High-Level Constitutive Secretory Expression and Low-Temperature Adaptation Engineering of the Acid Protease RMAP9 from the Mangrove-Derived Yeast Rhodotorula mucilaginosa

Acid proteases can improve nitrogen availability during ethanol fermentation by hydrolysing feedstock proteins, but their catalytic performance at relatively low temperatures may be limited. In this study, an acid protease-producing Rhodotorula mucilaginosa strain, TSM-6, was isolated from mangrove sediment, and an acid protease gene, rmap9, was identified by whole-genome sequencing and candidate-gene screening. RMAP9 was constitutively expressed in Komagataella phaffii GS115, and secretion was enhanced through EXG1-derived secretion-signal engineering, tandem assembly of three expression cassettes, and fermentation optimisation. Molecular dynamics simulations and Rosetta-based ΔΔG scoring were further used to identify candidate residues for temperature-adaptation engineering, followed by the site-saturation mutagenesis of Ser125. The optimised recombinant strain reached a maximum extracellular acid protease activity of 312.07 ± 18.37 U/mL, representing a 223% increase over the parental constitutive strain. The S125K variant showed a shift in optimum temperature from 50 to 40 °C and approximately 50% higher relative activity at 40 °C than wild-type RMAP9. Moreover, the low-temperature-favoured variants retained more than 80% residual activity after 72 h at 30 °C. These variants combined improved catalytic performance at relatively low temperatures with long-term stability at 30 °C, thereby providing enzyme properties compatible with temperature-related operational requirements for industrial ethanol fermentation.