School of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong, 510640, People's Republic of China.
College of Life Sciences, Anhui Normal University, Wuhu, Anhui, 241000, People's Republic of China.
Appl Biochem Biotechnol. 2022 Dec;194(12):6179-6193. doi: 10.1007/s12010-022-04022-3. Epub 2022 Jul 28.
Methanol can be used by Pichia pastoris as the carbon source and inducer to produce recombinant proteins in high-cell-density fermentations. However, methanol oxidation at high specific growth rates can lead to the reactive oxygen species (ROS) accumulation, resulting in cell damage. Here, we study the relationship between methanol feeding and ROS accumulation by controlling specific growth rate during the induction phase. A higher specific growth rate increased the level of ROS accumulation caused by methanol oxidation. While the cell growth rate was proportional to specific growth rate, maximum total protein production and highest enzyme activity were achieved at a specific growth rate of 0.05 1/h as compared to that of 0.065 1/h. Moreover, oxidative damage induced by over-accumulation of ROS in P. pastoris during the methanol induction phase caused cell death and reduced protein expression ability. ROS scavenging system analysis revealed that the higher specific growth rate, especially 0.065 1/h, resulted in increased intracellular catalase activity and decreased glutathione content significantly. Finally, Spearman's correlation analysis further revealed that the reduced glutathione might be beneficial for maintaining cell viability and increasing protein production under oxidative stress caused by ROS toxic accumulation. Our findings suggest an integrated strategy to control the feeding of the essential substrate based on analyzing its response to oxidative stress caused by ROS toxic accumulation, as well as develop a strategy to optimize fed-batch fermentation.
甲醇可以被巴斯德毕赤酵母用作碳源和诱导物，在高密度发酵中生产重组蛋白。然而，在高比生长速率下甲醇的氧化会导致活性氧（ROS）的积累，从而导致细胞损伤。在这里，我们通过在诱导阶段控制比生长速率来研究甲醇进料与 ROS 积累之间的关系。较高的比生长速率会增加甲醇氧化引起的 ROS 积累水平。虽然细胞生长速率与比生长速率成正比，但与 0.065 1/h 相比，在 0.051/h 的比生长速率下可实现最大总蛋白产量和最高酶活。此外，在甲醇诱导阶段，由于 ROS 的过度积累导致的氧化损伤会引起细胞死亡并降低蛋白质表达能力。ROS 清除系统分析表明，较高的比生长速率，特别是 0.065 1/h，会导致细胞内过氧化氢酶活性显著增加，谷胱甘肽含量显著降低。最后，Spearman 相关分析进一步表明，在 ROS 毒性积累引起的氧化应激下，还原型谷胱甘肽可能有利于维持细胞活力和提高蛋白质产量。我们的研究结果表明，基于分析其对由 ROS 毒性积累引起的氧化应激的反应，控制必需底物进料的综合策略，以及开发优化补料分批发酵的策略。
