The Study On Carbon Source Sensing In Celluase Induction Of Trichoderma Reesei

Ligocellulose is the most aboundant renewable resources in the world, hence, its utilization would relieve the increasing energy crisis to a great extent. It is very difficult to decompose cellulose into fermentable saccharides such as glucose which has become the bottleneck in ligocellulose utilization. Fortunately, there are some microbes processing the ability to metabolize cellulose naturally which provides us the opportunity of cellulose converse. Since its initial isolation as a decomposer of cellulosic materials, Trichoderma reesei has been developed into one of the most prolific cellulase producer in industry. When cellulose exsits, T. reesei can be induced to secret large amounts of cellulases rapidly.In the past decades, plenty of researches have been done on the physicochemical property, crystal structure and enzymolysis mechanism, but it is still remained unknown about how the microbes start cellulase secretion. Actually, many questions raised in the initial stage are still not answered such as what strategy microbes use to sense the insoluble cellulose and how the cellulose signal is transduced.Celloboise and cellodextrin are the main intermediates produced in the process of cellulose degradation and furthermore both of them are cellulase inducing carbon sources. Maybe it is cellobiose or cellodextrin that acts as the true inducer of cellulase secretion. In thesis researches have been done in the aspect of cellobiose transporter and cellobiose transceptor with the key point of carbon sources sensing. The main results obtained are as follows:1. Cellobise transporter screening system has been constructed and with which, the first cellobiose transporter of T. reesei was indentified.The wild type Saccharomyces cerevisiaeW303 was not able to metabolize cellobiose, but if both of β-glucosidase and cellobise transporter are expressed in W303 then it can grow with cellobiose as the sole carbon source. Based on this, we constructed the cellobiose transporter screening system. And with bioinformatics analysis, the first cellobiose transporter in T. reesei was found. Since the transporter indentified also possesses glucose and xylose transporting activity, we named it Sugar Transporter 1 with Stp1 for short.2. Deletion of stpl in T. reesei enhances cellulase secretion on Avicel but abolishes cellulase genes transcription on cellobiose.To analyse the function of Stp1 in cellulase induction, the gene stpl was knocked out in T. reesei with verification of anchor PCR and Southern blot. Agree with preconceive, lost of Stp1 decreased the growth on cellobise not only in growth rate but also in biomass accumulation. And on the other hand, deletion of stpl raises the cellulase activity when induced by Avicel but on the contrary, cellulase expression is abolished when grows on cellobiose.3. The increased extracelluar β-glucosidase activity is responsible for the noresponse to cellobiose but not the reason of high cellulase activity in ∧stpl grows on Avicel. Additionally, although Stpl is the homolog of glucose sensor Rgt2/Snf3, it does not participate in glucose catabolite repression in the process of cellulase secretion induced by Avicel.The noresponse to cellobiose of Astpl is caused by glucose catabolite repression resulting from cellobiose fast hydrolysis which is triggered off by rising extracellular P-glucosidase because deletion of bgll in Astpl restores cellulase expression on cellobiose. But differently, Bgll does not influence the efficient cellulase secretion of △stp1 on Avicel that although deletion of bgl1 in wild type strain impairs cellulase gene transcription, △stp1△bgl1 still keeps higher cellulase activity than wild type. What is more, lost of Stpl has no effect on glucose catabolite repression because not only the mix of Avicel and glucose but also addition of glucose after cellulase genes transcription started on Avicel impact cellulase induction of both wild type strain and △stp1.4. To further analyse the reason of raised activity of cellulose decomposing in △stp1, gene expressing profile of wild type strain and Astpl on Avicel was detected.The stp1 deletion strain showed a expression profile somewhat different from that ofto WT on Avicel. A total of 671 genes were differentially expressed （fold change≥2） as compared to WT on Avicel, including 139 up-regulated genes and 532 down-regulated.39 genes in the Stp1-downregulated group including 5 cellulase genes, 2 hemicellulase genes,2 genes encoding transporters,16 genes encoding hypothetical proteins, and 15 genes encoding other proteins In addition, a total of 1099 genes showed a moderate difference in the expression levels （1< fold change<2） including 166 moderately up-regulated genes and 933 moderately down-regulated genes in the absence of Stp1 expression levels than in WT.Of specific interest, the transporter （Tr₃405） upregulated in the absence of Stpl was annotated as MFS sugar transporters and the upregulation of Tr₃405 within the WT Avicel regulon was confirmed by Northern blot and quantitative RT-PCR.5. Over expression of Tr₃405 enhances the transcription of cellulase genes on Avicel and deletion of Tr₃405 destroies cellulase secretion almost on all the cellulase inducing carbon sources. In another word, gene Tr₃405 is fatal to cellulase expression in T.reesei, so it is named Cellulase Regulating Transporter 1 with Crtl for short.To know if it is Tr₃405 up-regulating that brings elevated cellulase activity in △stp, we overexpressed Tr₃405 in wild type strain driven by gpd promoter. Transcription of cellulase genes induced by Avicel was detected by QPCR and the data showed that overexpression of Tr₃405 can stimulate cellulase genes transcription to some degree. Futhermore, deletion of Tr₃405 in wild type or Astpl reveals coincident results with overexpression that no cellulase genes transcription was detected, no matter on Avicel, cellobise or sophorose by northern blot and QPCR.6. Orthologs of Crtl are widely distributed across filamentous ascomycete fungi. Phylogenetic analysis showed that Crtl roughly clustered with cellobiose transporters Cdtl and Cdt2 from N. crassa and had high identy with lactose permease LAC12 from K.lactis. Cdtl and Cdt2 have been shown to have a significant effect on cellobiose consumption, in contrast, a T.reesei strain carrying a deletion for crtl grew and consumed cellobiose at a similar rate with that of WT with cellobiose as the sole carbon source, indicating that the absence of crtl does not interfere with the assimilation of cellobiose. And also, deletion of crtl does not impact sophorose assimilation neither. All the above implies that Crtl regulates cellulase genes transcription through a way of transceptor like Rgt2/Snf3 in S.cerevisiae.7. The first monosaccharide derived cllulase inducer -gluconolactone was reported.Besides crystalline cellulose, several soluble sugars including cellobiosecan effectively induce cellulase formation. In this study, gluconolactone, previously reported as a β-glucosidase inhibitor, was demonstrated to be capable of inducing cellulase gene expression at a levelequivalent to that induced by cellobiose. Gluconolactone-induced formation of cellulase was abolishedin T. reesei strain lacking Xyrl or Crtl, two key regulators for cellulase gene expression. The inducedexpression of cellulase gene cbh I was eliminated in the absence of intracellular β-glucosidase Cell a ongluconolactone while it was hardly affected by the absence of extracellular β-glucosidase Bgll. Wefurther found that the absence of a cellobiose/glucose transporter Stpl compromised cellulaseproduction and led to a lower consumption of extracellular gluconolactone. These results suggest thatthe gluconolactone-derived inducing signal involves both its sensing at the membrane and itsintracellular delivery for further processing to initiate cellulase formation.