Functional Analysis Of Ras Signaling For Morphogenesis And Cellulase Synthesis In Trichoderma Reesei And Cellulase Optimization

Trichoderma ree.sei (anamorph of Hypocrea jecorina) is well known as one of the most widely spread filamentous fungi in the natural saprophytic habitat for its ability to degrade lignocellulose materials. The degradation of cellulose by T. reesei plays a key role in global carbon cycle. Because of its characteristics of great growth capability on cellulose substrate, T. reesei has been used as the model to study cellulose decomposition. Degradation of cellulose by T. reesei mainly depends on its powerful ability to secrete cellulase and hemicellulase. Although its genome encodes fewer cellulose-degrading enzymes, T. reesei is capable of responding to environmental cues to compete for nutrients in its natural habitat. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. The T. reesei cellulase and their expression characteristics have been studied for several decades. However, it is still unclear about the molecular mechanisms that T. reesei cells sense environmental signals, e.g., cellulose, to regulate development and cellulase gene expression. Ras signaling represents one of the most critical pathways involved in perception of environmental signals and subsequently regulation of gene expression in filamentous fungi. In this paper, we have studied the functions and regulatory mechanisms of Ras signaling mediated by Ras GTPases during fungal development and cellulase synthesis in T. reesei.In addition, T. reesei has been widely used for cellulase production and saccharification of cellulose due to its strong ability to secrete cellulase. Nevertheless, T. reesei cellulase still possesses many obstacles for its commercial application in enzymatic saccharification of lignocellulosic biomass, e.g., no ligninolytic enzymes and lower β-glucosidase concentration. To overcome these drawbacks, we have developed the T. reesei strains with heterologous expression of lignin-degrading enzyme laccase A or overexpression of the homologous P-glucosidase BGL1.1. Ras small GTPases modulate fungal development in T. reesei.Using the amino acid of Saccharomyces cerevisiae Ras1as the query, five putative Ras small GTPases, named TrRasl, TrRas2, TrRsr1, Tr107035and Tr66480, were identified in the genome database of T. reesei by a BLASTP search. Alignment of the amino acid sequences of these Ras small GTPases along with that of their orthologue Saccharomyces cerevisiae Rasl revealed that all the conserved domains are included, including GTP or GDP binding site, GAP effector binding site, the GTPase domain and the CAAX box for membrane association. Expression of these five Ras small GTPases could be detected in the T. reesei hyphal cells. Using gene targeting and overexpression-based manipulations, we have found that TrRasl, TrRas2and TrRsrl were involved in morphogenesis and carbon source utilization. Specially, deletion of TrRasl resulted in small and dense yeast-like colonies with highly branched, swollen and misshapen hyphal cells. Similarly, TrRas2was also involved in modulation of polarized apical growth and hyphal branch formation. In contrast to the wild-type, the ATrRas2mutant formed hyphae with a hyper-branching phenotype while dominant activation of the TrRas2resulted in fewer hyphal branches and enhanced polarized apical growth. The ATrRas2mutant exhibited reduced colonies with irregular boundaries and greatly decreased aerial hyphae. The dominant activated TrRas2G16V strain also showed reduced colonies with no conidia and aerial hyphae, but their colonies exhibited regular borders. In general, TrRasl is more dominant than TrRas2during controlling the morphogenesis of hyphae. Although deletion of TrRsrl resulted in decreased growth of T. reesei, the formation of hyphae, colony and conidiation in ΔTrRsrl showed no differences with that of the wild-type strain. In addition, we found that deletion of either TrRas2or TrRsr1could decrease the growth of T. reesei on lactose and CMC-Na, suggesting these two Ras small GTPases also play important roles in carbon source utilization. Specially, cultivation of the ΔTrRas2strain on cellulose plate led to no clear cellulolytic zone formation, which indicated that TrRas2might be involved in regulation of cellulase expression. Having found the critical roles of TrRas1and TrRas2in polarized apical growth, we wonder what the signaling mechanisms that regulated by TrRas1and TrRas2during filamentous growth of T. reesei are. In our study, we found that TrRasl and TrRas2play similar roles in increasing cAMP level, while TrRasl is more dominant. In comparison with the parental strain, the cAMP level in the ATrRasl strain decreased by41.4%, while expression of the dominant activated TrRas2G16V allele resulted in34%and50%increase in cAMP levels on glucose and cellulose respectively. However, the cAMP level in ΔTrRas2showed no changes neither on cellulose nor glucose. Furthermore, addition of exogenous cAMP could rescue the filamentous growth and the aerial hyphae growth defects of the ATrRasl mutant, which indicated that TrRasl might regulate filamentation program through cAMP signalling pathway. Therefore, we subsequently studied the interaction between TrRas1/TrRas2and adenylate cyclase (Acy1) to further explore the Ras regulation during filamentation in T. reesei. The Co-IP results showed that either TrRasl or TrRas2could interact with the Ras associated domain of Acyl, indicating that TrRasl/TrRas2might function via modulating the activity of Acyl in T. reesei.Moreover, transcriptome analysis revealed that TrRasl and TrRas2regulate the expression of genes associated with energy metabolism and amimo acid/lipid synthesis, thus influence the cell membrane building and growth, and subsequently modulate the development of T. reesei.2. Signalling pathway mediated by TrRas2modulates cellulase gene expressionStrikingly, we found that TrRas2is involved in modulation of cellulase gene expression. Deletion of TrRas2resulted in considerably decreased transcription of cellulolytic genes upon growth on cellulose, while the expression of the major cellulase genes cbhl and cbh2increased by73%and128%respectively in the dominant activated TrRas2G16V strain. Although the strain carrying a constitutively activated TrRas2G16V allele exhibited increased cellulase gene transcription, the cbhl and cbh2expression in this mutant still strictly depended on cellulose, indicating TrRas2does not directly mediate the transmission of the cellulose signal. Furthermore, our data suggested that the effect of TrRas2on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1. Moreover, TrRas2also possesses negative influences on the expression of the other two cellulase transcriptional factors Acel and Cre1.Previous reports showed that cAMP signaling pathway is involved in regulation of cellulase expression in T. reesei. Meantime, our data suggested that TrRas2could improve intracellular cAMP concentration. However, deletion of TrRas2led to significant decrease in cellulase expression but no cAMP level changes. Therefore, we concluded that the positive influence of TrRas2on cellulase expression could be transmitted by increasing cAMP content. Meanwhile, other pathways may also act downstream of TrRas2to modulate cellulase expression besides cAMP signaling. In addition, our results also excluded the possibility that TrRas2signaling directly transmits the effect of light signal on cellulase expression.In addition, the genome-wide transcriptional analysis showed that TrRas2positively influences the transcription of most of the lignocellulose degrading enzymes in T. reesei, which also indicated that TrRas2play an important role in lignocellulose decomposition.Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the powerful competitive ability of plant cell wall degrading fungi in nature.3. Improved saccharification efficiency through tailoring cellulase properties with genetic manipulationCommercial cellulase produced by T. ressei has been widely applied in enzymatic saccharification of lignocellulosic biomass for bioethanol and chemical intermediates production. However, it is generally believed that the insufficiency of P-glucosidase or ligninolytic enzymes in the cellulase complex of T. reesei is the bottleneck in efficient lignocellulose hydrolysis. Here, the extracellular β-glucosidase BglⅠ was overexpressed in T. reesei under the control of the modified four-copy cbhl promoter. The transformants with successful integration of the bgll gene expression cassette exhibited1.8-to3.7-fold increase in β-glucosidase activity than the parental strain. Furthermore, saccharification of corncob residue with the crude enzyme dosages showed that the reducing sugar yields of BglⅠ-overexpression strains were11-29%higher than that of the parental strain. In addition, the lignin-degrading enzyme Trameles sp. AH28-2Laccase A fused to cellobiohydrolase I signal peptide was heterologously expressed in T. reesei. The lacA cDNA was under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter. Native PAGE analysis indicated that the Laccase-expression transformants were able to secrete recombinant laccase A, and the laccase activities corresponding to ABTS oxidation could reach3.62IU/ml. Most of the characteristics of the recombinant laccase were similar to those of the native enzyme. Glucose yields of the Laccase-expression transformants obtained from saccharification of corn residue by crude enzyme increased by30.8-45.3%compared to the host strain. Together, these data indicate that tailoring cellulase properties with critical lignocellulose degrading enzymes through genetic manipulation would be a feasible strategy to improve saccharification efficiency of biomass by cellulase preparation.