Transcriptional Downregulation of Methanol Metabolism Key Genes During Yeast Death in Engineered Pichia pastoris

ABSTRACT Pichia pastoris possesses the unique ability to utilize methanol as its sole carbon source, which makes it a proper host for producing various high‐value‐added products via metabolic engineering. Nevertheless, cell death has been observed during the fermentation of modified P. pastoris , with limited literature elucidating the underlying causes and mechanisms. Understanding the death mechanisms during methanol‐based fermentation is crucial for optimizing fermentation strategies, enhancing the accumulation of target products, and reducing production costs. Here, we first sought to eliminate the potential causes of cell death during fermentation, such as inadequate inorganic salts and toxic by‐product accumulation. The elimination of these potential causes was achieved efficiently utilizing the high‐throughput fermentation equipment. Subsequently, we established a correlation between yeast cell death and the duration of the methanol metabolism period by monitoring the growth of the yeast at different fermentation stages. A critical revelation from this work came from analyzing the yeast's transcriptomic data at various stages of methanol metabolism. It was observed that a significant characteristic of yeast cell death during fermentation was the marked down‐regulation of transcript levels of key enzymes involved in the methanol assimilation pathway and genes related to their biosynthesis process. The findings of this work are crucial for better understanding the causes and mechanisms of cell death for engineered P. pastoris during methanol‐utilized fermentation.