Enhanced secretion of thermostable phytases from Myceliophthora thermophila by Komagataella phaffii.

Phytases catalyze the hydrolysis of phytic acid to release inorganic phosphate, thereby improving phosphorus bioavailability. Recombinant phytases hold great potential as additives in the feed, food, pharmaceutical, and chemical industries. However, their enzymatic stability and activity can be compromised during industrial processing, particularly during high-temperature treatments such as tableting. In this study, three putative phytase genes (designated MtPhyA1, MtPhyA2, and MtPhyA3) were identified from the thermotolerant fungus Myceliophthora thermophila through in silico sequence analysis. The genes were codon-optimized, cloned, and heterologously expressed in the methylotrophic yeast Komagataella phaffii. To enhance secretion efficiency, the native signal sequences were truncated and exchanged for the truncated S. cerevisiae α-mating factor signal peptide (Δ57–70). Among the three variants, the strain expressing MtPhyA2 exhibited the highest production and enzymatic activity, with an optimal pH of 6.0 and temperature of 65 °C and specific activities reaching 147.7 ± 1.56 U mg− 1 at 37 °C, and 210.5 ± 4.23 U mg− 1 at 65 °C. To further enhance secretion, the pre-signal sequence of the α-mating factor signal peptide was replaced with alternative pre-sequences. The K. phaffii Ost1, Kluyveromyces lactis Ost1, and K. phaffii Gcw28 pre-sequences, when combined with the truncated S. cerevisiae α-factor pro-sequence (Δ57–70), improved phytase secretion by up to 60%. Upscaling in Eppendorf Minifors bioreactors further led to phytase production of 1.6 g L− 1, demonstrating a promising strategy for the efficient and economical production of thermostable phytases.