Squalene-Tetrahymanol Cyclase Expression Enables Sterol-Independent Growth of Saccharomyces cerevisiae

Abstract

Biosynthesis of sterols, which are considered essential components of virtually all eukaryotic membranes, requires molecular oxygen. Anaerobic growth of the yeast Saccharomyces cerevisiae therefore strictly depends on sterol supplementation of synthetic growth media. Neocallimastigomycota are a group of strictly anaerobic fungi which, instead of sterols, contain the pentacyclic triterpenoid ‘sterol surrogate’ tetrahymanol, which is formed by cyclization of squalene. Here, we demonstrate that expression of the squalene-tetrahymanol cyclase gene TtTHC1 from the ciliate Tetrahymena thermophila enables synthesis of tetrahymanol by S. cerevisiae. Moreover, expression of TtTHC1 enabled exponential growth of anaerobic S. cerevisiae cultures in sterol-free synthetic media. After deleting the ERG1 gene from a TtTHC1-expressing S. cerevisiae strain, native sterol synthesis was abolished and sustained sterol-free growth was demonstrated under anaerobic as well as aerobic conditions. Anaerobic cultures of TtTHC1-expressing S. cerevisiae on sterol-free medium showed lower specific growth rates and biomass yields than ergosterol-supplemented cultures, while their ethanol yield was higher. This study demonstrates that acquisition of a functional squalene-tetrahymanol cyclase gene offers an immediate growth advantage to S. cerevisiae under anaerobic, sterol-limited conditions and provides the basis for a metabolic engineering strategy to eliminate the oxygen requirements associated with sterol synthesis in yeasts. Importance The laboratory experiments described in this study simulate a proposed horizontal gene transfer event during the evolution of strictly anaerobic fungi. The demonstration that expression of a single heterologous gene sufficed to eliminate anaerobic sterol requirements in the model eukaryote Saccharomyces cerevisiae therefore contributes to our understanding of how sterol-independent eukaryotes evolved in anoxic environments. This study provides a proof-of-principle for a metabolic engineering strategy to eliminate sterol requirements in yeast strains that are applied in large-scale, anaerobic industrial processes. The sterol-independent yeast strains described in this study provide a valuable platform for further studies on the physiological roles and impacts of sterols and sterol surrogates in eukaryotic cells.

Engineering the thermotolerant industrial yeastKluyveromyces marxianusfor anaerobic growth

“…However, specific growth rates and biomass yields of tetrahymanol-expressing K. marxianus in anaerobic cultures were lower than those of wild-type S. cerevisiae strains. A similar phenotype of tetrahymanol-producing S. cerevisiae was proposed to reflect an increased membrane permeability 46 . Additional membrane engineering or expression of a functional sterol transport system is therefore required for further development of robust, anaerobically growing industrial strains of K. marxianus 56 .…”

“…Expression of a squalene-tetrahymanol cyclase from Tetrahymena thermophila ( TtSTC1 ), which catalyzes the single-step oxygen-independent conversion of squalene into tetrahymanol (Fig. 5a), was recently shown to enable sterol-independent growth of S. cerevisiae 46 .…”

“…The oven was programmed to start at 80 °C for 1 min, ramp first to 280 °C with 60 °C•min -1 and secondly to 320 °C with a rate of 10 °C•min -1 with a final temperature hold of 15 min. Spectra were compared to separate calibration lines of squalene, ergosterol, α-cholestane, cholesterol and tetrahymanol as described previously 46 .…”

A squalene-hopene cyclase inSchizosaccharomyces japonicusrepresents a eukaryotic adaptation to sterol-independent anaerobic growth

“…In anaerobic laboratory cultures of yeasts, biosynthetic oxygen requirements are easily obscured by unintended entry of minute amounts of oxygen 19,20,37,38 . Furthermore, upon transfer from aerobic to anaerobic conditions, some yeasts continue growing on media without sterols or UFAs by mobilizing intracellular reserves 20,37 . To check if such complications affected conclusions from an early literature report on sterol- and UFA-independent anaerobic growth of Sch.…”

“…As opposed to Neocallimastigomycetes, no evolutionary adaptations to sterol-independent anaerobic growth have hitherto been reported for yeasts, or for ascomycete and basidiomycete fungi in general. We recently demonstrated that expression of an STC gene from the ciliate Tetrahymena thermophila supported tetrahymanol synthesis and sterol-independent growth of S. cerevisiae 20 . This result inspired us to re-examine a 1971 publication in which Bulder 21 reported sterol- and UFA-independent growth of the dimorphic fission yeast Schizosaccharomyces japonicus .…”