Yarrowia lipolytica, Komagataella phaffii and secretory proteins: Recombinant laccase as a case study

For decades, Komagataella phaffii has been the reference host for recombinant secretory protein (rsProt) production. However, secretion bottlenecks associated with the limited processing capacity of the endoplasmic reticulum restrict the secretion efficiency under high protein loads. Besides, Yarrowia lipolytica possesses a secretion pathway resembling that of filamentous fungi and naturally secretes large amounts of hydrolytic enzymes, making it a promising alternative host. In this study, we systematically investigated the impact of pre- and pro-sequences on the secretion of LacVader, an evolved laccase with industrial applications. In Y. lipolytica, nine constructs combining four pre-sequences (αPre, Yps3Pre, Lip2Pre, SoAmyPre) and two pro-sequences (αPro, Lip2Pro) were integrated at the LIP2 locus and expressed under the constitutive P_TEF promoter. Comparative analysis revealed that most pre-pro constructs resulted in higher laccase specific activity compared to K. phaffii expressing the enzyme under the canonical P_AOX1 promoter and the S. cerevisiae α-factor signal peptide. Notably, both the pre- and pro-sequences had a strong influence on laccase secretion in Y. lipolytica. The Lip2Pro sequence consistently enhanced secretion, with the Yps3Pre-Lip2Pro construct yielding the highest activity, eightfold greater than that obtained in K. phaffii. These findings highlight the crucial role of secretion signal optimization in rsProt production and confirm the superior potential of Y. lipolytica over K. phaffii as a robust host for industrial enzyme secretion.