Molecular screening of lactic acid bacteria enzymes and their regulation under oenological conditions

Aims: To screen Oenococcus oeni strains of South African wine origin for the presence of genes encoding enzymes of oenological relevance using a PCR-based detection approach. Methods and Results: A total of 24 O. oeni strains isolated in South Africa from brandy base wines and from red grape and wine samples were investigated in this study. The reference strains incorporated were NCDO 1894, NCDO 2122 and DSMZ 20252. Using a molecular approach, O. oeni strains were subjected to PCR analysis with primers targeting the mleA, bgl, estA, prtP, pad, citD, citE, citF, maeP, alsD, alsS, metK, metB, metC, gshR, arcA, arcB and arcC genes. As expected, all the strains possessed all the genes investigated, with the notable exception of arcB, arcC and pad. It is the first time that the pad gene encoding a phenolic acid decarboxylase is detected in O. oeni, as two of our strains possessed this gene. The identity of PCR-generated fragments, representing all the genes tested for, was confirmed by sequencing. Homology searches were performed by comparing nucleotide sequences of IWBT B040 strain to other DNA sequences available in GenBank database. The highest sequence identities (98–100%) were recorded with those of three other O. oeni strains (PSU-1, ATCC BAA-1163 and AWRI B429) published in GenBank. Phylogenetic trees were also constructed based on DNA sequences of the alsS, estA, metK and mleA genes, which revealed that all O. oeni strains are phylogenetically distinct from other closely related species of the genus Leuconostoc. Conclusions: We have shown using a molecular approach that O. oeni strains possess a vast array of enzyme-encoding genes of relevance in winemaking. From this study, it was worth noting that most genes are conserved amongst the South African strains, and a worldwide scale comparison would provide more insights into the genetic diversity of O. oeni. Significance and Impact of the Study: The findings reported in this work provide a better insight on the possible role that O. oeni strains can have in modulating wine organoleptic quality with regards to the presence of genes encoding enzymes involved in various metabolic pathways. This study should therefore be followed by the expression analysis of