| Biofuel derived from lignocellulose is a clean, renewable energy that can partially replace our current demand for fossil fuels. However, degrading the insoluble plant biomass into fermentable monosaccharaides represents a main bottleneck for biofuel production. Trichoderma reesei secretes large amounts of (hemi) cellulase that could hydrolyze plant biomass into soluble oligosaccharides, and is now one of the most important industrial strains to produce cellulases and hemicellulases. The mechanism of cellulase induction is still indistinct and the best inducers of the cellulase for T. reesei are preferably insoluble cellulose, hemicellulose, and plant biomasses. Currently it is generally believed that small molecules such as cellobiose and sophorose released from these insoluble substrates are the real inducers.In addition to cellobiose and sophorose, lactose can also induce the production of cellulase, however currently we still do not understand the mechanism underlying this induction. There are some experimental evidences showing that lactose transport and subsequent intracellular hydrolysis or conversion may play important roles in the induction process. T. reesei lacks typical intracellular β-galactosidase which belongs to glycoside hydrolase family2, and the intracellular protein(s) involved in intracellular lactose hydrolysis and conversion has (have) not yet been identified.There are lines of evidence that β-glucosidases play a vital role in the process of rapid response to cellulose and the induced synthesis of cellulase in T. reesei. For example, deletion of the major extracellular β-glucosidase encoding gene bgll/cl3a delayed cellulase expression on cellulose, while the bgll/cel3a overexpressing strain had an elevated expression of cellulase not only on cellulose but also on sophorose (a potential inducer of cellulase). T. reesei contains11P-glucosidases, among which Bgll/Cel3a is an extracellular β-glucosidase, belonging to glycoside hydrolase family3, and Cell a and Cellb are intracellular enzymes both belonging to glycoside hydrolase family1. Transcription of these three β-glucosidases on cellulose and lactose is significantly raised in T. reesei. Additionally, in some cellulase-high-yielding strains, transcription of these three β-glucosidases is higher than that of the wild type, suggesting that they may be involved in the process of cellulase induction. It is a common phenomenon that β-glucosidases of glycoside hydrolase family1also possess side β-galactosidase activity, making them important candidates for intracellular conversion of lactose. Whether the two GH1P-glucosidases Cel1a and Cel1b of T. reesei have β-galactosidase activity and their roles in lactose induction need further study.In order to illustrate the roles of β-glucosidases in cellulase induction of T. reesei, three main β-glucosidase genes bgl1/cel3a, Cel1a and Cel1b were knockout singly and multiply. Cellulase expression capacity of these β-glucosidase-deficient strains was studied on different carbon sources such as cellulose, cellobiose, sophorose and lactose. Besides, the two intracellular β-glucosidases Cel1a and Cellb were heterologously expressed to study their activities on lactose and their roles in cellulase induction by lactose. The main innovative results of this study are as follows:1. A series of β-glucosidases deletion strains were constructed by gene replacement to study the in vivo function of these proteins in cellulase induction.In order to study the in vivo function of different β-glucosidases in T. reesei, we constructed a number of β-glucosidase deletion strains by means of homologous recombination, including△cel1a,△cel1b,△cel1a△cel1b and△triβG. Anchored PCR and Southern blot were used to verify gene knockout and single copy integration. During culture on cellulose, intracellular β-glucosidase activity of Acel1b was slightly lower than that of wild type strain, while it fell75%in the Acella strain, the extracellular β-glucosidase activity of Bgll deletion strain dramatically declined. In summary, Cel3a accounts for the major extracellular β-glucosidase activity, while Cel1a is the major intracellular β-glucosidase, Cel1b contributes only a small part to the total intracellular β-glucosidase activity in T. reesei.2. Deletion of cella, cellb delayed cellulase expression on cellulose, but not on sophorose, suggesting that intracellular Cella and Cellb contribute to the induction of cellulase genes probably through participating in the formation of cellulase inducer.To further analyze the influence of P-glucosidases on cellulase expression we studied the kinetics of cbhl gene transcription in different β-glucosidase-mutant strains using Northern blot/qRT-PCR. This analysis showed that in comparison to the rapid induction in the WT strain, which occurred as early as3h upon induction by cellulose, productive activation of transcription of cbhl was delayed by about3h and21h in the△cel1b and△cel1a strains, respectively. This lag in gene expression was further extended to36h in the△cel1a△cel1b strain, although the final level of transcription was almost the same. Western blot of CBHI protein in the fermentation broth also showed similar results. However, the induction effects by sophorose were not affected in the P-glucosidase mutant strains. In summary, all these data suggests that β-glucosidases are vital in rapid cellulase induction, and may be involved in inducer synthesis.3. Deletion of major β-glucosidases in T. reesei resulted in higher expression levels of cellulase on cellobiose suggesting that lowering the degree of cellobiose hydrolysis may facilitate cellulase synthesis.As the main degradation products of cellulose, cellobiose at a low concentration can induce the expression of cellulase and it has been shown that cellobiose could be transformed into sophorose by β-glucosidases in T. reesei. We therefore analyzed the impact of β-glucosidases on cellulase induction by cellobiose. The cellulase expression on cellobiose were significantly elevated in△cel1a△cel1b and△triβG strains suggesting that lowering the degree of cellobiose hydrolysis may facilitate cellulase synthesis and the conversion of cellobiose by the main intracellular and extracellular β-glucosidases may not account for the rapid cellulase expression on cellulose. By exogenously adding a certain proportion of cellobiose to cellulose medium, we found that the cellulase induction capacity of β-glucosidase-deficient strains on cellulose recovered. These data indicated that the productive induction defect on cellulose as observed in the absence of the major β-glucosidases may to a larger extent be caused by the insufficient cellobiose initially available for triggering the induction cascade.4. Intracellular β-glucosidases Cell a and Cellb affect expression of cellulase and Xyrl by lactose. Constitutive expression of Xyrl could not restore the efficient induction of β-glucosidase-deficient strains.We further analyze the effect of β-glucosidases on induction of cellulase by lactose. By analyzing the cellulase expression of△cella,△cel1b and△cel1a△cel1b strains cultured on lactose, we found that single deletion of intracellular β-glucosidase lead to lower cellulase expression on lactose, while double deletion of cel1a and cel1b almost abolished it. Further analysis of cbhl transcription by qRT-PCR revealed that the decrease of the cellulase expression occurred at the transcriptional level. These results show that intracellular β-glucosidases are essential for efficient induction of cellulase by lactose. Xyrl is the key transcription factor regulating cellulase expression, whose absence abolished cellulase expression on cellulose and lactose. We found transcription of xyrl decreased in the△cel1a△cel1b strain cultured on lactose, however constitutively expression of xyrl could not restore its efficient cellulase expression, suggesting that expression of xyrl alone is not sufficient to activate the cellulolytic transcription by lactosein the absence of Cel1a and Cel1b.5. Intracellular β-glucosidase Cel1a and Cel1b exhibited apparent hydrolytic activity toward ONPG and lactose.Enzymes involved in intracellular metabolism of lactose have not so far been identified in T. reesei. Most β-glucosidases belonging to GH1also possess β-galactosidase activity. Besides this, our previous data showed that intracellular β-glucosidases played central roles in the progress of cellulase induction by lactose. All these data make us speculate whether Cel1a and Cel1b of T. reesei possess P-galactosidase activity. So we heterologously expressed and purified Cel1a and Cel1b in E. coli and analyzed their substrate specificity. We found that Cel1a and Cel1b can hydrolyze not only cellobiose and pNPG, but also lactose and ONPG, suggesting that they possess β-galactosidase activity. Analyzing the lactose hydrolyzing products by HPLC, we found oligosaccharides in addition to D-glucose and D-galactose were also produced, suggesting that β-glucosidases of T. reesei also possess transglycosylation activity toward lactose.6. Both Cel1a and its associated hydrolytic activity are responsible for the efficient induction of cellulase genes by lactose.To gain insight into the possibility that Cel1a-associated glycoside hydrolytic activity is involved in the induction process, we retransformed△cel1a strain with wild-type Cella and a mutant protein Cella (Q367A) which bears no activity towards cellobiose and lactose. While constitutive expression of WT Cel1a resulted in a relatively higher induced expression of cbhl as compared with the WT strain, cells expressing the Cella (Q367A) displayed the same kinetic of cbhl induction as in the△cel1a strain on lactose. These results suggest that hydrolytic activity of Cel1a was required for cellulase expression. To further test Cel1a-associated hydrolytic activity toward lactose is fully responsible for cellulase induction, β-galactosidase LAC4from Kluyveromyces lactis was expressed in the Acel1a strain. Analysis of the induced expression of cbh1revealed no restoration of induced cbhl expression in the△cel1aCElac4strain. Altogether, these data suggest that intracellular processing of lactose mediated by Cella and Cellb beyond sole hydrolysis may account for their important roles in cellulase induction by lactose. |
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