Biochemical exploration of family GH119 reveals a single α-amylase specificity and confirms shared catalytic machinery with GH57 enzymes.

While hundreds of starch- and glycogen-degrading enzymes have been characterized experimentally in historical families such as GH13, GH14, GH15, GH57 and GH126 of the CAZy database (www.cazy.org), the α-amylase from Bacillus circulans is the only enzyme that has been characterized in family GH119. Since glycosidase families have been shown to often group enzymes with different substrates or products, a single characterized enzyme in a family is insufficient to extrapolate enzyme function based solely on sequence similarity. Here we report the rational exploration of family GH119 through the biochemical characterization of five GH119 members. All enzymes shared single α-amylase specificity but display distinct product profile. We also report the first kinetic constants in family GH119 and the first experimental validation of previously predicted catalytic residues in family GH119, confirming that families GH119 and GH57 can be grouped in the novel clan GH-T of the CAZy database.

Glucuronan lyases from family PL7 use a Tyr/Tyr syn ß-elimination catalytic mechanism for glucuronan breakdown.

TpPL7A and TpPL7B, members of CAZy family PL7, act as ß-glucuronan lyases. TpPL7A diverges by lacking the catalytic histidine, identified as the Brønsted base in PL7 alginate lyases. Our research, including TpPL7A's crystal structure, and mutagenesis studies, reveals a shared syn-ß-elimination mechanism with a single tyrosine serving as both base and acid catalyst. This mechanism may extend to subfamily PL7_4 glucuronan lyases.

Characterization and structural study of a novel ß-N-acetylgalactosaminidase from Niabella aurantiaca.

We report here the identification, characterization and three-dimensional (3D) structure determination of NaNga, a newly identified ß-N-acetylgalactosaminidase from the Gram-negative soil bacterium Niabella aurantiaca DSM 17617. When recombinantly expressed in Escherichia coli, the enzyme selectively cleaved 4-nitrophenyl-N-acetyl-ß-d-galactosamine (pNP-ß-d-GalpNAc). The X-ray crystal structure of the protein was refined to 2.5 Å and consists of an N-terminal ß-sandwich domain and a (ß/α)8 barrel catalytic domain. Despite a mere 22% sequence identity, the 3D structure of NaNga is similar to those previously determined for family GH123 members, suggesting it also employs the same substrate-assisted catalytic mechanism. Inhibition by N-acetyl-galactosamine thiazoline (GalNAc-thiazoline) supports the suggested mechanism. A phylogenetic analysis of its proximal sequence space shows significant clustering of unknown sequences around NaNga with sufficient divergence with previously identified GH123 members to subdivide this family into distinct subfamilies. Although the actual biological substrate of our enzyme remains unknown, examination of the active site pocket suggests that it may be a ß-N-acetylgalactosaminide substituted by a monosaccharide at O-3. Analysis of the genomic context suggests, in turn, that this substituted ß-N-acetylgalactosaminide may be appended to a d-arabinan from an environmental Actinomycete.

Polyphenol Oxidase Products Are Priming Agents for LPMO Peroxygenase Activity.

Polyphenol oxidases catalyze the hydroxylation of monophenols to diphenols, which are reducing agents for lytic polysaccharide monooxygenases (LPMOs) in their degradation of cellulose. In particular, the polyphenol oxidase MtPPO7 from Myceliophthora thermophila converts lignocellulose-derived monophenols, and under the new perspective of the peroxygenase reaction catalyzed by LPMOs, we aim to differentiate the role of the catalytic products of MtPPO7 in priming and fueling of LPMO activity. Exemplified by the activity of MtPPO7 towards guaiacol and by using the benchmark LPMO NcAA9C from Neurospora crassa we show that MtPPO7 catalytic products provide the initial electron for the reduction of Cu(II) to Cu(I) but cannot provide the required reducing power for continuous fueling of the LPMO. The priming reaction is shown to occur with catalytic amounts of MtPPO7 products and those compounds do not generate substantial amounts of H2 O2 in situ to fuel the LPMO peroxygenase activity. Reducing agents with a low propensity to generate H2 O2 can provide the means for controlling the LPMO catalysis through exogenous H2 O2 and thereby minimize any enzyme inactivation.

A GH115 α-glucuronidase structure reveals dimerization-mediated substrate binding and a proton wire potentially important for catalysis.

Xylan is a major constituent of plant cell walls and is a potential source of biomaterials, and the derived oligosaccharides have been shown to have prebiotic effects. Xylans can be highly substituted with different sugar moieties, which pose steric hindrance to the xylanases that catalyse the hydrolysis of the xylan backbone. One such substituent is α-D-glucuronic acid, which is linked to the O2' position of the ß-1,4 D-xylopyranoses composing the main chain of xylans. The xylan-specific α-glucuronidases from glycoside hydrolase family 115 (GH115) specifically catalyse the removal of α-D-glucuronic acid (GlcA) or methylated GlcA (MeGlcA). Here, the molecular basis by which the bacterial GH115 member wtsAgu115A interacts with the main chain of xylan and the indirect involvement of divalent ions in the formation of the Michaelis-Menten complex are described. A crystal structure at 2.65 Šresolution of wtsAgu115A originating from a metagenome from an anaerobic digester fed with wastewater treatment sludge was determined in complex with xylohexaose, and Asp303 was identified as the likely general acid. The residue acting as the general base could not be identified. However, a proton wire connecting the active site to the metal site was observed and hence a previous hypothesis suggesting a Grotthuss-like mechanism cannot be rejected. Only a single molecule was found in the asymmetric unit. However, wtsAgu115A forms a dimer with a symmetry-related molecule in the crystal lattice. The xylohexaose moieties of the xylohexaose are recognized by residues from both protomers, thus creating a xylohexaose recognition site at the dimer interface. The dimer was confirmed by analytical size-exclusion chromatography in solution. Kinetic analysis with aldouronic acids resulted in a Hill coefficient of greater than 2, suggesting cooperativity between the two binding sites. Three Ca2+ ions were identified in the wtsAgu115A structures. One Ca2+ ion is of particular interest as it is coordinated by the residues of the loops that also interact with the substrate. Activity studies showed that the presence of Mg2+ or Mn2+ resulted in a higher activity towards aldouronic acids, while the less restrictive coordination geometry of Ca2+ resulted in a decrease in activity.

The Endo-α(1,4) Specific Fucoidanase Fhf2 From Formosa haliotis Releases Highly Sulfated Fucoidan Oligosaccharides.

Fucoidanases are endo-fucoidanases (also known as endo-fucanases) that catalyze hydrolysis of α-glycosidic linkages in fucoidans, a family of sulfated fucose-rich polysaccharides primarily found in the cell walls of brown seaweeds. Fucoidanases are promising tools for producing bioactive fucoidan oligosaccharides for a range of biomedical applications. High sulfation degree has been linked to high bioactivity of fucoidans. In this study, a novel fucoidanase, Fhf2, was identified in the genome of the aerobic, Gram-negative marine bacterium Formosa haliotis. Fhf2 was found to share sequence similarity to known endo-α(1,4)-fucoidanases (EC 3.2.1.212) from glycoside hydrolase family 107. A C-terminal deletion mutant Fhf2∆484, devoid of 484 amino acids at the C-terminus, with a molecular weight of approximately 46 kDa, was constructed and found to be more stable than the full-length Fhf2 protein. Fhf2∆484 showed endo-fucoidanase activity on fucoidans from different seaweed species including Fucus evanescens, Fucus vesiculosus, Sargassum mcclurei, and Sargassum polycystum. The highest activity was observed on fucoidan from F. evanescens. The Fhf2∆484 enzyme was active at 20-45°C and at pH 6-9 and had optimal activity at 37°C and pH 8. Additionally, Fhf2∆484 was found to be calcium-dependent. NMR analysis showed that Fhf2∆484 catalyzed hydrolysis of α(1,4) linkages between L-fucosyl moieties sulfated on C2 (similar to Fhf1 from Formosa haliotis), but Fhf2∆484 in addition released oligosaccharides containing a substantial amount of 2,4-disulfated fucose residues. The data thus suggest that the Fhf2∆484 enzyme could be a valuable candidate for producing highly sulfated oligosaccharides applicable for fucoidan bioactivity investigations.

Discovery of a Novel Glucuronan Lyase System in Trichoderma parareesei.

Glucuronan lyases (EC 4.2.2.14) catalyze depolymerization of linear ß-(1,4)-polyglucuronic acid (glucuronan). Only a few glucuronan lyases have been characterized until now, most of them originating from bacteria. Here we report the discovery, recombinant production, and functional characterization of the full complement of six glucuronan specific polysaccharide lyases in the necrotic mycoparasite Trichoderma parareesei. The enzymes belong to four different polysaccharide lyase families and have different reaction optima and glucuronan degradation profiles. Four of them showed endo-lytic action and two, TpPL8A and TpPL38A, displayed exo-lytic action. Nuclear magnetic resonance revealed that the monomeric end product from TpPL8A and TpPL38A underwent spontaneous rearrangements to tautomeric forms. Proteomic analysis of the secretomes from T. parareesei growing on pure glucuronan and lyophilized A. bisporus fruiting bodies, respectively, showed secretion of five of the glucuronan lyases and high-performance anion-exchange chromatography with pulsed amperometric detection analysis confirmed the presence of glucuronic acid in the A. bisporus fruiting bodies. By systematic genome annotation of more than 100 fungal genomes and subsequent phylogenetic analysis of the putative glucuronan lyases, we show that glucuronan lyases occur in several ecological and taxonomic groups in the fungal kingdom. Our findings suggest that a diverse repertoire of glucuronan lyases is a common trait among Hypocreales species with mycoparasitic and entomopathogenic lifestyles. IMPORTANCE This paper reports the discovery of a set of six complementary glucuronan lyase enzymes in the mycoparasite Trichoderma parareseei. Apart from the novelty of the discovery of these enzymes in T. parareesei, the key importance of the study is the finding that the majority of these lyases are induced when T. parareesei is inoculated on Basidiomycete cell walls that contain glucuronan. The study also reveals putative glucuronan lyase encoding genes in a wealth of other fungi that furthermore points at fungal cell wall glucuronan being a target C-source for many types of fungi. In a technical context, the findings may lead to controlled production of glucuronan oligomers for advanced pharmaceutical applications and pave the way for development of new fungal biocontrol agents.

Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases.

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6-12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d/l-, α/ß-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.

Specificities and Synergistic Actions of Novel PL8 and PL7 Alginate Lyases from the Marine Fungus Paradendryphiella salina.

Alginate is an anionic polysaccharide abundantly present in the cell walls of brown macroalgae. The enzymatic depolymerization is performed solely by alginate lyases (EC 4.2.2.x), categorized as polysaccharide lyases (PLs) belonging to 12 different PL families. Until now, the vast majority of the alginate lyases have been found in bacteria. We report here the first extensive characterization of four alginate lyases from a marine fungus, the ascomycete Paradendryphiella salina, a known saprophyte of seaweeds. We have identified four polysaccharide lyase encoding genes bioinformatically in P. salina, one PL8 (PsMan8A), and three PL7 alginate lyases (PsAlg7A, -B, and -C). PsMan8A was demonstrated to exert exo-action on polymannuronic acid, and no action on alginate, indicating that this enzyme is most likely an exo-acting polymannuronic acid specific lyase. This enzyme is the first alginate lyase assigned to PL8 and polymannuronic acid thus represents a new substrate specificity in this family. The PL7 lyases (PsAlg7A, -B, and -C) were found to be endo-acting alginate lyases with different activity optima, substrate affinities, and product profiles. PsAlg7A and PsMan8A showed a clear synergistic action for the complete depolymerization of polyM at pH 5. PsAlg7A depolymerized polyM to mainly DP5 and DP3 oligomers and PsMan8A to dimers and monosaccharides. PsAlg7B and PsAlg7C showed substrate affinities towards both polyM and polyG at pH 8, depolymerizing both substrates to DP9-DP2 oligomers. The findings elucidate how P. salina accomplishes alginate depolymerization and provide insight into an efficient synergistic cooperation that may provide a new foundation for enzyme selection for alginate degradation in seaweed bioprocessing.

Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum.

Fusarium graminearum produces an α-l-fucosidase, FgFCO1, which so far appears to be the only known fungal GH29 α-l-fucosidase that catalyzes the release of fucose from fucosylated xyloglucan. In our quest to synthesize bioactive glycans by enzymatic catalysis, we observed that FgFCO1 is able to catalyze a transglycosylation reaction involving transfer of fucose from citrus peel xyloglucan to lactose to produce 2'-fucosyllactose, an important human milk oligosaccharide. In addition to achieving maximal yields, control of the regioselectivity is an important issue in exploiting such a transglycosylation ability successfully for glycan synthesis. In the present study, we aimed to improve the transglycosylation efficiency of FgFCO1 through protein engineering by transferring successful mutations from other GH29 α-l-fucosidases. We investigated several such mutation transfers by structural alignment, and report that transfer of the mutation F34I from BiAfcB originating from Bifidobacterium longum subsp. infantis to Y32I in FgFCO1 and mutation of D286, near the catalytic acid/base residue in FgFCO1, especially a D286M mutation, have a positive effect on FgFCO1 transfucosylation regioselectivity. We also found that enzymatic depolymerization of the xyloglucan substrate increases substrate accessibility and in turn transglycosylation (i.e., transfucosylation) efficiency. The data include analysis of the active site amino acids and the active site topology of FgFCO1 and show that transfer of point mutations across GH29 subfamilies is a rational strategy for targeted protein engineering of a xyloglucan-active fungal α-l-fucosidase.

Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis.

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37-40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V.

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10-13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

Proteomic enzyme analysis of the marine fungus Paradendryphiella salina reveals alginate lyase as a minimal adaptation strategy for brown algae degradation.

We set out to investigate the genetic adaptations of the marine fungus Paradendryphiella salina CBS112865 for degradation of brown macroalgae. We performed whole genome and transcriptome sequencing and shotgun proteomic analysis of the secretome of P. salina grown on three species of brown algae and under carbon limitation. Genome comparison with closely related terrestrial fungi revealed that P. salina had a similar but reduced CAZyme profile relative to the terrestrial fungi except for the presence of three putative alginate lyases from Polysaccharide Lyase (PL) family 7 and a putative PL8 with similarity to ascomycete chondroitin AC lyases. Phylogenetic and homology analyses place the PL7 sequences amongst mannuronic acid specific PL7 proteins from marine bacteria. Recombinant expression, purification and characterization of one of the PL7 genes confirmed the specificity. Proteomic analysis of the P. salina secretome when growing on brown algae, revealed the PL7 and PL8 enzymes abundantly secreted together with enzymes necessary for degradation of laminarin, cellulose, lipids and peptides. Our findings indicate that the basic CAZyme repertoire of saprobic and plant pathogenic ascomycetes, with the addition of PL7 alginate lyases, provide P. salina with sufficient enzymatic capabilities to degrade several types of brown algae polysaccharides.

Loop engineering of an α-1,3/4-l-fucosidase for improved synthesis of human milk oligosaccharides.

The α-1,3/4-l-fucosidases (EC 3.2.1.111; GH29) BbAfcB from Bifidobacterium bifidum and CpAfc2 from Clostridium perfringens can catalyse formation of the human milk oligosaccharide (HMO) lacto-N-fucopentaose II (LNFP II) through regioselective transfucosylation of lacto-N-tetraose (LNT) with 3-fucosyllactose (3FL) as donor substrate. The current work exploits structural differences between the two enzymes with the aim of engineering BbAfcB into a more efficient transfucosidase and approaches an understanding of structure-function relations of hydrolytic activity vs. transfucosylation activity in GH29. Replacement of a 23 amino acids long α-helical loop close to the active site of BbAfcB with the corresponding 17-aminoacid α-helical loop of CpAfc2 resulted in almost complete abolishment of the hydrolytic activity on 3FL (6000 times lower hydrolytic activity than WT BbAfcB), while the transfucosylation activity was lowered only one order of magnitude. In turn, the loop engineering resulted in an α-1,3/4-l-fucosidase with transfucosylation activity reaching molar yields of LNFP II of 39 ±â€¯2% on 3FL and negligible product hydrolysis. This was almost 3 times higher than the yield obtained with WT BbAfcB (14 ±â€¯0.3%) and comparable to that obtained with CpAfc2 (50 ±â€¯8%). The obtained transfucosylation activity may expand the options for HMO production: mixtures of 3FL and LNT could be enriched with LNFP II, while mixtures of 3FL and lacto-N-neotetraose (LNnT) could be enriched with LNFP III.

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.

Characterization of the First α-(1→3) Branching Sucrases of the GH70 Family.

Leuconostoc citreumNRRL B-742 has been known for years to produce a highly α-(1→3)-branched dextran for which the synthesis had never been elucidated. In this work a gene coding for a putative α-transglucosylase of the GH70 family was identified in the reported genome of this bacteria and functionally characterized. From sucrose alone, the corresponding recombinant protein, named BRS-B, mainly catalyzed sucrose hydrolysis and leucrose synthesis. However, in the presence of sucrose and a dextran acceptor, the enzyme efficiently transferred the glucosyl residue from sucrose to linear α-(1→6) dextrans through the specific formation of α-(1→3) linkages. To date, BRS-B is the first reported α-(1→3) branching sucrase. Using a suitable sucrose/dextran ratio, a comb-like dextran with 50% of α-(1→3) branching was synthesized, suggesting that BRS-B is likely involved in the comb-like dextran produced byL. citreumNRRL B-742. In addition, data mining based on the search for specific sequence motifs allowed the identification of two genes putatively coding for branching sucrases in the genome ofLeuconostoc fallaxKCTC3537 andLactobacillus kunkeeiEFB6. Biochemical characterization of the corresponding recombinant enzymes confirmed their branching specificity, revealing that branching sucrases are not only found inL. citreumspecies. According to phylogenetic analyses, these enzymes are proposed to constitute a new subgroup of the GH70 family.

Inventory of the GH70 enzymes encoded by Leuconostoc citreum NRRL B-1299 - identification of three novel α-transglucosylases.

Leuconostoc citreum NRRL B-1299 has long been known to produce α-glucans containing both α-(1→6) and α-(1→2) linkages, which are synthesized by α-transglucosylases of the GH70 family. We sequenced the genome of Leuconostoc citreum NRRL B-1299 to identify the full inventory of GH70 enzymes in this strain. Three new genes (brsA, dsrM and dsrDP) putatively encoding GH70 enzymes were identified. The corresponding recombinant enzymes were characterized. Branching sucrase A (BRS-A) grafts linear α-(1→6) dextran with α-(1→2)-linked glucosyl units, and is probably involved in the α-(1→2) branching of L. citreum NRRL B-1299 dextran. This is the first report of a naturally occurring α-(1→2) branching sucrase. DSR-M and DSR-DP are dextransucrases that are specific for α-(1→6) linkage synthesis and mainly produce oligomers or short dextrans with molar masses between 580 and 27 000 g·mol(-1) . In addition, DSR-DP contains sequences that diverge from the consensus sequences that are typically present in enzymes that synthesize linear dextran. Comparison of the genome with five other L. citreum genomes further revealed that dsrDP is unique to L. citreum NRRL B-1299. The presence of this gene in a prophage represents the first evidence of phage-mediated horizontal transfer of genes encoding such enzymes in lactic acid bacteria. Finally, brsA and dsrM are located in a chromosomal region in which genes encoding strain-specific GH70 enzymes are consistently located. This region may be a good target on which to focus in order to rapidly evaluate the diversity of GH70 enzymes in L. citreum strains.

Complete Genome Sequence of Leuconostoc citreum Strain NRRL B-742.

Leuconostoc citreum belongs to the group of lactic acid bacteria and plays an important role in fermented foods of plant origin. Here, we report the complete genome of the Leuconostoc citreum strain NRRL B-742, isolated in 1954 for its capacity to produce dextran.

Exopolysaccharide (EPS) synthesis by Oenococcus oeni: from genes to phenotypes.

Oenococcus oeni is the bacterial species which drives malolactic fermentation in wine. The analysis of 50 genomic sequences of O. oeni (14 already available and 36 newly sequenced ones) provided an inventory of the genes potentially involved in exopolysaccharide (EPS) biosynthesis. The loci identified are: two gene clusters named eps1 and eps2, three isolated glycoside-hydrolase genes named dsrO, dsrV and levO, and three isolated glycosyltransferase genes named gtf, it3, it4. The isolated genes were present or absent depending on the strain and the eps gene clusters composition diverged from one strain to another. The soluble and capsular EPS production capacity of several strains was examined after growth in different culture media and the EPS structure was determined. Genotype to phenotype correlations showed that several EPS biosynthetic pathways were active and complementary in O. oeni. Can be distinguished: (i) a Wzy-dependent synthetic pathway, allowing the production of heteropolysaccharides made of glucose, galactose and rhamnose, mainly in a capsular form, (ii) a glucan synthase pathway (Gtf), involved in ß-glucan synthesis in a free and a cell-associated form, giving a ropy phenotype to growth media and (iii) homopolysaccharide synthesis from sucrose (α-glucan or ß-fructan) by glycoside-hydrolases of the GH70 and GH68 families. The eps gene distribution on the phylogenetic tree was examined. Fifty out of 50 studied genomes possessed several genes dedicated to EPS metabolism. This suggests that these polymers are important for the adaptation of O. oeni to its specific ecological niche, wine and possibly contribute to the technological performance of malolactic starters.

Optimizing the production of an α-(1→2) branching sucrase in Escherichia coli using statistical design.

Experimental design and Response Surface Methodology (RSM) were used to optimize the production of ∆N123-GBD-CD2, an α-(1 → 2) branching sucrase previously reported as mainly produced in inclusion bodies. The ∆N123-GBD-CD2 encoding gene was cloned into two expression vectors in fusion with 6xHis tag or Strep tag II encoding sequences at 5' and 3' ends of the gene and expressed in five Escherichia coli strains. Three host-vector combinations were first selected on the basis of the amount of soluble enzyme produced. RSM with Box-Behnken design was used to optimize the expression conditions in an auto-inducible medium. Five factors were considered, i.e. culture duration, temperature and the concentrations of glycerol, lactose inducer and glucose repressor. The design consisted of three blocks of 45 assays performed in deep well microplates. The regression models were built and fitted well to the experimental data (R (2) coefficient >94 %). The best response (production level of soluble enzyme) was obtained with E. coli BL21 Star DE3 cells transformed with the pET-55 vector. Using the predicted optimal conditions, 5,740 U L(-1) of culture of soluble enzyme was produced in microtiter plates and more than 12,000 U L(-1) of culture in Erlenmeyer flask, which represents a 165-fold increase compared to the production levels previously reported.