We demonstrated previously that expression of Rhizomucor miehei lipase (RML) in Pichia pastoris could be significantly increased by addition of gene propeptide, optimized signal peptide codons and manipulation of gene dosage. In this study, effects of various strategies on the protein synthesis and secretion pathways were analyzed. Using nine strains previously constructed, we evaluated cell culture properties, enzymatic activities, and analyzed transcriptional levels of nine genes involved in protein synthesis and secretion pathways by qPCR. We observed that (i) Addition of propeptide decreased lipase folding stress by down-regulated four UPR-related genes. (ii) Signal peptide codons optimization had no effect on host with no change in the nine detected genes. (iii) Folding stress and limited transport capacity produced when rml gene dosage exceed 2. Different limiting factors on lipase expression in strains with different construction strategies were identified. This study provides a theoretical basis for further improving RML by transforming host.
Keywords: lipase; gene dosage; propeptide; protein synthesis and secretion pathway; signal peptide.