Yeast Heterologous The Table Dagro Trichoderma Reesei Cellulase Characteristics

Ethanol is the most potential liquid fuel of renewable resources. The production of ethanol from food is greatly limited as the rapid growth of the world population and the shortage of food. Transfermation of cellulose directly to ethanol by recombinant yeast has become hotspot of recent studies, since the equipments required and technology are easier, and the cost is lower.Yeast is a good host for heterogenous expression because of its high expression and transformation efficiency. Many fungal cellulases had been expressed in Saccharomyces. cerevisiae and Pichia pastoris, and many recombinant proteins were obtained. However,the recombinant cellulases have very low expression level and low enzyme activities. Cel7A, the major component of the Hypocrea cellulase system (representing about 60% of the total cellulase protein produced by the fungus on a mass basis, and is the key enzyme acting on crystalline regions of cellulose), were found to be hyperglycosylated expressed in Saccharomyces cerevisiae and Pichia pastoris, and either had no activity, or had significantly lower activity than that of the wild type enzyme. The heterogenously expression level of Cel5A which accounts for most of the endoglucanase activity produced by Hypocrea jecorina (syn. Trichoderma reesei) in yeast is significantly lower than in H. jecorina.To understand the effect of hyperglycosylation on recombinant Cel7A in P. pastoris, asparagines of the four N-glycosylation sites were replaced by serines using site-directed mutagenesis and expressed, and the higher activity mutant enzyme was abtained; we constructed Saccharomyces cerevisiae mutator strains which heterogenously expressed Cel5A, and we select the mutants with elevated expression level of Cel5A. These works not only improve the activity and expression level of cellulases of H. jecorina expressed in yeast, but also offer useful information and platform to cellulases protein engineering using yeast as hosts.The main results of the thesis are as follows:1. The N-glycosylation of Cel7A expressed in P. pastoris Asparagines of the four N—glycosylation sites, Asn45, Asn64, Asn270 and Asn384 of Cel7A were replaced by serines using site-directed mutagenesis. These four mutants and the Cel7A gene from H. jecorina were transformed into P. pastoris, then the enzymes expressed were purified and analyzed. Data showed that the activity of mutant enzyme N64S was 7 folds higher than that of rCel7A expressed in P. pastoris. Analysis of the three-dimentional structure of Cel7A indicates that Asn64 is almost inside the protein, and that the space surrounding Asn64 is crowded, and there is little space to accommodate the glycosylation. These comparisons suggest that the neighboring local structure of the heterogeneously expressed Cel7A would be evidently changed if the Asn64 site is N-glycosylated. It would even affect the whole structure and enzyme activity of the protein. Comparing the activities of Cel7A from different mutants and native Cel7A, experimental data showed that hyperglycosylation is only a subordinate reason for the low activity of recombinant Cel7A in P. pastoris. We speculate that the differences in cotranslational N-linked glycosylation of proteins may affect protein folding, especially the formation of the disulfide bridges and the tunnel structure, resulting in the reduction of the enzyme secretion and activity.2.construction fo imitator strains and the acquirement of mutants with high Cel5A expression.We constructed three mutator strains RDKY3615(ΔCTT),RDKY3615 (ΔSOD) and RDKY3615 (ΔSRX) by knocking out genectt1,sod1 and srx1 gene which act in preventing oxidative damage, respectivly. We then measured their growth and mutation rate indirectly by measuring the reversion mutation rate of kanamycin resistance gene and researches about mutagen H₂O₂'s effect of different processing time and concentration on mutation rate. Endoglucanase Cel5A was then expressed in mutator strains. After H₂O₂ treatment and selection, several bigger hydrolyzed transparency circle mutants were abtained. 15 mutants, consist of 11 RDKY3615(ΔCTT)-Cel5A mutants and 4 RDKY3615 (ΔSOD) -Cel5A mutants, with with elevated enzyme activity were selected. The highest enzyme activity is 10.1 folds than that of wild type. The protein assignment and sequence of the mutants' Cel5A gene show that the reasons of enzyme activity elevation of the mutants were heritable mutations of Saccharomyces cerevisiae genome.