Heterologous Expression Of Trichoderma Reesei Cellobiohydrolase Ⅰ In Filamentous Fungi

The degradation of crystalline cellulose is the key problem to improve the efficiency of cellulose utilization. Trichoderma reesei secretes series cellulolytic enzymes known to participate synergistically in the degradation of biomass,60% of total extracellular protein is CBH I. CBH I can hydrolyze the crystalline cellulose from the reducing end to release cellobiose and is considered to be the key enzyme component of cellulases in the degradation of crystalline cellulose. However, the enzymatic efficiency of cellulases is fairly low in the degradation of the raw cellulose materials in nature, which could not meet the need of industrialization transformation. With the development of gene engineering and protein engineering, we can use methods such as site-directed mutagenesis in the research of the key amino acids and the catalytic mechanism of cellulases and analyze the limiting factors of the efficiency of cellulose hydrolysis. Theses studies would provide the basis for improving the efficiency of cellulose hydrolysis.The dual domain structure, posttranslational modifications during secretion and the complicated folding process of CBH I are considered to be the main reason for the difficulty of heterologolous expression of CBH I. E. coli and yeast have been proven to be not the suitable host for the expression of CBH I. CBH I was produced as insoluble inclusion bodies in E. coli owing to the lack of necessary post-translational modification while in yeast CBH I was hyperglycosylated with lower activity. In mammalian expression system, the recombinant protein production is low, the culturing time is long and the cost is higher. With high protein secretion capacity, eukaryote protein translation, folding ability and post-translational modification, filamentous fungi are recognized as being the suitable expression host for cellulases including CBH I.In this study, we tried to express CBH I in Aspergillus niger and Trichoderma reesei, thus attempts to establish the effective filamentous fungi expression system for cellulases. We first constructed a highly efficient transformation system of Aspergillus niger. We studied different factors that might affect the preparation of protoplasts of Aspergillus niger including enzymes, culturing condition and spawn age. Digesting Aspergillus niger MGG029 mycelium with Lywallzyme could generate 107/ml protoplasts, but snailase could produce a small amount of protoplasts about 104/ml. Lysing enzyme which is effieient in the preparation of Trichoderma reesei protoplasts had no effect and cellulase also had no effect The optimum enzymes to prepare the Aspergillus niger protoplast is the Lywallzyne and Snailase mixed enzyme system. High-speed shaking culture (300 rpm) and static culture can obtain loose mycelium hence more protoplasts. The high-speed shaking culture (300 rpm) could generate more protoplasts than static culture. Spawn age is an important factor in protoplast preparation. The largest number of protoplasts was obtained with 14 h mycelium while the largest number of regenerative protoplasts was obtained with 16 h mycelium. The younger the spawn is, the more protoplasts can be obtained but with low regenerative activity. We selected the spawn age of 16 h as the best spawn age. Taking all the above results together, the optimal preparing conditions for the Aspergillus niger protoplasts is as follows:The 16 h mycelium was obtained with the high-speed shaking condition at 300 rpm and digested with 10 mg/ml lywallzyme+10 mg/ml snailase in the osmotic stabilizer (1M sorbitol, 10mM KH2PO4-K2HPO4 pH 5.8) for 2 h under 30℃. With this method we could obtained 107-108 protoplasts/ml. Using the PEG-CaCl2-mediated transformation method, we successfully transformed the Aspergillus niger MGG029 with the plasmid pAB4-1 (containing pyrG gene). The transformation efficiency was 200 transformants/μg DNA.After construction of Aspergillus niger transformation system, the expression vector (pAN56-CBH-pyrG) was constructed based on the pAN56-2 plasmid. His-tag and HA-tag were fused to the C-terminus of CBH I. cbh1 gene was then fused to glaA promoter which is a strong promoter in Aspergillus niger. The pyrG expression cassete was further inserted in the plasmid. The CBH I expression vector (pAN56-CBH-pyrG) was transformed into Aspergillus niger MGG029 with the PEG-CaCl2-mediated transformation method and 200 transformants were obtained. The transformants that had integrated the cbh1 gene were selected by PCR while the expression of CBH I was further confirmed by western blot. The recombinant CBH I was primarily purified. Its molecular weight was between 58 and 80k Da. The activity of the recombinant CBH I was determined using pNPC and Avicel pH101 as substrate, the result showed that the recombinant CBH I are active.The different glycosylation forms of Trichoderma reesei and Aspergillus niger caused the over-glycosylation phenomenon in the recombinant expression of CBH I in Aspergillus niger, which may affecte the activity of CBH I. Therefore, we further constructed the Trichoderma reesei expression system. We use the pyruvate kinase promoter (pki) which is constitutive promoter and cbh2 terminator to construct the Trichoderma reesei expression vector (pRLMex-CBHⅠ-pyr4). The vector was transformed into the uridine-deficient host Trichoderma reesei TU6. The transformants were confirmed by PCR. The result showed that the transformants had integrated the cbh1 gene.