Elucidation and Engineering Mitochondrial Respiratory-related Genes for Improving Bioethanol Production at High Temperature in

Industrial manufacturing of bioproducts, especially bioethanol, can benefit from high-temperature fermentation, which requires the use of thermotolerant yeast strains. Mitochondrial activity in yeast is closely related to its overall metabolism. However, the mitochondrial respiratory changes in response to adaptive thermotolerance are still poorly understood and have been rarely utilized for developing thermotolerant yeast cell factories. Here, adaptive evolution and transcriptional sequencing, as well as whole-genome-level gene knockout, were used to obtain a thermotolerant strain of . Furthermore, thermotolerance and bioethanol production efficiency of the engineered strain were examined. Physiological evaluation showed the boosted fermentation capacity and suppressed mitochondrial respiratory activity in the thermotolerant strain. The improved fermentation produced an increased supply of adenosine triphosphate required for more active energy-consuming pathways. Transcriptome analysis revealed significant changes in the expression of the genes involved in the mitochondrial respiratory chain. Evaluation of mitochondria-associated gene knockout confirmed that or were the key factors for the adaptive evolution of thermotolerance in the engineered yeast strain. Intriguingly, overexpression of with promoter regulation led to a 10.1% increase in ethanol production at 42 °C. The relationships between thermotolerance, mitochondrial activity, and respiration were explored, and a thermotolerant yeast strain was developed by altering the expression of mitochondrial respiration-related genes. This study provides a better understanding on the physiological mechanism of adaptive evolution of thermotolerance in yeast.