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.