Molecular Cloning, Expression and Characterization of Oenococcus oeni Priming Glycosyltransferases

Abstract

Oenococcus oeni is the main bacterial species that drives malolactic fermentation in wine. Most O. oeni strains produce capsular exopolysaccharides (EPS) that may contribute to protect them in the wine hostile environment. In O. oeni genome sequences, several genes are predicted to encode priming glycosyltransferases (pGTs). These enzymes are essential for EPS formation as they catalyze the first biosynthetic step through the formation of a phosphoanhydride bond between a hexose-1-phosphate and a lipid carrier undecaprenyl phosphate. In many microorganisms, mutations abolishing the pGT activity also abolish the EPS formation. We first made an in silico analysis of all the genes encoding putative pGT over 50 distinct O. oeni genome sequences. Two polyisoprenyl-phosphate-hexose-1-phosphate transferases, WoaA and WobA, and a glycosyltransferase (It3) were particularly examined for their topology and amino acid sequence. Several isoforms of these enzymes were then expressed in E. coli, and their substrate specificity was examined in vitro. The substrate specificity varied depending on the protein isoform examined, and several mutations were shown to abolish WobA activity but not EPS synthesis. Further analysis of woaA and wobA gene expression levels suggests that WoaA could replace the deficient WobA and maintain EPS formation.

Characterization of Pediococcus ethanolidurans CUPV141: A β-D-glucan- and Heteropolysaccharide-Producing Bacterium

“…It is known that the first enzyme in the process is the p-GTF, which transfers the first phospho-sugar residue from an activated nucleotide sugar to the undecaprenyl phosphate-lipid carrier embedded in the membrane ( Lebeer et al, 2009 ). The pGTF from O. oeni has been recently characterized ( Dimopoulou et al, 2017 ), but no studies in P. ethanolidurans have been performed until now. Thus, the detection of the p-GTF coding gene was approached by PCR amplification of the highly conserved C-terminal sugar transferase domain of the enzyme, using degenerate primers previously described ( Provencher et al, 2003 ).…”

Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance

“…Furthermore, many single constraints relevant in wine modify the corresponding mRNA concentrations particularly after 24 h. The absence of significant change in certain glycosyltransferase genes expression after short period of time of stress application is in accordance with previous transcriptomic studies (Olguín et al, 2015 ; Bastard et al, 2016 ; Margalef-Català et al, 2016 ; Liu et al, 2017 ). Moreover, their increased expression after 24 h suggests that the eps genes are not really involved in the immediate response to stress, but rather in adaptation to new environmental conditions (Guzzo et al, 2000 ; Bourdineaud et al, 2003 ; Da Silveira et al, 2003 ; Maitre et al, 2014 ; Dimopoulou et al, 2017 ). Consequently, the EPS liberation is stimulated in acidic or alcohol containing media and we observed increased specific production rates after 2 weeks of incubation.…”

Polysaccharide Production by Wine Lactic Acid Bacteria: Negative Trait or Potential Advantage? A Review

“…The production of the EPS capsule seems directly correlated to the presence of a non-truncated eps2 gene cluster, while the function of cluster eps1 remains unclear. Nevertheless, a recent study suggests that complementation phenomena between the eps1 and eps2 clusters contribute to maintain the capsule biosynthesis [55]. Furthermore, the very large diversity of O. oeni eps2 gene clusters recalls what is described in pathogenic bacteria such as S. pneumoniae, in which the eps operons direct the capsule synthesis, a proven pathogenicity factor [59].…”

“…(iv) It3 is able to transfer glucose and galactose but its contribution to EPS biosynthesis remains unclear, and the activity of It4 was not explored [55].…”