Abstract
Esters are important flavor and fragrance compounds that are present in many food and beverage products. Many of these esters are produced by yeasts and bacteria during fermentation. While ester production in yeasts through the alcohol acyl transferase reaction has been thoroughly investigated, ester production through alcoholysis has been completely neglected. Here, we further analyze the catalytic capacity of the yeast Eat1 enzyme and demonstrate that it also has alcoholysis and thiolysis activities. Eat1 can perform alcoholysis in an aqueous environment in vitro, accepting a wide range of alcohols (C2–C10) but only a small range of acyl donors (C2–C4). We show that alcoholysis occurs in vivo in several Crabtree negative yeast species but also in engineered Saccharomyces cerevisiae strains that overexpress Eat1 homologs. The alcoholysis activity of Eat1 was also used to upgrade ethyl esters to butyl esters in vivo by overexpressing Eat1 in Clostridium beijerinckii. Approximately 17 mM of butyl acetate and 0.3 mM of butyl butyrate could be produced following our approach. Remarkably, the in vitro alcoholysis activity is 445 times higher than the previously described alcohol acyl transferase activity. Thus, alcoholysis is likely to affect the ester generation, both quantitatively and qualitatively, in food and beverage production processes. Moreover, mastering the alcoholysis activity of Eat1 may give rise to the production of novel food and beverage products.
Highlights
The α/β-hydrolase fold superfamily of enzymes belongs to one of the largest groups of structurally related enzymes sharing a typical α/β-sheet with a conserved active site
Together with the ability of the enzyme to use esters and thioesters as substrates, we hypothesized that Eat1 should be capable of catalyzing alcoholysis
In this study we show that the α/β-hydrolase Eat1 can catalyze both alcoholysis and thiolysis reactions
Summary
The α/β-hydrolase fold superfamily of enzymes belongs to one of the largest groups of structurally related enzymes sharing a typical α/β-sheet with a conserved active site. It is hypothesized that the role of Eat is to regenerate the free CoA pool in the cell during iron or oxygen limitation through ethyl acetate production (Fredlund et al, 2004; Urit et al, 2012; Löser et al, 2014; Löser et al, 2015; Kruis et al, 2018) This hypothesis was strengthened by the localization of Eat in the mitochondria of K. lactis and K. marxianus and its upregulated expression in W. anomalus during iron-limited conditions (Kruis et al, 2017, 2018; Löbs et al, 2018)
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