Robust Pichia kudriavzevii for open production of methylotrophic protein.

Methanol has emerged as a promising substrate in biomanufacturing, where high tolerance and efficient assimilation are critical to overcome the cytotoxic effects of methanol and its intermediate metabolites. In this study, a Pichia kudriavzevii P22 strain (genome size: 17.3 Mb) was isolated from fermented camel milk. Remarkably, this strain tolerated up to 15% (v/v) methanol and survived under multiple stress conditions, including pH 2, 47 °C, 5 g/L acetic acid, and 100 g/L NaCl. In a 5-L fed-batch fermentation system operated under open, non-sterile conditions with 7.5% (v/v) methanol as the sole carbon source, P22 remained viable for 78 h. The maximum cell dry weight (CDW) reached 27.56 g/L, with proteins accounting for 54%. Furthermore, a preliminary investigation of the methanol metabolic pathway in P. kudriavzevii P22 was performed. Collectively, this work identifies P. kudriavzevii P22 as a potential eukaryotic chassis for developing methylotrophic yeast cell factories. Importantly, it demonstrates an open, unsterilized methanol-based fermentation process at 5 L scale, offering a potential strategy for scale-up fermentation with simplified operation and reduced cost. KEY POINTS: • A P. kudriavzevii P22 strain capable of tolerating 15% (v/v) methanol was isolated from fermented camel milk. • In an open, non-sterile 5-L fermentation system with methanol as the sole carbon source, P. kudriavzevii P22 achieved 27.56 g/L CDW with 54% protein content.