Abstract
BackgroundA key pathway for ester biosynthesis in yeast is the condensation of an alcohol with acetyl-CoA by alcohol-O-acetyltransferase (AATase). This pathway is also prevalent in fruit, producing short and medium chain volatile esters during ripening. In this work, a series of six AATases from Saccharomyces and non-Saccharomyces yeasts as well as tomato fruit were evaluated with respect to their activity, intracellular localization, and expression in Saccharomyces cerevisiae and Escherichia coli cell hosts. The series of AATases includes Atf1 and Atf2 from S. cerevisiae, as well as AATases from S. pastorianus, Kluyveromyces lactis, Pichia anomala, and Solanum lycopersicum (tomato).ResultsWhen expressed in S. cerevisiae, Atf1, Atf2, and an AATase from S. pastorianus localized to lipid droplets, while AATases from non-Saccharomyces yeasts and tomato fruit did not localize to intracellular membranes and were localized to the cytoplasm. All AATases studied here formed intracellular aggregates when expressed in E. coli, and western blot analysis revealed that expression levels in E. coli were upwards of 100-fold higher than in S. cerevisiae. Fermentation and whole cell lysate activity assays of the two most active AATases, Atf1 from S. cerevisiae and an AATase from tomato fruit, demonstrated that the aggregates were enzymatically active, but with highly reduced specific activity in comparison to activity in S. cerevisiae. Activity was partially recovered at lower expression levels, coinciding with smaller intracellular aggregates. In vivo and in vitro activity assays from heterologously expressed Atf1 from S. cerevisiae, which localizes to lipid droplets under homologous expression, demonstrates that its activity is not membrane dependent.ConclusionsThe results of these studies provide important information on the biochemistry of AATases under homologous and heterologous expression with two common microbial hosts for biochemical processes, S. cerevisiae and E. coli. All studied AATases formed aggregates with low enzymatic activity when expressed in E. coli and any membrane localization observed in S. cerevisiae was lost in E. coli. In addition, AATases that were found to localize to lipid droplet membranes in S. cerevisiae were found to not be membrane dependent with respect to activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0221-9) contains supplementary material, which is available to authorized users.
Highlights
A key pathway for ester biosynthesis in yeast is the condensation of an alcohol with acetyl-CoA by alcohol-O-acetyltransferase (AATase)
We have previously shown that Atf1 and −2 from S. cerevisiae localize to lipid droplets (LDs) via N- and C-terminal amphipathic helices [19]
Preliminary activity screening from S. cerevisiae whole cell lysates with overexpressed AATases revealed that Atf1-S.c has the highest activity towards C2 to C5 alcohols with acetyl-CoA (Additional file 1: Table S1)
Summary
A key pathway for ester biosynthesis in yeast is the condensation of an alcohol with acetyl-CoA by alcohol-O-acetyltransferase (AATase) This pathway is prevalent in fruit, producing short and medium chain volatile esters during ripening. During yeast fermentation and fruit ripening short chain linear and branched alcohols are converted to their corresponding acetate esters by alcohol-O-acetyltransferase (AATase; EC 2.3.1.84; Figure 1). These volatile esters produce sweet and fruity fragrances: phenyl ethyl acetate smells of flowers, isoamyl acetate (isopentyl acetate). The hydrophobic nature of these enzymes and varied intracellular localization of orthologs in their native hosts present complicating factors for heterologous expression in engineered hosts [8,18,19]
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