PT  - JOURNAL ARTICLE
AU  - Jonna Bouwknegt
AU  - Sanne J. Wiersma
AU  - Raúl A. Ortiz-Merino
AU  - Eline S. R. Doornenbal
AU  - Petrik Buitenhuis
AU  - Martin Giera
AU  - Christoph Müller
AU  - Jack T. Pronk
TI  - A squalene-hopene cyclase in <em>Schizosaccharomyces japonicus</em> represents a eukaryotic adaptation to sterol-independent anaerobic growth
AID  - 10.1101/2021.03.17.435848
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.03.17.435848
4099  - http://biorxiv.org/content/early/2021/03/17/2021.03.17.435848.short
4100  - http://biorxiv.org/content/early/2021/03/17/2021.03.17.435848.full
AB  - Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report (Bulder (1971), Antonie Van Leeuwenhoek, 37, 353–358) that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop-17(21)-ene, hop-21(22)-ene and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and supported ergosterol-independent anaerobic growth, thus confirming that one or more of the hopanoids produced by SjShc1 can act as ergosterol surrogate in anaerobic yeast cultures. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast sterol-independent anaerobic growth of Sch. japonicus is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.Significance statement Biosynthesis of sterols requires oxygen. This study identifies a previously unknown evolutionary adaptation in a eukaryote, which enables anaerobic growth in absence of exogenous sterols. A squalene-hopene cyclase, proposed to have been acquired by horizontal gene transfer from an acetic acid bacterium, is implicated in a unique ability of the yeast Schizosaccharomyces japonicus to synthesize hopanoids and grow in anaerobic, sterol-free media. Expression of this cyclase in S. cerevisiae confirmed that at least one of its hopanoid products acts as sterol-surrogate. The involvement of hopanoids in sterol-independent growth of this yeast provides new leads for research into the structure and function of eukaryotic membranes, and into the development of sterol-independent yeast cell factories for application in anaerobic processes.Competing Interest StatementJB, SJW and JTP are co-inventors on a patent application that covers parts of this work