Improved ethanol tolerance and production of Saccharomyces cerevisiae by global transcription machinery engineering via directed evolution of the SPT8 gene

ABSTRACT

Ethanol tolerance involves complex interactions of several genes in the Saccharomyces cerevisiae genome that encodes proteins that are the subunits of the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex, particularly those that interact with the TATA-binding protein. Global transcription machinery engineering has been used to reprogram the transcription levels of multiple genes to change cellular phenotypes important for technological applications by altering key proteins by error-prone polymerase chain reaction (epPCR) mutations. In this study, we produced S. cerevisiae mutants from the wild-type wine yeast Y01 strain by directed evolution of the SPT8 (suppressor of ty insertions 8) gene using epPCR combined with construction and overexpression of the constructed pUPST mutant library for improved ethanol tolerance and production. We found that the mutant strains gave 8.9% higher ethanol tolerance and 10.8% higher ethanol production than the wild-type. The desired phenotype resulted from the combined effect of two separate mutations in SPT8, which caused serine to replace phenylalanine (Asn¹⁵⁶His) and glycine to replace serine (Gly⁵⁸⁵Ser) in the encoded SPT8 protein.

KEYWORDS:

• Error-prone PCR
• ethanol tolerance
• gTME
• overexpression
• Saccharomyces cerevisiae
• SPT8

Acknowledgments

We thank Margaret Biswas, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. This study was funded by the Natural Science Foundation of Fujian Province (Grant No. 2017J01622), the Sugar Crop Research System (Grant No. CARS-170501), and the Education Department Project of Fujian Province (Grant No. JAT160114).

Conflict of Interest

This article is an original work and no portion of the study has been published or is under consideration for publication elsewhere. None of the authors has any potential conflict of interest related to this manuscript. All authors have contributed to the work, and they have agreed to submit the manuscript.

Additional information

Funding

This work was supported by the Natural Science Foundation of Fujian Province [2017J01622];Education Department Project of Fujian Province [JAT160114];Sugar Crop Research System [CARS-170501].