High-Level Production of a Rhizomucor miehei Lipase Variant in Komagataella phaffii via Gene Dosage Optimization and Ribosomal Engineering for Biodiesel Synthesis

Abstract Rhizomucor miehei lipase (pRML) is an efficient biocatalyst for biodiesel synthesis, but its production remains insufficient for industrial use. In this study, expression of a previously reported propeptide mutant (S7E/P34S/P46A-pRML) in a five-copy Komagataella phaffii strain yielded 7,650 U/mL, and deleting the ribosomal protein gene rps25 further increased it to 8,600 U/mL. Three protein forms were observed in the supernatant: glycosylated S7E/P34S/P46A-pRML-gly, nonglycosylated S7E/P34S/P46A-pRML-nogly, and propeptide-free form mRML. S7E/P34S/P46A-pRML-gly showed the lowest kcat/Km, indicating that glycosylation reduces catalytic efficiency. Molecular dynamics simulations showed stronger hydrogen bonds in its propeptide (K52–G54) and the C-terminal lid loop (I159–F164), restricting lid opening. SDS-PAGE suggested a more prominent presence of high catalytic efficiency forms after rps25 deletion, likely contributing to higher extracellular activity. The high-yield lipase maintained efficiency, converting 92.9% of Nannochloropsis gaditana oil to biodiesel within 9 h. These findings offer new insights into optimizing protein expression in Komagataella phaffii.