Expression And Molecular Modification Of Rhizomucor Miehei Lipase In Pichia Pastoris

Lipases are biotechnologically important enzymes with applications in food, pharmaceutical, detergent and synthesis industries. The structure and catalytic mechanisms of Rhizomucor miehei lipase have been widely studied in an industrial setting, and the enzyme activity has been drastically improved through directed evolution and protein structure analysis in the upstream process and through optimization of fermentation conditions and reaction conditions in the downstream process. Native RML consists of a24-amino acid signal peptide, a70-amino acid propeptide and a269-amino acid functional region. The molecular weight of mature protein is about30KDa. The present work chose the gene containing the propeptide and the mature region as the target for expression and engineering.Many studies have demonstrated that the properties of enzymes expressed in eukaryotes can be affected by the position and extent of glycosylation in the enzyme. In this study, WT of RML was cloned into pPIC9K, linearized with SalI and expressed in Pichia pastoris. The enzyme activity of the WT expressed was1700±250U/mg, which was about6.7times higher than that WT expressed in E. coli. Two potential glycosylation sites (the8th and the58th asparagine) were identified and the effect of propeptide glycosylation on the Rhizomucor miehei lipase (RML) expressed in P. pastoris was investigated. To better understand the effect of glycosylation on the activity of RML, three mutants (M1, generated by N8A; M2, generated by N58A; and M3, generated by N8A and N58A) were designed to generate deglycosylated enzymes. The results showed that deglycosylated RML exhibited a twofold higher activity compared to the wild type. However, it was also found that glycosylation in the propeptide was important for the removal of the propeptide by Kex2protease and thus the secretion of the enzyme. Thus, our study provided a further insight into the role of glycosylation in enzyme function.Four mutants (G1:L57V, G2:L57V/V67A/I111T, G3:L57V/V67A/I111T/S168P, G4:L57V/V67A/I111T/S168P/S65A) were generated from the wild type RML (WT) in Escherichia coli through directed evolution. The enzyme activity was increased stepwise from G1to G4, and the mutants were cloned into pPIC9K to generate pPIC9K-WT, pPIC9K-G1, pPIC9K-G2, pPIC9K-G3, pPIC9K-G4. The resulting plasmids were linearized with SalI and expressed in P. pastoris. The results showed that the activities of the four mutants were not increased when expressed in expressed in P. pastoris. The results demonstrated that the mutants generated through directed evolution in E. coli could not be adapted to P. pastoris, which suggests that the law of directed evolution in prokaryotes is not applicable to eukaryotes due to the different post-translational modification systems.To enhance the activity by directed evolution in the yeast, four new recombinants were constructed with a-factor and INU signal peptides (a-factor+WT+pESC-URA+BY4741, INU+WT+pESC-URA+BY4741,a-factor+WT+pESC-URA+EBY100,INU+WT+pESC-UR A+EBY100), and secretive RML was successfully expressed in Saccharomyces cerevisiae. However, due to the low expression level and enzyme activity in S. cerevisiae, we chose P. pastoris as a host for RML directed evolution, using the method of rhodamine B plate for high-throughput screening. We conducted a round of evolution and achieved two mutants, M8-4(R41C) and M8-15-1(A115V/D313E), the activities of which were both25%higher than that of WT.To improve the production of RML, the optimal conditions of fermentation were explored. A three-stage process was utilized for RML production:In the first stage, the engineered strain was batch-cultured in a simple defined medium containing glycerol to accumulate biomass. After40hours, glycerol was completely consumed and OD600reached100. The second stage was a fed-batch transition phase in which glycerol was fed to the culture to further increase the biomass concentration and to prepare the cells for induction, this stage lasted from40-72hours, and OD600could reach up to320. The third stage, induction phase, was initiated by adding methanol at a slow rate, and then the methanol feeding rate was periodically increased until the desired growth rate was reached. After132hours, OD600was300, the protein concentration was0.375mg/mL, and the enzyme activity was233U/mL.This work related to the upstream process such as gene engineering and downstream process containing fermentation conditions. The study will lay the foundation for future industrial production of RML.