Elucidation and engineering mitochondrial respiratory-related genes for improving bioethanol production at high temperature in Saccharomyces cerevisiae

Abstract

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 Saccharomyces cerevisiae. 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 ADK1, DOC1, or MET7 were the key factors for the adaptive evolution of thermotolerance in the engineered yeast strain. Intriguingly, overexpression of DOC1 with TEF1 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.

Keywords

Saccharomyces cerevisiae / Adaptive evolution / Transcriptional sequencing / Mitochondrial respiratory / Thermotolerance

Data Availability Statement

The RNA sequencing raw data are deposited in the NCBI Gene Expression Omnibus (GEO) under the accession number GSE96829. The S. cerevisiae S288c genome as a reference can be accessed at https://www.ncbi.nlm.nih.gov/assembly/GCF_000146045.2/.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Xianni Qi: Investigation, Methodology, Writing - original draft. Zhen Wang: Investigation, Methodology, Writing - original draft, Writing - review & editing, Funding acquisition. Yuping Lin: Supervision, Data curation, Writing - original draft. Yufeng Guo: Methodology, Visualization, Formal analysis. Zongjie Dai: Supervision. Qinhong Wang: Supervision, Funding acquisition, Conceptualization, Writing - review & editing.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2021YFC2103300), Research Equipment Program of Chinese Academy of Sciences (YJKYYQ20170023), National Natural Science Foundation of China (32071423, 31470214, and 32200067), Natural Science Foundation of Hebei Province (C2020204013), and Development Program Projects of Hebei Province (22322905D). Qinhong Wang and Yuping Lin were supported by Tianjin Industrial Synthetic Biology Innovation Team.

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