Genes and mechanisms responsible for β-glucoside metabolism in the oenologically important lactic acid bacterium Oenococcus oeni.

Abstract

The lactic acid bacterium Oenococcus oeni plays a pivotal role in winemaking by carrying out malolactic fermentation (MLF), which results in the decarboxylation of L-malic acid to L-lactic acid. It is the species commonly inoculated for MLF but also it will often spontaneously develop after alcoholic fermentation because of its superior tolerance to wine conditions such as high alcohol (up to 16% v/v), low pH (from 3.0 to 4.0) and little or no residual sugar. A marked increase in aroma has been reported after the completion of MLF. This increase has been principally attributed to enzymatic modifications by lactic acid bacteria. In accordance with this O. oeni has been reported to possess β-glucosidase activity. The hydrolysis of β-glucosides in wine can have a significant impact on the sensory profile of a wine by conferring an increase in aroma. Many aroma compounds in wine and must are found in the glycosidic form (i.e. linked to a sugar) and are only perceivable in their nonglycosidic form. For this reason it is of interest to characterise such activities, particularly in O. oeni. Comparative sequence analyses of lactic acid bacteria suggest that six open reading frames (AG1 and ORFs 1 to 5) from the sequenced O. oeni PSU-1 are involved in the hydrolysis of β-glucosides. The ORFs 1 to 3 demonstrated homology to glycosyl hydrolase family (GHF) 1 β-glucosidase/β-glucanase/phospho-β-glucosidase N-terminal and active site signature sequences, whilst AG1 and ORF 4 were lacking the N-terminal signature sequence. Glycosyl hydrolase family 3 β-glucosidase signature sequences were identified in ORF 5. ORF 1 (subsequently designated bglD) was characterised as a GHF 1 phospho-β-glucosidase and found to be part of a phosphoenolpyruvate phosphotransferase system (PEP-PTS) β-glucoside metabolising operon, bgl. Site directed mutagenesis identified a single amino acid responsible for the affinity of BglD towards phosphorylated substrates, providing insight to the catalytic mechanism for all GHF 1 enzymes. ORF 2 and 3 (designated celD and celC) were also characterised as GHF 1 phospho-β-glucosidases and are components of a second PEP-PTS β-glucoside metabolising operon, cel. Neither AG1 nor ORF 4 could be expressed as soluble proteins and it is speculated that the lack of the GHF 1 N-terminal signature sequence is responsible for this. ORF 5 was found to be a GHF 3 β- glucosidase. Transcriptional analysis indicates that these β-glucosidase metabolising operons may be regulated by carbon catabolite repression and transcriptional anti-termination. Given the potential impact of β-glycosidases on the sensory profile of wine, it is hoped that the characterization of β-glycosidase systems from O. oeni will provide information to aid winemakers in tailoring wine aroma, colour and overall complexity where grape quality may otherwise be compromised due to adverse weather conditions or poor viticultural practices.