(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Eukaryota: NE > Opisthokonta: NE > Fungi: NE > Dikarya: NE > Ascomycota: NE > saccharomyceta: NE > Saccharomycotina: NE > Saccharomycetes: NE > Saccharomycetales: NE > Phaffomycetaceae: NE > Wickerhamomyces: NE > Wickerhamomyces anomalus: NE > Wickerhamomyces anomalus NRRL Y-366-8: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MFFTKVLNNQVANGLKQLPVHKRVQMAYDLHIPNKTVNPNLNIRSHEPIV FVHGIFGSKKNYRHDCQKIANVTHTPVYTIDLRNHGQSMHALPFDYETLA QDVTDFCEDHGLKKVNLIGYSLGAKICMLTMLQNPDLVRSGVIIDNSPIE QPHIEIFLTQFIKSMLHVLNSTKIRADDKDWKSKANQAMRRYIPNGGIRD YLLANLINKVPKGYKSPVINYDDGYIHFQNPVRHMTEVAVKNVSAWPTEH VKGLKFEGQVRFLKGTKSAFIDEKGLEAIKEYFPNYSLSELNATHFILNE RPQEYVKLICDFIKVNRYKSLQEHIRHVENFSSAELEARHNAEHERQMEE LRQLTQTTPTAEQVKTIDNLASKVDLSATERQQQQNKEITV
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.
Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the alpha/beta hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains.
