Enhanced Production of Recombinant Thermophilic Xylanase X11P in Ogataea polymorpha via In‐Silico Signal Peptide Discovery and Fed‐Batch Fermentation

Efficient secretion of heterologous proteins is essential for advancing yeast-based bioprocesses, yet signal peptide (SP) optimization in the thermotolerant methylotrophic yeast Ogataea polymorpha remains limited. This study integrates in-silico SP discovery, experimental validation, and bioprocess engineering to enhance secretion of the thermophilic xylanase X11P under sucrose-inducible expression. Genome-wide screening of 5184 O. polymorpha proteins using SignalP, Phobius, DeepLoc, WoLF PSORT, and ProP identified 11 high-confidence SP candidates. Comparative analysis with Komagataella phaffii endogenous proteins guided selection of seven SPs for experimental evaluation. Among these, the novel O. polymorpha α-mating factor-like peptide FUN_005010 exhibited strong secretion-promoting activity, with its prepro-sequence yielding the highest extracellular xylanase levels and outperforming the classical Saccharomyces cerevisiae α-MF. To evaluate industrial applicability, sucrose-based fermentation strategies were systematically optimized in a 5-L bioreactor. Controlled sucrose feeding and balanced C/N ratios were found to be critical for maximizing maltase (MAL) promoter-driven expression. A stepwise increasing sucrose feed combined with induction at 30°C enabled X11P titers up to 770 U/mL, representing a 15-fold improvement over shake-flask cultures. This work demonstrates that the combination of SP evaluation and optimized sucrose-inducible fed-batch operation significantly enhances X11P production in O. polymorpha. The identified FUN_005010-prepro SP and the refined process framework provide valuable tools for developing O. polymorpha as a high-performance industrial expression platform.