| In recent years, the use of lignocellulosic conversion of the production of biofuels is becoming a research hotspot. However, hydrolysis enzymes were still a major cost factor in the production of biofuels from lignocellulosic biomass. And making it had no competitive compared with traditional fossil fuels.The key solving this problem was to in crease the production of cellulase. Lignocellulase hypersecretion has been achieved in industrial fungal workhorses such as Trichoderma reesei RUT-C30; however, the underlying mechanism associated with this process is not well understood. Previous comparative genomic studies revealed that the mutagenic T. reesei strain RUT-C30 harbors hundreds of mutations in comparison with its parental strain QM6a; which mutations are present and how they actually contribute to the hypersecretion phenotype remain to be elucidated. Given that Neurospora crassa has a close phylogenetic relationship with T. reesei and possesses a nearly complete set of genome-wide gene deletion mutants, thereby making it a powerful tool for use in genetic studies, we reasoned that comparative genomic screening of N. crassa mutants could be applied as an alternative approach to study functions of mutated genes in T. reesei RUTC-30.Hydrolysis of lignocellulosic requires the synergistic action of three kinds of enzymes. Efficient endoglucanase and β-glucosidase could be significantly improved cellulose hydrolysis efficiency, reducing the cost of lignocellulose degradation, thus contributing to promote the use of biofuels. Therefore, in this study, we cloned and expressed one endoglucanase gene Agcel5 A and one β- glucosidase gene Ncbgl2. In order to obtain some efficient cellulases, to improve the efficiency of cellulose hydrolysis, and thus to reduce the cost of cellulase.The classical cellulase system was composed of endoglucanase, exoglucanase and β-glucosidase. Whether require another component to disrupt polymer packing in the substrate, thereby increasing its accessibility for hydrolytic enzymes? Lytic polysaccharide monooxygenases(LPMO) previously called GH61, now belong to the AA9 family, could increase the cocktail activity significantly. Recently, serious studies shown that this group proteins interaction with cellobiose dehydrogenase(CDH) or other low moleular weight reducing agents(such as ascorbic acid), take place a redox reaction and cleave glycosidic linkages of cellulose chain, to destroy cellulose structure and improve the efficiency of traditional cellulase activities. Myceliophthora thermophila is a thermophilic fungus, with very efficient cellulose degradation capability. Many enzyme with great characteristics for biotechnology has been cloned from this fungus. In this study, we cloned, purified and characterized two Lpmo genes from M. thermophila. In order to improve the original cellulase enzyme hydrolysis activity, reduce the cost of cellulase enzymes, which promote the purpose of sustainable development of biofuels.The main findings of this study were as follows:(1) we used gene deletion mutant stocks of N. crassa to investigate the function of orthologs of T. reesei genes whose mutations might contribute to the hypersecretion phenotype of the RUT-C30 strain. 12 mutants showed production of lignocellulases that was more than 25% in comparison with the wild-type strain, while secretion was nearly 25% lower in 4 strains. We found deletion of Ncap3m(NCU03998), which encodes the μ subunit of adaptor protein complex 3(AP-3 adaptor complex) in N. crassa, led to the most significant increase in lignocellulase secretion. Moreover, strains lacking the β subunit of the AP-3 adaptor complex, encoded by Ncap3b(NCU06569), had a similar phenotype to ΔNcap3m. The typical RESS(repressed expression of secreted sequences) phenomenon attenuated in these two mutants. All these results were suggesting the AP-3 adaptor complex is involved in lignocellulase secretion in N. crassa.(2) Successfully expressed an efficient endoglucanase Ag Cel5 A and a β-glucosidase Nc BGL2 in Pichia pastoris. Both of them secreted only one major protein to the fermentation broth. We found that Ag Cel5 A was a thermostability and halotolerance emzyme, and also showed a broad p H range of stability. The optima reaction conditions of Ag Cel5 A was at p H 5.0, 55 °C. Co2+ had great positive effects on enzyme activity. The recombinant β-glucosidase Nc BGL2 displayed its highest activity at p H 5.6 and 60 °C. The activty of Nc BGL2 increased by more than 80% with the addition of 10% ethanol. The purified Nc BGL2 showed a heightened ability to convert the major soybean isoflavone glycosides into their corresponding aglycone forms.(3) In the process of cellulase hydrolysis, when Mt LPMO9 A or Mt LPMO9 B was added into the cellulases cocktail, it could make the dosage of cellulase reduced 40%. Owing to the expression of two lpmo genes in N. crassa, the component of extracellular cellulase cocktail scereted by recombinant strains has been changed, and the activity of cellulase cocktail was increased. But the secretion amount of LPMO proteins need to ensure enough.In summary, we cloned two efficient cellulose hydrolysis enzyme, an endoglucanase Ag Cel5 A and a β-glucosidase Nc BGL2, both of them could get good used in biofuel production. We also found that LPMO protein(Mt LPMO9 A and Mt LPMO9A) had a synergistic effect with cellulase, which could improve the original cellulase hydrolysis efficiency and promote hydrolysis of cellulose. Using the model cellulolytic fungus N. crassa, we explored potential hypersecretion-related mutations in T. reesei strain RUT-C30. Particularly, we confirmed that the function of AP-3 adaptor complex associated with secretory pathway. This study provides a novel view of lignocellulases secretion and a new strategy for future fungal strain improvement. And all these research would help to reduce the cost on cellulase, and had both scientific significance and application values on biofuel production and application. |
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