Yeast Yeast 2004; 21: 303–312. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1076 Research Article Expression of a plant serine O-acetyltransferase in Saccharomyces cerevisiae confers osmotic tolerance and creates an alternative pathway for cysteine biosynthesis Jose M. Mulet 1 , Beatriz Alemany 1 , Roc Ros 2 , Juan J. Calvete 3 and Ramon Serrano 1 * 1 Instituto de Biolog´ ıa Molecular y Celular de Plantas, Universidad Polit´ ecnica de Valencia-CSIC, Avda de los Naranjos S/N, 46022 Valencia, Spain 2 Departament de Biologia Vegetal, Facultat de Ci` encies Biol´ ogiques, Universitat de Valencia, 46100 Burjassot (Valencia), Spain 3 Instituto de Biomedicina de Valencia-CSIC, C/Jaime Roig 11, 46010 Valencia, Spain *Correspondence to: Ramon Serrano, Instituto de Biolog´ ıa Molecular y Celular de Plantas, Universidad Polit´ ecnica de Valencia-CSIC, Avda de los Naranjos S/N, 46022 Valencia, Spain. E-mail: rserrano@ibmcp.upv.es Received: 1 September 2003 Accepted: 10 November 2003 Abstract Screening of a sugar beet (Beta vulgaris cv. Dita) cDNA library for clones able to confer osmotic tolerance to the osmosensitive gpd1 mutant of Saccharomyces cerevisiae identified a novel serine O-acetyltransferase (BvSAT; EC 2.3.1.30). This enzyme is involved in cysteine biosynthesis in plants and bacteria, producing O- acetylserine, which is converted into cysteine in a reaction catalysed by O-acetylserine sulphydrylase (EC 4.2.99.8). This pathway is not conserved in yeast, where cys- teine is synthesized in a four-step pathway starting with homoserine and having O-acetylhomoserine, homocysteine and cystathionine as intermediates. Expression of BvSAT in yeast takes advantage of the activity of yeast O-acetylhomoserine sul- phydrylase (MET15/MET17/MET25 ; EC 4.2.99.10) with O-acetylserine as substrate and induces an alternative cysteine biosynthesis pathway. Our results imply that the resulting increase in cysteine production confers enhanced resistance against osmotic stress in the osmosensitive yeast strain. These data demonstrate that cysteine biosyn- thesis is a limiting factor in osmotic stress tolerance in yeast. Copyright  2004 John Wiley & Sons, Ltd. Keywords: Saccharomyces cerevisiae : osmotic stress; cysteine biosynthesis; heterol- ogous expression Introduction Under natural conditions Saccharomyces cerevisiae cells must withstand severe osmotic changes, from rain water to the high sugar concentration found in grape juice. To cope such an osmotically unsta- ble environment, S. cerevisiae has developed a complex regulatory stress response pathway. One aspect of the yeast response to osmotic stress is the synthesis and accumulation of the osmolite glycerol (Serrano, 1996; Hohmann, 2002). In yeast glycerol is produced by the glycerol phosphate dehydro- genase pathway, thus a yeast strain deficient in the major isoform (GPD1 ) is osmosensitive and is unable to perform a proper osmotic adjustment (Larsson et al., 1993; Albertyn et al., 1994; Ansell et al., 1997). Therefore this strain constitutes a good genetic background to screen for plant genes involved in the osmotic stress response. The yeast overexpression approach was devel- oped in our laboratory as an alternative to the in planta approaches to identify plant genes (Kan- honou et al., 2001; Forment et al., 2002; Rausell et al., 2003). The assumption is made that, at the cellular level, the mechanisms of stress tol- erance are similar in yeast and plants and there- fore that plant stress genes can be identified by random overexpression in yeast and functional Copyright  2004 John Wiley & Sons, Ltd.