Improved production of recombinant <i>Rhizomucor miehei</i> lipase by coexpressing protein folding chaperones in <i>Pichia pastoris</i>, which triggered ER stress

<i>Rhizomucor miehei</i> lipase (RML) is a biocatalyst that widely used in laboratory and industrial. Previously, RML with a 70-amino acid propeptide (pRML) was cloned and expressed in <i>P. pastori</i><i>s</i>. Recombinant strains with (strain containing 4-copy <i>prml</i>) and without ER stress (strain containing 2-copy <i>prml</i>) were obtained. However, the effective expression of pRML in <i>P. pastoris</i> by coexpressing ER-related elements in pRML-produced strain with or without ER stress has not been reported to date. In this study, an efficient way to produce functional pRML was explored in <i>P. pastoris</i>. The coexpression of protein folding chaperones, including <i>PDI</i> and <i>ERO1</i>, in different strains with or without ER stress, was investigated. <i>PDI</i> overexpression only increased pRML production in 4-copy strain from 705 U/mL to 1430 U/mL because it alleviated the protein folded stress, increased the protein concentration from 0.56 mg/mL to 0.65 mg/mL, and improved enzyme-specific activity from 1238 U/mg to 2186 U/mg. However, <i>PDI</i> coexpression could not improve pRML production in the 2-copy strain because it increased protein folded stress, while <i>ERO1</i> coexpression in the two strains all had a negative effect on pRML expression. We also investigated the effect of the propeptide on the substrate specificity and the condition for pRML enzyme powder preparation. Results showed that the relative activity exceeded 80% when the substrates C8–C10 were detected at 35°C and pH 6, and C8–C12 at 45°C and pH 8. The optimal enzyme powder preparation pH was 7, and the maximum recovery rate for pRML was 73.19%.