Abstract
Current large-scale, anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Responses of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for anaerobic industrial applications.
Highlights
In terms of product volume (87 Mton y− 1) (“Annual World Fuel Ethanol Production, 2020; Jansen et al, 2017), anaerobic conversion of carbohydrates into ethanol by the yeast Saccharomyces cerevisiae is the single largest process in industrial biotechnology
The chemostats were operated at a D of 0.10 h− 1, which is used as a reference value in many yeast physiology studies (Tai et al, 2005; Van Eunen et al, 2010)
Identifying and eliminating oxygen re quirements of these yeasts is essential to unlock their industrially rele vant traits for application. This challenge was addressed for the thermotolerant yeast K. marxianus, using a systematic approach based on chemostat-based quantitative physiology, genome and transcriptome analysis, sterol-uptake assays and genetic modification
Summary
In terms of product volume (87 Mton y− 1) (“Annual World Fuel Ethanol Production, 2020; Jansen et al, 2017), anaerobic conversion of carbohydrates into ethanol by the yeast Saccharomyces cerevisiae is the single largest process in industrial biotechnology. The high maximum growth temperature of thermotolerant yeasts, such as Kluyveromyces marxianus (up to 50 ◦C as opposed to 39 ◦C for S. cerevisiae), could enable lower cooling costs (Choudhary et al, 2016; Hong et al, 2007; Laman Trip and Youk, 2020) It could reduce the required dosage of fungal polysaccharide hydrolases during simultaneous saccharification and fermentation (SSF) processes (Mejía-Barajas et al, 2018; Thorwall et al, 2020). As yet unidentified oxygen requirements hamper implementation of Kluyveromyces species in large-scale anaerobic pro cesses (Kiers et al, 1998; Merico et al, 2007; Snoek and Steensma, 2006; Visser et al, 1990)
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