V regulatory module showed a better effect on cellulase production than the expression of Xyr1A824V alone.In order to increase the transcription level of the heterologous cellulase genes in the regulatory module,we replaced the promoter of the T.reesei cellulase genes by the promoters of the P.oxalicum cellulase genes.The transcription level of heterologous cellulase genes was significantly improved,and the cellulase activity of the strain was also significantly enhanced.In addition,we increased the production of cellulase and hemicellulase significantly by co-expressing Nxlr-1A828V derived from N.crassa and the cellulase genes Ncbh1 and Neg1.It was shown that co-expression of transcription factors and their target genes is feasible in genetic engineering of strains.4.Construction of cellulolytic enzyme high-producing strainsBy means of Rec/six self-excising marker recycling system,the XlnR regulatory modules derived from P.oxalicum,T.reesei and N.crassa were accumulatively overexpressed on the basis of strain DB2,and the production of lignocellulose-degrading enzymes of strain were enhanced significantly.Compared with the original strain M12,the FPase activity and the xylanase activity of high-yield strain RE-4-2 increased 5.1 times and 28 times,respectively.Crude enzyme produced by RE-4-2 also showed 93%higher saccharification efficiency on pretreated corn stover compared with that of the original strain M12.In order to further improve the ability of the strain to degrade hemicellulose,we overexpressed the point mutated transcription activator AraRA731V on the basis of the high-yield strain RE-4-2,and obtained the strain RE-4-2-AraRA731V.Compared with RE-4-2,the arabinofuranosidase and xylosidase activities increased by 7.2 times and 1.2 times in RE-4-2-AraRA731V,respectively.The saccharification efficiency of the enzymes produced by strain RE-4-2-AraRA731V on corn stover was increased by 13%when compared with RE-4-2. |
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