Abstract
Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H+ symporter, with Km 0.45±0.07 mM and Vmax 0.57±0.02 mmol h−1 (gdw) −1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.
Highlights
Zygosaccharomyces rouxii and Z. bailii are food spoilage yeasts that can grow under harsh conditions that are restrictive for most yeast species due to their extreme osmotolerance and resistance to weakacid preservatives [1]
Symporter In our previous work, we characterized the first two low-affinity fructose transporters from the fructophilic yeast Z. rouxii CBS 732: the fructose-specific facilitator ZrFfz1, which is the major system responsible for the high fructose transport capacity of Z. rouxii; and ZrFfz2, which facilitates the uptake of both fructose and glucose [14]. When both the ZrFFZ1 and ZrFFZ2 genes in Z. rouxii are deleted, the obtained strain still grows on glucose and fructose media meaning that other functional hexose transporters are present in this yeast
We performed a BLASTP search against known sugar transporters belonging to the Sugar Porter family and, besides the four putative hexose transporters similar to S. cerevisiae Hxts and the one similar to the hexose sensor ScSnf3, which was reported by Palma et al [29], we found one putative transporter similar to the Kluyveromyces lactis glucose/fructose/galactose transporter Hgt1 and another similar to the S. pastorianus fructose/H+ symporter Fsy1
Summary
Zygosaccharomyces rouxii and Z. bailii are food spoilage yeasts that can grow under harsh conditions that are restrictive for most yeast species due to their extreme osmotolerance and resistance to weakacid preservatives [1]. Z. bailii and Z. rouxii strains were isolated from high-sugar environments, 72 and 90% (w/v) glucose, respectively. These yeasts are fructophilic, i.e. they consume fructose faster than glucose, whereas the main fermentative yeast Saccharomyces cerevisiae exhibits glucophilic behavior [2]. The fructophily of these Zygosaccharomyces is based on the kinetics and substrate specificity of their sugar transporters, mainly those mediating the uptake of sugars via facilitated diffusion (facilitators).
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