Homology analysis of 35 ß-glucosidases in Oenococcus oeni and biochemical characterization of a novel ß-glucosidase BGL0224.

ß-Glucosidases play an important role in food industry. Oenococcus oeni are typical lactic acid bacteria that initiate malolactic fermentation of wines. 35 ß-glucosidases from O. oeni were selected and their conserved domains and evolutionary relationships were further explored in this study. The homology analysis results indicated that 35 ß-glucosidases were basically derived from GH1 and GH3 family. A novel ß-glucosidase was successfully expressed and characterized. The recombinant protein, referred to as BGL0224, consisted of a total 480 amino acids with an apparent molecular weight of 55.15 kDa and was classified as GH1 family. It achieved the highest activity at pH 5.0 and 50 °C. The activity and stability were significantly increased when 12% ethanol was supplemented to the enzyme. Using p-NPG as substrate, the Km, Vmax and Kcat of BGL0224 were 0.34 mM, 382.81 U/mg and 351.88 s-1, respectively. In all, BGL0224 has good application prospects in food industry.

Assessment of ß-D-glucosidase activity and bgl gene expression of Oenococcus oeni SD-2a.

Glycosidases enhance flavor during wine-making by mediating the enzymatic release of aroma molecules. In order to better understand the aroma enhancement potential of Oenococcus oeni SD-2a, ß-D-glucosidase (ßG) activities in the culture supernatant, whole cells, and disrupted cell lysate were assessed at mid log, late log and stationary growth phase. The enzymatic activity was also compared further from cell cultures with 5 different carbon sources (glucose, cellobiose, arbutin, glucose and cellobiose, glucose and arbutin) at late log phase. Correspondingly, expression levels of 3 bgl genes, OEOE-0224, OEOE-1210, and OEOE-1569 were investigated from cell cultures of the 3 growth phases, and the 5 cell cultures with different carbon sources. Finally, the volatile aroma compounds released by O. oeni SD-2a in synthetic wines with natural glycosides were evaluated by GC-MS. Results showed ßG of O. oeni SD-2a was not extracellular enzyme, and the location of it didn't change with the change of growth phase and carbon source studied. ßG activities in the whole cells and disrupted cell lysate were similar and constant at the 3 growth phases. As for the carbon sources, ßG activities of whole cells and disrupted lysate were positively affected by cellobiose. While arbutin displayed positive and negative effect on ßG activity of whole cells and disrupted lysate, respectively. It is probably that bgl genes OEOE-0224 and OEOE-1210 were related to ßG activity of SD-2a whole cells, while OEOE-1569 was responsible for ßG activity of disrupted lysate. More kinds of volatile compounds and higher total concentration were released by SD-2a in synthetic wine compared with control. Thus, SD-2a showed a great potential for flavor enhancement under wine-like conditions. This study provides more information for further study of ßG activity from O. oeni SD-2a.

l-Malate (-2) Protonation State is Required for Efficient Decarboxylation to l-Lactate by the Malolactic Enzyme of Oenococcus oeni.

Malolactic fermentation (MLF) is responsible for the decarboxylation of l-malic into lactic acid in most red wines and some white wines. It reduces the acidity of wine, improves flavor complexity and microbiological stability. Despite its industrial interest, the MLF mechanism is not fully understood. The objective of this study was to provide new insights into the role of pH on the binding of malic acid to the malolactic enzyme (MLE) of Oenococcus oeni. To this end, sequence similarity networks and phylogenetic analysis were used to generate an MLE homology model, which was further refined by molecular dynamics simulations. The resulting model, together with quantum polarized ligand docking (QPLD), was used to describe the MLE binding pocket and pose of l-malic acid (MAL) and its l-malate (-1) and (-2) protonation states (MAL- and MAL2-, respectively). MAL2- has the lowest ∆Gbinding, followed by MAL- and MAL, with values of -23.8, -19.6, and -14.6 kJ/mol, respectively, consistent with those obtained by isothermal calorimetry thermodynamic (ITC) assays. Furthermore, molecular dynamics and MM/GBSA results suggest that only MAL2- displays an extended open conformation at the binding pocket, satisfying the geometrical requirements for Mn2+ coordination, a critical component of MLE activity. These results are consistent with the intracellular pH conditions of O. oeni cells-ranging from pH 5.8 to 6.1-where the enzymatic decarboxylation of malate occurs.

Heterologous expression of the puuE from Oenococcus oeni SD-2a in Lactobacillus plantarum WCFS1 improves ethanol tolerance.

Oenococcus oeni is the main bacteria extensively used in malolactic fermentation due to its high tolerance against stress factors in wine production. Among these, ethanol is one of the main challenges to O. oeni, and its ethanol tolerance mechanism remains unclear. In this study, the puuE gene related to ethanol tolerance from O. oeni SD-2a was heterologously expressed in Lactobacillus plantarum WCFS1. Results showed that the recombinant strain (W-pMG36epuuE) exhibited better growth performance and survival rate compared to the control strain (W-pMG36e) under ethanol-stress conditions. In addition, it was found that the activities of superoxide dismutase and the concentration of glutathione of W-pMG36epuuE were significantly higher than those of W-pMG36e. This resulted in the decrease of intracellular reactive oxygen species (ROS) accumulation (10.34% lower than control). Moreover, heterologous expression of puuE in WCFS1 exhibited improved activities of two ATPases in membrane, increasing the cell membrane integrity (37.67% higher than control). These results revealed the role of the puuE gene in improving ethanol tolerance in O. oeni by decreasing ROS accumulation and enhancing cell membrane integrity.

A dextran with unique rheological properties produced by the dextransucrase from Oenococcus kitaharae DSM 17330.

A gene encoding a novel dextransucrase was identified in the genome of Oenococcus kitaharae DSM17330 and cloned into E. coli. With a kcat of 691s-1 and a half-life time of 111h at 30°C, the resulting recombinant enzyme -named DSR-OK- stands as one of the most efficient and stable dextransucrase characterized to date. From sucrose, this enzyme catalyzes the synthesis of a quasi linear dextran with a molar mass higher than 1×109g·mol-1 that presents uncommon rheological properties such as a higher viscosity than that of the most industrially used dextran from L. mesenteroides NRRL-B-512F, a yield stress that was never described before for any type of dextran, as well as a gel-like structure. All these properties open the way to a vast array of new applications in health, food/feed, bulk or fine chemicals fields.

Influence of glycosides on behavior of Oenococcus oeni in wine conditions: growth, substrates and aroma compounds.

Autochthonous Oenococcus oeni strains (MS9, MS20 and MS46) with good malolactic performance and yielding adequate diacetyl levels, were selected to investigate the effect of synthetic and grape glycosides on bacterial growth, substrate utilization and ß-glucosidase (ßGlu), α-arabinofuranosidase (αAra) and α-rhamnopyranosidase (αRha) activities in a wine-like medium containing 6% ethanol, pH 4.0 (WBM). Then, changes in the volatile compounds profile were evaluated at the end of malolactic fermentation (MLF) carried out by the MS46 strain in WBM containing 1 mg L-1 of natural glycoside. All strains grew and efficiently degraded L-malic acid in WBM where ßGlu and αAra activities were found but not αRha. In presence of a synthetic glycoside (eriodictyol 7-O-ß-rutinoside) ßGlu activity was significantly enhanced for two of the cultures tested (MS20 and MS460) while a low αRha activity was induced, presenting MS46 the better performance. Glycosides extracted from fermented grape musts under different conditions allowed maximum growths, L-malic acid utilization rates and glycosidase activities in the MS46 strain. Thus, ßGlu, αAra and αRha activities increased between 30-50 and 3-11% respectively. This indirectly correlated to significant changes in total esters and higher alcohols at the end of MLF, which increased by up to 140 and 30% respectively. Moreover, ethyl and acetate esters formed up to 100-fold than alcohols or esters degraded highlighted the main role of this microorganism in the esters synthesis. Results obtained encourage the potential use of selected indigenous O. oeni strains as a tool to enhance wine complexity through MLF, mainly on highly fruity aroma.

Molecular Cloning, Expression and Characterization of Oenococcus oeni Priming Glycosyltransferases.

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.

Growth and metabolism of Oenococcus oeni for malolactic fermentation under pressure.

Malolactic fermentation is a biological deacidification process of wine, characterized by the transformation of l-malic acid to l-lactic acid and CO2 . Oenococcus oeni is able to perform malolactic fermentation and to survive under wine harsh conditions, representing great interest for wine industry. The aim of this work was to evaluate the effect of high pressure on the metabolism of O. oeni growing in culture media, regarding malolactic fermentation, sugars metabolism and bacterial growth. A pressure stress of 50 MPa during 8 h did not result in significant modifications in bacterial metabolism. In contrast, a stress of 100 MPa during 8 h resulted in lower amounts of l-lactic acid, while higher amounts of d-lactic acid were also registered, indicating changes in bacterial metabolism. A pressure stress of 0·5 MPa during 300 h resulted in complete inactivation of O. oeni, but malolactic fermentation was still observed at some extent, showing that malolactic enzyme was not completely inactivated at these conditions. It was concluded that high pressure causes modification of O. oeni metabolism, and possibly in enzyme activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that high pressure affects the viability and metabolism of Oenococcus oeni on a culture medium, depending on the pressure intensity and holding time applied. These effects were particularly noteworthy on malolactic fermentation. After high pressure (HP)-stress of 100 MPa for 8 h, modifications in the activity of malolactic enzyme were detected, possibly due to a change in specificity. After a HP-stress of 300 MPa for 0·5 h, malolactic enzyme showed some residual activity, although O. oeni was completely inactivated. This study provides relevant information about the impact of high pressure on malolactic fermentation, opening interesting possibilities to the improvement of biocatalytic processes.

Genetically engineered Oenococcus oeni strains to highlight the impact of estA2 and estA7 esterase genes on wine ester profile.

Besides deacidifying wine, Oenococcus oeni bring significant changes in the chemical composition of wine by releasing esters by the action of their own esterases. The impact of O. oeni esterases remains relatively unexplored. Four esterase genes were identified from O. oeni genome (estA2, estA7, estC, and estB). The dual objective of this study was, first to use a genetic tool enabling the expression of esterase genes in enological conditions and, second, to investigate the impact of O. oeni esterase gene expression during winemaking on wine aromatic profile. Both estA2 and estA7 genes were successfully cloned and expressed in O. oeni and recombinant strains were inoculated in Aligoté wine to initiate malolactic fermentation (MLF). Ester profile of experimental wine was established by SPME-GC-MS. EstA2 caused significant decreases in the concentrations of isoamyl acetate, ethyl hexanoate, isobutyl acetate, and hexyl acetate, by 42.7%, 23.4%, 51.5%, and 28.9%, respectively. EstA2 has preferential hydrolytic activity toward acetate esters from higher alcohols. EstA7 has synthetic activity toward hexyl acetate with a significant 22.7% increase. This study reports the first efficient expression system enabling the production of a functional protein in O. oeni in enological conditions.

Arginine deiminase pathway genes and arginine degradation variability in Oenococcus oeni strains.

Trace amounts of the carcinogenic ethyl carbamate can appear in wine as a result of a reaction between ethanol and citrulline, which is produced from arginine degradation by some bacteria used in winemaking. In this study, arginine deiminase (ADI) pathway genes were evaluated in 44 Oenococcus oeni strains from wines originating from several locations in order to establish the relationship between the ability of a strain to degrade arginine and the presence of related genes. To detect the presence of arc genes of the ADI pathway in O. oeni, pairs of primers were designed to amplify arcA, arcB, arcC and arcD1 sequences. All strains contained these four genes. The same primers were used to confirm the organization of these genes in an arcABCD1 operon. Nevertheless, considerable variability in the ability to degrade arginine among these O. oeni strains was observed. Therefore, despite the presence of the arc genes in all strains, the expression patterns of individual genes must be strain dependent and influenced by the different wine conditions. Additionally, the presence of arc genes was also determined in the 57 sequenced strains of O. oeni available in GenBank, and the complete operon was found in 83% of strains derived from wine. The other strains were found to lack the arcB, arcC and arcD genes, but all contained sequences homologous to arcA, and some of them had also ADI activity.

Identification and Localization of ß-D-Glucosidase from Two Typical Oenococcus oeni Strains.

ß-D-glucosidase (ßG) gene from Oenococcus oeni SD-2a and 31MBR was cloned, sequenced and analyzed, also intracellular ßG of the two strains was further localized. The results showed that ßG gene of the two strains was in high homology (> 99%) to reported ßG gene, confirming both strains possess ßG activity at the molecular level. Intracellular ßG of SD-2a is a mainly soluble protein, existing mostly in the cytoplasm and to some extent in the periplasm. While for 31MBR, intracellular ßG is mainly insoluble protein existing in the cytoplasmic membrane. This study provides basic information for further study of the metabolic mechanism of ßG from O. oeni SD-2a and 31MBR.

Cyclopropane fatty acid synthase from Oenococcus oeni: expression in Lactococcus lactis subsp. cremoris and biochemical characterization.

Bacterial cyclopropane fatty acid synthases (CFA synthases) catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the double bond of a lipid chain, thereby forming a cyclopropane ring. CFAs contribute to resistance to acidity, dryness, and osmotic imbalance in many bacteria. This work describes the first biochemical characterization of a lactic acid bacterium CFA synthase. We have overexpressed Oenococcus oeni CFA synthase in E. coli in order to purify the enzyme. The optimum cyclopropanation activity was obtained at pH 5.6 and 35.8 °C. The high K(m) (AdoMet) value obtained (2.26 mM) demonstrates the low affinity of O. oeni enzyme toward the L. lactis subsp. cremoris unsaturated phospholipids. These results explain the partial complementation of the L. lactis subsp. cremoris cfa mutant by the O. oeni cfa gene and suggest a probable substrate specificity of the O. oeni enzyme. The current study reveals an essential hypothesis about the specificity of O. oeni CFA synthase which could play a key function in the acid tolerance mechanisms of this enological bacterium.

Improving Oenococcus oeni to overcome challenges of wine malolactic fermentation.

Oenococcus oeni is crucial for winemaking, bringing stabilization, deacidification, and sensory impacts through malolactic fermentation (MLF) to most wine styles. The poor nutritional make-up of wine together with typically low processing temperatures and pH and high ethanol content and sulfur dioxide (SO2) hinder O. oeni growth and activity. Production delays and interventions with starter cultures and nutritional supplements have significant cost and quality implications; thus, optimization of O. oeni has long been a priority. A range of optimization strategies, some guided by detailed characterization of O. oeni, have been exploited. Varying degrees of success have been seen with classical strain selection, mutagenesis, gene recombination, genome shuffling, and, most recently, directed evolution (DE). The merits, limitations, and future prospects of each are discussed.

NMR study of histidine metabolism during alcoholic and malolactic fermentations of wine and their influence on histamine production.

The metabolic pathways of amino acids play a crucial role in the organoleptic and hygienic quality in wines. In particular, histidine is one of the most studied amino acids of wines due to histamine toxicity in humans, a biogenic amine derived from histidine by enzymatic decarboxylation. The development of new tools to increase knowledge on metabolism that produces histamine in wine is critical. This study investigated by using nuclear magnetic resonance (NMR) spectroscopy the transformation of histidine into histaminol and histamine during alcoholic and malolactic fermentations. The transformations of histidine into histaminol during alcoholic fermentation and into histamine during malolactic fermentation were observed. This paper highlights the importance of selecting lactic acid bacteria for malolactic fermentation to avoid the production of biogenic amines such as histamine.

Ester synthesis and hydrolysis in an aqueous environment, and strain specific changes during malolactic fermentation in wine with Oenococcus oeni.

Previous work has shown that Oenococcus oeni produces esterases that are capable of hydrolysing artificial substrates. Using SPME-GCMS, this study provides evidence that purified O. oeni esterases have the ability to both synthesise and hydrolyse esters. Two purified esterases (EstA2 and EstB28) synthesised ethyl butanoate and ethyl hexanoate to varying degrees. Both purified esterases hydrolysed ethyl butanoate, ethyl hexanoate and ethyl octanoate. Once this dual activity was confirmed, malolactic fermentation (MLF) trials were conducted in wine with O. oeni strains that had been previously observed to have either high or low esterase activity against artificial substrates. Strain specific differences were observed and strains with low esterase hydrolysis activity against artificial substrates had a higher level of total esters measured after MLF. The results demonstrate the impact that O. oeni has on wine aroma and relates this to the ester hydrolysis and synthesis abilities of O. oeni strains.

Surface display of malolactic enzyme from Oenococcus oeni on Saccharomyces cerevisiae.

In order to display malolactic enzyme (MLE) on the cell surface of Saccharomyces cerevisiae, a yeast cell surface display plasmid pADH1-AGG was constructed by fusing the α-factor signal encoding sequence (267 bp) and the C-terminal half of α-agglutinin encoding sequence (1,645 bp) into the plasmid pADH1. The pADH1-AGG could successfully express and anchor the enhanced green fluorescent protein (EGFP) onto the yeast cell surface when the EGFP was used to verify its function. Then the pADH1-MLE was constructed by inserting the MLE encoding sequence (1,600 bp) into the pADH1-AGG and introduced into S. cerevisiae cells. The positive strain carrying pADH1-MLE was confirmed by use of the 6× His monoclonal antibody and fluorescein isothiocyanate-conjugated goat anti-mouse IgG. All results indicated that the MLE was displayed successfully on the cell surface of positive transformant. The MLE activity of genetically engineered yeast strain could turn 21.11 % L-malate into lactic acid after 12 h reaction with L-malate. The constructed yeast strain might be used to conduct malolactic fermentation (MLF) in wine to solve the important issues of sluggish MLF, microbial spoilage, and adverse metabolic substances produced by the lactic acid bacteria.

Distribution and functions of phosphotransferase system genes in the genome of the lactic acid bacterium Oenococcus oeni.

Oenococcus oeni, the lactic acid bacterium primarily responsible for malolactic fermentation in wine, is able to grow on a large variety of carbohydrates, but the pathways by which substrates are transported and phosphorylated in this species have been poorly studied. We show that the genes encoding the general phosphotransferase proteins, enzyme I (EI) and histidine protein (HPr), as well as 21 permease genes (3 isolated ones and 18 clustered into 6 distinct loci), are highly conserved among the strains studied and may form part of the O. oeni core genome. Additional permease genes differentiate the strains and may have been acquired or lost by horizontal gene transfer events. The core pts genes are expressed, and permease gene expression is modulated by the nature of the bacterial growth substrate. Decryptified O. oeni cells are able to phosphorylate glucose, cellobiose, trehalose, and mannose at the expense of phosphoenolpyruvate. These substrates are present at low concentrations in wine at the end of alcoholic fermentation. The phosphotransferase system (PTS) may contribute to the perfect adaptation of O. oeni to its singular ecological niche.

Structural and biochemical characterisation of a NAD⁺-dependent alcohol dehydrogenase from Oenococcus oeni as a new model molecule for industrial biotechnology applications.

Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni(2+)-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni(2+), activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD(+)/NADH, whereas no activity has been observed with NADP(+)/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn(2+). The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni(2+)-dependent activity towards 1,3-propanediol.

Structure of alanine racemase from Oenococcus oeni with bound pyridoxal 5'-phosphate.

The crystal structure of alanine racemase from Oenococcus oeni has been determined at 1.7 Šresolution using the single-wavelength anomalous dispersion (SAD) method and selenium-labelled protein. The protein exists as a symmetric dimer in the crystal, with both protomers contributing to the two active sites. Pyridoxal 5'-phosphate, a cofactor, is bound to each monomer and forms a Schiff base with Lys39. Structural comparison of alanine racemase from O. oeni (Alr) with homologous family members revealed similar domain organization and cofactor binding.

Synthesis of fruity ethyl esters by acyl coenzyme A: alcohol acyltransferase and reverse esterase activities in Oenococcus oeni and Lactobacillus plantarum.

AIMS: To assess the abilities of commercial wine lactic acid bacteria (LAB) to synthesize potentially flavour active fatty acid ethyl esters and determine mechanisms involved in their production. METHODS AND RESULTS: Oenococcus oeni AWRI B551 produced significant levels of ethyl hexanoate and ethyl octanoate following growth in an ethanolic test medium, and ester formation generally increased with increasing pH (4.5 > 3.5), anaerobiosis and precursor supplementation. Cell-free extracts of commercial O. oeni strains and Lactobacillus plantarum AWRI B740 were also tested for ester-synthesizing capabilities in a phosphate buffer via: (i) acyl coenzyme A: alcohol acyltransferase (AcoAAAT) activity and (ii) reverse esterase activity. For both ester-synthesizing activities, strain-dependent variation was observed, with AcoAAAT activity generally greater than reverse esterase. Reverse esterase in O. oeni AWRI B551 also esterified 1-propanol to produce propyl octanoate, and deuterated substrates ([(2)H(6)]ethanol and [(2)H(15)]octanoic acid) to produce the fully deuterated ester, [(2)H(5)]ethyl [(2)H(15)]octanoate. CONCLUSIONS: Wine LAB exhibit ethyl ester-synthesizing capability and possess two different ester-synthesizing activities, one of which is associated with an acyl coenzyme A: alcohol acyltransferase. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that wine LAB exhibit enzyme activities that can augment the ethyl ester content of wine. This knowledge will facilitate greater control over the impacts of malolactic fermentation on the fruity sensory properties and quality of wine.