Studies On Cellulase Transcriptional Factors And Application Of Cellulase

Currently, the increasing depletion of oil resources led to a new rise of the renewable energy industry, and bioethanol production from lignocelluloses becomes more and more attention. The defect of the first-generation bioethanol industry is that it has the competion of food with people. Therefore, the second-generation bioethanol technology emerges. It uses straw, corn cobs and other lignocellulosic materials to produce bioethanol instead of food. Cellulase plays important roles in degradation of lignocellulose. The cellulases are mainly produced by filamentous fungi such as Trichoderma reesei and Penicililum oxalicum. The biggest problem in second-generation bioethanol industry is the high cost. There are two reasons for this phenomenon. Firstly, the low production yield of cellulase, and secondly, the low conversation efficiency of cellulase. In order to reduce the cost of production of cellulase, one hand the regulatory mechanism of cellulase are studied, and on the other hand, cellulose enzyme complex to improve the conversion efficiency of cellulase, reduce the cost of cellulase are also investigated.T. reesei is a major strain of cellulase production. The regulation of cellulase is a very complex process. It is affected by the environment factors such as carbon sources, light condition and pH. The transcriptional level of cellulase is mainly regulated by cellulase regulators:transcriptional activators Xyr1, Ace2 and Clr2; transcriptional repressors Cre1 and Ace1. Cre1 is the major transcriptional repressor. In our research, we study the function and phosphorylation of Crel. However, these significant advances in knowledge on regulation do not rule out the participation of other factors in cellulase expression, as cellulase induction pathways have been shown to be complex and interactive in filamentous fungi. In this study, we found a new family-velvet family protein plays important roles in regulation of cellulase. At the same time, in this study, we degrade the potato residues using cellulase enzymes and convert the glucose to gluconic acid in cooperation with other lab.The main contents in this study are listed in the following text.1) The function of velvet family proteins in T. reesei.Using the method of BLAST, we found three velvet-domain-containing proteins. Phylogenetic analysis showed that they are VeA, VelB and VelC proteins and we named them Ve1, Vel2 and Vel3. We constructed Δve1, Δvel2 and Δvel3 stains using homologous recombination and research the change of morphogenesis, sporulation and cellulase expression under light and dark condition. We cultivated the velvet mutants on glucose, glycerol and lactose plates under light and dark condition and found that velvet family proteins participant in maintence of morphogenesis and secondary metabolism:velvet mutants produced more short branches under light and dark condition. The diameter of Δvel2 became smaller and irregular. Δvel2 did not produce but Δvel3 produced more yellow pigments. The biomass of Δveland Δvel3 kept unchanged but in Δvel2 it decreased drastically. Deletion of vel or vel2 led to the disappear of spores and in Δvel3 produced less spores under light and dark condition compared with the parent strain indicating that vel and vel2 play important roles and vel3 act auxiliary. Further research found that velvet family regulated sporulation by regulating the key genes during sporulation such as wet A and abaA. The transcriptional levels of cellulase coding genes in velvet mutants were significantly down regulated under light and dark condition compared with the parent strain. Measurements of FPase, CMCase, pNVCase and pNPGase activities further confirmed the roles of velvet family in cellulase production. Meanwhile, we found that velvet family proteins regulated cellulase expression by regulating the transcription of cellulase activator xyrl. Furthermore, we found that velvet family proteins had no relationship with env which play key roles in light-dependent cellulase regulation and new transcriptional factor clr2. The velvet family proteins did not participate in cellulase secretion. Previous reports showed that the velvet domain was a new kind of transcription factor which could bind to brlA. In order to investigate the mechanism of regulation of cellulase expression by velvet family, the EMS A experiment will be carried out to detect the combination between velvet family protein and cellulase transcriptional activator xyrl or cellulase transcriptional repressor crel. Reports showed that Vel could be phosphorylated. The mechanism and the effects of phosphorylation need to be further researched.2) Study of carbon catabolite repressor Crel in T. reesei.The expression of cellulase in filamentous fungi was reguated by cellulase transcriptional activator and cellulase transcriptional repressor. The carbon catabolite repressor Crel is the main repressor of cellulase expression in T. reesei and the full expression of cellulase transcriptional activator xyrl is dependent on the existence of Crel. In our study, we study the phosphorylation of Cre1. Deletion of crel led to serious damage of growth and longer growth time. Previous reports showed that Cre1 could be phosphorylation. Therefore, in this study we research the effects of phosphorylation of Cre1 on expression of cellulase. Using the method of yeast two-hybrid, we proved the protein interaction between Cre1 and casein kinase CKII catalytic subunit al. The conserved sequence of CKII in Crel is HSNDEDD and S241 is the phosphorylation site and it must be in acidic condition. In order to study the function of the conserved sequence in cellulase expression, we constructed 7 mutants:S241V (S), D243V (D), E244V (E), D243V/E244V (DE), S241V/D243V (SD), S241V/E244V (SE), S241V/D243V/E244V (SDE). The biomass of these mutants had no change compared with the parent strain.Parent strain and 7 mutants were cultivated in glucose, lactose and cellobiose. The expression of cellulase was significant down regulated in S strain in three carbon sources. In D, SD, SDE mutants, the expression of cellulase were up regulated to 10 fold in glucose medium compared with the parent strain and had no change in lactose and cellobiose. Mutation of E did not affect the expression of cellulase expression but may had effects on the function of S and D sites as the change of cellulase expression is E<DE<D and SE<SDE<SD. The mechanism remained to be researched. The expression of cellulase in glucose was higher than that in lactose and cellobiose. At the same time, we successfully got two proteins that may have protein interaction with Cre1 in glucose condition using the method of co-immunoprecipitation. Structure predication of Crel showed that there were two Zn2Cys6 zinc fingers at the N-terminus. Second structure analysis showed that there were only a little a-helix and P-sheet in Cre1. The hydrophilic amino acid was about 80%, indicating that Crel was a hydrophilic protein. In order to research the structure of Crel, we successfully expressed Crel in E. coli but failed because thrombin not only cut the linkage between Crel and GST, but also cut Crel into pieces. In future study, we should exchange the expression vector. At the same time, we constructed the Cre1+HIS strains in order to research the structure of phosphorylated Cre1, but failed to purify the correct Cre1 protein.3) Degradation of potato residues using cellulase.The potato pulp was the by-product of starch industury. In this study, the concentration of substrate was 25%. Component analysis showed that the main components of potato residues were starch, pectin and cellulose. It did not contain hemicelluloses and had a little lignin. We choose Penicillium oxalicum A10-1 and T. reesei TX as P. oxalicum A10-1 had higher FPase and pectinase activities and T. reesei TX had higher β-glucosidease activities. The high content of pectin could hider the degradation of potato pulp, so in this study addition of pectinase was essential for promoting the degradation of potato pulp. In this study, we degraded potato residues combined cellulase enzymes with pectinase. The glucose conversion rate was higher when degraded high temperature treated potato residues compared with untreated material using T. reesei TX. The glucose conversion rate was almost consistent when degraded high temperature treated potato residues and untreated material using P.oxalicum A10-1. In this study, we degraded treated potato residues by adding 2000PGU/g dry substrate pectinase plus different FPU/g dry substrate P.oxalicum A10-1 or T. reesei TX and by adding 8FPU/g dry substrate P.oxalicum A10-1 or T. reesei TX plus different PGU/g dry substrate pectinase. The best result was got by adding 8FPU/g dry substrate P.oxalicum A10-1 plus 1000PGU/g dry substrate and the glucose conversion rate reached 80% and the glucose concentration was 83.4 mg/ml. The glucose was converted into gluconic acid. The overall conversion yield from glucose to gluconic acid was 94.9% and the productivity was 4.07 g/L/h. The gluconic acid concentration was 81.4 mg/ml.