Unveiling the thermotolerance mechanism of Pichia kudriavzevii LC375240 through transcriptomic and genetic analyses.

Abstract

Background: Thermotolerance is a critical trait for yeasts employed in industrial settings, and the utilization of unconventional yeasts has gained notable attention in recent years. However, the mechanisms underlying thermotolerance in unconventional yeasts, particularly Pichia spp., remain insufficiently elucidated.

Results: This study focuses on the thermotolerance of a non-traditional yeast strain Pichia kudriavzevii LC375240, renowned for its remarkable thermotolerance. Through transcriptomic analysis of both short-term and long-term heat shock exposures, we uncovered an intricate regulatory response in P. kudriavzevii. During long-term heat treatment, the yeast exhibited elevated expression of genes involved in the tricarboxylic acid (TCA) cycle and suppressed expression of genes in the pentose phosphate pathway (PPP). Additionally, long-term heat treatment led to an upregulation of heat shock proteins (HSPs) and an increase in trehalose, glutathione (GSH), and superoxide dismutase (SOD) levels, along with a reduction in the intracellular NADPH/NADP⁺ ratio and pyruvate content. These changes collectively contribute to the thermotolerance of P. kudriavzevii. CRISPR-Cas9-mediated knockout experiments further highlighted the critical roles of HSPs, antioxidases, and the trehalose metabolic pathway in the yeast's response to high temperatures.

Conclusions: Taken together, this study demonstrates that P. kudriavzevii adapts to thermal stress through a combination of enhanced TCA cycle, reduced PPP, increased HSPs, trehalose, GSH, and SOD levels. These findings provide a comprehensive understanding of the molecular mechanisms underlying thermotolerance in P. kudriavzevii.