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Abstract <p>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 <i>Pichia kudriavzevii</i> P22 strain (genome size: 17.3&#xa0;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&#xa0;°C, 5&#xa0;g/L acetic acid, and 100&#xa0;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&#xa0;h<i>.</i> The maximum cell dry weight (CDW) reached 27.56&#xa0;g/L, with proteins accounting for 54%. Furthermore, a preliminary investigation of the methanol metabolic pathway in <i>P. kudriavzevii</i> P22 was performed. Collectively, this work identifies <i>P. kudriavzevii</i> 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.</p> Key points <p>•&#xa0;<i>A P. kudriavzevii P22 strain capable of tolerating 15% (v/v) methanol was isolated from fermented camel milk.</i></p> <p>•&#xa0;<i>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.</i></p>

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Robust Pichia kudriavzevii for open production of methylotrophic protein

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月24日

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  • Wenyu Gao,
  • Ruiqi Wang,
  • Jing Xu,
  • Meiling Zhang,
  • Guo Yin,
  • Yuxian Wang,
  • Haifeng Chen,
  • Zheyi Kuang,
  • Zhaoxu Xu,
  • Haitao Yue

摘要

Abstract

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.