Mechanism Underlying Cellulases Synthesis Induced By Soluble Inducers In Trichoderma Reesei And Improvement Of Its Cellulolytic Enzyme Production

The gradual depletion of fossil fuels and concern on their environmental influence have generated great interest in exploring renewable and environmentally friendly fuels.Biofuels produced from lignocellulosic feedstocks such as corn stover through biorefinery are alternative solution for addressing these challenges.A general scheme for the biorefinery includes biomass pretreatment,enzymatic hydrolysis of the cellulose component and microbial fermentation to prodece targeted products.However,the high cost of cellulases production is still one of the bottlenecks restricting their industrial applications,making the development of robust cellulases—producing strains a necessity.Strains for cellulases production are predominantly derived Trichoderma reesei.Cellulases produced by the fungal species mainly consist of endoglucanase,cellobiohydrolase and(3-glucosidase.and cellulose degradation is a synergy of these enzyme components.The expression of genes encoding cellulases and xylanases in T.reesei is coordinately regulated by various transcription factors,among which Xyrl is the key transcription activator and Crel is the major negative regulator mediating carbon catabolite repression?CCR?.Abundant transcriptomic and proteomic data have enriched understanding of the regulation of cellulases synthesis,and led to discovery of novel genes and transcription factors.On the other hand,cellulases production by T.reesei requires induction,and sophorose has been acknowledged as the most efficient inducer,however,its high price hinders industrial use.In our previous study.the mixture of glucose-disaccharides?MGD?synthesized from glucose through the transglycosylation reaction catalyzed by(?—glucosidase was validated as efficient and low-cost inducer,but it is still not clear on its inducing mechanism,particularly compared to that observed in the commonly used soluable inducer lactose.In this study,the transcriptome of T.reesei Rut C30 induced by MGD was analysed using lactose induction as the control.The results showed that the transcription of genes encoding glycoside hydrolases?GHs?was different.While the total amount of transcription of GH genes induced by MGD was higher than that induced by lactose.the transcription of GH5,GH6 and GH7 encoding major cellulases was 1.4-fold higher than that induced by lactose.but the expression of GH11 encoding a major xylanase was downregulated.The expression of cellulolytic and xylanolytic genes in T.reesei was coordinately regulated by transcription factors?TFs?and transporters responsible for carbon signal transduction.It was found that MGD induction affected the transcription of genes encoding TFs and transporters,and the major transcription activator Xyrl was significantly upreglulated by 1.6 times compared to that observed in lactose induction.Meanwhile.MGD induction upregulated the mitogen-activated protein kinase?MAPK?pathway for signal transduction,which might indirectly affect the expression of cellulase genes.In addition,the upregulation of cellulase genes activated mRNA regulatory pathway and accelerated RNA transport process and endoplasmic reticulum protein processing pathway,which might lead to unfolded protein response?UPR?.The transcription of irel encoding transmembrane kinase that senses unfolded proteins in the cell and hacl encoding UPR—specific transcription factors were significantly upregulated by MGD,which might relieve UPR.The protein secretion pathway plays a crucial role in the production of extracellular cellulases by T.reesei.Therefore,three genes including shsp?TrireC30₁00314?encoding small molecular weight heat shock protein family,nef?TrireC30₁24246?encoding nucleotide exchange factor and gst?TrireC30₁0865?encoding glutathione transferase were selected and overexpressed in T.reesei Rut C30,respectively.The selected transformants sHSP,NEF and GST didn't show significant increase in the production of extracellular cellulases and proteins,but the overexpression of these genes conferred thermotolerance to T.reesei,since the growth of the transformants was almost not affected at the high temperature of 40?,but the growth of the wild type strain was compromised by approximately 50%.Moreover,the expression of irel and hacl in the transformants was significantly downregulated at the early stage?24 h?of fermentation compared with T.reesei Rut C30.Then,the transcription of UPR-related genes was upregulated due to increased cellulases production.The strains were treated with dithiothreitol?DTT?to prevent disulfide bond formation for ER stress,and it was observed that colonies of the transformants sHSP and NEF were formed at 84 h,while the transformant GST and the wild type strain formed colonies at 120 h when 10 mM DTT was added.The above results indicated that the overexpression of shsp and nef might partially relieve T.reesei from endoplasmic reticulum stress during cellulases producton.Many sites for Xyrl and Crel binding exist in the promoters of major cellulases and xylanases genes.In T,reesei Rut C30,crel was mutated from binding onto the sites,which eliminated the carbon catabolite repression.However,cellulases production was detected in extremely low level in the culture with glucose.Therefore,a chimeric transcription activator containing Crel binding domain and Xyrl effector and binding domain was constructed,which could bind onto both Xyrl and Crel binding sites to activate gene expression through Xyrl effector domain.The Xyrl-Crelb was overexpressed in T.reesei Rut C30 directed by the strong and constitutive pdcl promoter.As a result,genes enconding major cellulases,xylanses and auxiliary activity proteins were activated under non-induction conditions,since a recombinant strain T.reesei zxy-2 displayed a constitutive cellulases and xylanases secretion in the glucose-based culture,producing 12.88 and 61.89 folds of cellulases and xylanases in flask culture,compared to T.reesei Rut C30.Furthermore,the titers of cellulases and xylanases 2.63 and 108.72 IU/mL were achieved at 48 h when cultured in 7-L fermenter for batch fermentation.However,the cellualse activity of the transformants was not enhanced by the induction of MGD,lactose and cellulose.Additionally,the crude enzyme was evaluated by hydrolyzing pretreated corn stover and Scenedesmus obliquus,and high glucose yield of 99.2%and 65.9%was obtained,respectively,when supplemented with ?-glucosidase properly.The results will provide basis for further exploring mechanism of cellulases biosynthesis,as well as development of novel strategies for metabolic engineering of T.reesei to efficiently produce cellulases for the biorefinery of cellulosic biomass.