Construction Of Multi-enzyme Co-display System Using Scaffoldins Of Cellulosomes

Artificial mini-scaffoldin of cellulosomes was anchored on the yeast cell surface of the host strain Pichia. pastoris KM71 H via fusing the cell wall protein Sed1 p from Saccharomyces cerevisiae EBY100. Formate dehydrogenase and cytochrome P450_BM-3 monooxygenase were co-displayed in ratio of 1:1 on the cell surface of the host strain P. pastoris KM71 H. The engineered KM71 H cells equipped with the co-display system were constructed based on the protein-protein interaction mediated by cohesin module and dockerin module from the specific cellulosomes. Due to the direct couple reaction of formate dehydrogenase and cytochrome P450_BM-3 monooxygenase on the yeast cell surface, NAD-NADH coenzyme system was simply and efficiently recycled without adding extra coenzymes. The major results of this work were listed below:1. Compared to C. boidinii, E. coli host strains preferred the high GC content in its coding DNA. The gene of formate dehydrogenase of C. boidinii（Cbfdh） was codon-optimized based on the E. coli codon usage, its CAI value was significantly improved from 0.307 to 0.723, which is more suitable for E. coli expression system. Fused enzyme CbFDH-Ccdockerin was expressed in E.coli in soluble fraction, and its size was 42 kDa in the monomer form. At room temperature and pH 7.4, its specific activity was 4.15 U/mg using sodium formate and NAD as substrate and coenzyme. Ccdockerin module showed no obvious impact on the enzymatic activity of CbFDH.2. The crystal structure 4dql_A of P450 reductase was chosen by SWISS-MODEL workspace as the reference model for the coenzyme specificity mutation of cytochrome P450_BM-3 monooxygenase. Residues S965、R966、K972 and W1046 were associated with the coenzyme specificity of cytochrome P450_BM-3 monooxygenase. The triplet mutant P450tri（R966L/K972L/W1046H） significantly enhanced its NADH utilization efficiency. At room temperature and pH 7.4, its specific activity was 0.15 U/mg using oleic acid and NADH as substrate and coenzyme. Mutants mainly disrupted the polar interaction with the phosphate moiety of NADPH; and alleviate the steric hindrance of electron flow between NADPH and FAD.3. The DNA of artificial mini-scaffoldin（mixa3） was properly codon-optimized based on the P. pastoris codon usage. Expression recombinant vectors pTrc99a-mixa3 and pET20b-mixa3 were successfully constructed. Compared to E. coli TOP10/pTrc99a-mixa3, E. coli BL21（DE3）/ pET20b-mixa3 heterologously expressed the artificial mini-scaffoldin mixA3. The expression differences indicate that E. coli BL21（DE3）/pET20b-mixa3 is more suitable for the mixa3 expression in E. coli.4. Hydrophobic residues accounted for 50% amino acid composition and formed the hydrophobic internal cores in Ctcohesin、Rfcohesin and Cccohesin. Besides, two repeats were detected in Ctdockerin、Rfdockerin and Ccdockerin. Fused enzymes CbFDH-Ccdockerin and CYP102A1-Ctdockerin were attached to the mixA3-His and formed the cohesin::dockerin complex by the recognition and binding of dockerin to cohesion.5. Reporter protein eGFP was expressed in the recombinant strain KM71H/pPICZaA-egfp, and secreted to the culture medium after methanol inducement. For its intracellular expression, compared to KM71H/pPICZB-egfp, green fluorescence was only detected in KM71H/pPICZB-iniegfp cells. These results indicate that the initial codon AUG of egfp interacts with the flanking codons forming a local hairpin structure, which causes ribosome minor subunit failing to recognize the initial codon AUG, resulting in the translation failure.6. Kex2 is a protease located in the golgi apparatus in P. pastoris, it can recognize and cleavage dibasic amino acids Lys-Arg, and its homologs are widely distributed in fungi. Based on the structural information, Lys214-Arg215 were mutated into Ser214-Ser215. Mutated eGFP was used as the reporter for mixA3 anchoring on the host cell surface. Hydropathy and mutation analyses indicated that the last 40 amino acids in the C terminus of Sed1 p was the anchor signal. The formate dehydrogenase and cytochrome P450_BM-3 monooxygenase were co-displayed on the P. pastoris cell surface, and recycled the NAD-NADH coenzyme regeneration system.