| Depleting reserve of crude oil and environmental pollution and climate changes associated with the over-consumption of petroleum-based foels and chemicals present challenges for sustainable socio-economic development.Lignocellulosic biomass such as residues from agriculture and forestry is renewable and abundantly available,with cellulose and hemicelluloses as major components,which can be hydrolyzed into sugars for microbial fermentation to produce biofuels and bio-based chemicals.Such a biorefinery is an alternative to petrolum-dependant economy,and attracts worldwide attention.While hemicelluloses are easily hydrolyzed,cellulose as the major protect component of plant cell walls has been evolved with recalcitrance to degradation,particularly to enzyme attack.Therefore,low cost and high efficient cellulase has become one of the bottlenecks for the sugar platform based on cellulose hydrolysis and the biorefinery as well.Cellulase composes mainly of endoglucanase,cellobiohydrolase and β-glucosidase,and its degradation needs a synergy of these enzyme components.Trichoderma reesei Rut-C30 is one of the best cellulase-producer,which has been applied for commercial cellulase production.However,low cellulase titer in the culture of the fungal strain makes cellulase too costly for biorefinery.Studies on mechanism for cellulase biosynthesis showed that cellulase production in T.reesei is mainly regulated by several innate transcription factors,yet other unknown factors affecting cellulase biosynthesis remain unexplored.Zinc finger proteins(ZFPs)with zinc finger binding domains are regulatory factors in different living organisms,and artificial zinc finger protein(AZFP)library has been designed for engineering microbial strains such as Escherichia coli and yeast.However,no report on their applications for engineering filamentous fungi is available.In this work,improvement of cellulase production by AZFP overexpression was explored.The AZFP library was constructed and transformed into T.reesei Rut-C30.and mutants with improved cellulase production and protein secretion were selected and studied.Plasmids for engineering filamentous fungi were constructed,and protoplast transformation and Agrobacterium tumefaciens mediated transformation were explored.A.tumefaciens mediated transformation was validated to be more efficient,and transformation parameters were further optimized:growing A.tumefaciens(AGL-1)to an OD660 of 0.8,and co-incubating with T.reesei Rut-C30 at pH 5.3 and 24℃ for 48 h with 200 μM acetosyringone supplemented.The transformation efficiency was roughly 200 transformants per 106 conidia under these conditions.The AZFP library was further constructed to transform T.reesei Rut-C30,and about 600 transformants were obtained,in which a mutant U3 with improved cellulase production was selected through cellulosic plate and flask cultures.Cellulase production by U3 was further studied.Compared to the host strain,an improvement of 55%in cellulase activity(FPase)was observed.Real-time quantitative RNA analysis of genes and measurement of activities for cellulase components were performed,and the results indicated that p-glucosidase activity in the crude enzyme produced by U3 was 8 times higher than that produced by the host strain,and in the meantime endoglucanase activity was increased by 28%.When alkali pretreated corn stover was hydrolyzed,115%increase in glucose yield was observed in the hydrolysis catalyzed by crude enzyme produced by U3.These results validated that the AZFP transformation in U3(AZFP-U3)altered cellulase composition,and consequently improved the biomass hydrolysis efficiency.Analysis of the putative target genes of AZFP-U3 was performed,and four genes 7183,46633,88814 and 125610 with putative transcription factors,mitochondrial inner membrane transporter and glycoside hydrolase were selected for further study.Overexpression plasmids carrying these genes were constructed,and transformed into T.reesei Rut-C30.Compared to the host strain,extracellular cellulase produced by the mutant overexpressing gene 125610 was increased by 200%,and extracellular protein secretion increased by 219%.Another transformant U5 produced even more cellulase than U3,and the PFase activity of its cellulase was 112%higher than that produced by the host strain,and the secretion of extracellular proteins was increased by 86%.However,degradation of alkali pretreated com stover with crude enzyme produced by U5 showed an increase of only 33.9%in glucose yield compared to that achieved with crude enzyme produced by the host strain,lower than that achieved with crude enzyme produced by U3.Comparative transcriptomic analysis was performed for cellulase produced by U5 and the host strain to reveal the underlying mechanisms of improved cellulase production.The transcription of genes encoding glycoside hydrolases,transporters,transcription factors and eukaryotic basic transcription factor for RNA polymerase Ⅱ was significantly upregulated at 24 h within U5,and genes related to key enzymes in mRNA surveillance pathway,endoplasmic reticulum(ER)protein processing and protein degradation were also upregulated,indicating that extracellular protein secretion was enhanced at gene transcription,protein processing and secretion for cellulase.In addition,the transcription of genes encoding peroxisomes and key enzymes in the pathways of CoA synthesis,N-glycoside synthesis,aminoacyl-tRNA synthesis,nicotinamide metabolic and fatty acid metabolism was also upregulated to provide more amino acid precursors,coenzymes and energy for cellulase transcription and protein processing.Meanwhile,repression under secretion stress specific in filamentous fungi was activated obviously in U5,especially at 48 h,resulting in the degradation of mRNA encoding for extracellular secretion proteins,but the correlation between RESS and cellulase production remained unclear.Increased transcription of major glycoside hydrolases and ER proteins related to protein processing was significant at 48 h in U5,indicating an enhanced effect of AZFP-U5 in glycoside hydrolases expression and post-translational modification.Changes in the putative target genes of AZFP-U5 were also observed,which might involve in the post-translational modication,signal transduction,and amino acid metabolism.The transcriptomic analysis presented here provides a basis for further study of the regulatory mechanism of ZFPs in T.reesei,the key metabolic pathways and regulatory genes related to cellulase production.The progress of this work showed that cellulase production and protein secretion can be improved by metabolic engineering of T.reesei using the AZFP strategy.The results obtained in this study provide basis for further breeding high cellulase-producing strains from T.reesei,and also applications of AZFP in engineering other filamentous fungi. |
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