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

Author

Maria Dimopoulou, Marlène Vuillemin, Hugo Campbell-Sills, Patrick M Lucas, Patricia Ballestra, Cécile Miot-Sertier, Marion Favier, Joana Coulon, Virginie Moine, Thierry Doco, Maryline Roques, Pascale Williams, Melina Petrel, Etienne Gontier, Claire Moulis, Magali Remaud-Simeon, and Marguerite Dols-Lafargue

Subjects
Medicine, Science
Abstract

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 β-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 β-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.

Published
2014
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3. A dextran with unique rheological properties produced by the dextransucrase from Oenococcus kitaharae DSM 17330

Author

Marguerite Dols-Lafargue, Claire Moulis, Florent Grimaud, Magali Remaud-Simeon, Pierre Monsan, Catherine Garnier, Patrick Lucas, Marlène Vuillemin, Marion Claverie, Agnès Rolland-Sabaté, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Sécurité et Qualité des Produits d'Origine Végétale (SQPOV), Avignon Université (AU)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), Institut National de la Recherche Agronomique (INRA), Unité de Recherche Oenologie [Villenave d'Ornon], Institut National de la Recherche Agronomique (INRA)-Université de Bordeaux (UB)-Institut des Sciences de la Vigne et du Vin (ISVV), Toulouse White Biotechnology (TWB), ANR-10-ALIA-0003,OENOPOLYS,Les polysaccharides de la bactérie lactique Oenococcus oeni, de l'élucidation de leurs structures et voies de biosynthèse à leur valorisation technologique(2010), ANR-14-CE27-0011,ASPIRE,Exploration des relations structure-propriétés de copolymères biosourcés(2014), Institut National de la Recherche Agronomique (INRA)-Avignon Université (AU), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), ANR-10-ALIA-0003, ANR-14-CE27-0011-01, Avignon Université (AU)-Institut National de la Recherche Agronomique (INRA), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Unité de Recherche Oenologie [Villenave d'Ornon] (OENO)

Subjects
0301 basic medicine, Sucrose, Polymers and Plastics, plavolex, Glucansucrase, gène codant, 010402 general chemistry, Polysaccharide, 01 natural sciences, Dextransucrase, sucrose 6-glucosyltransferase, 03 medical and health sciences, chemistry.chemical_compound, Materials Chemistry, Carbohydrate Conformation, spreadability, Enzyme kinetics, Amino Acid Sequence, Databases, Protein, chemistry.chemical_classification, Molar mass, biology, sulfate de dextrane, propriété rhéologique, Chemistry, Viscosity, dextransucrase, Organic Chemistry, Bacterial polysaccharide, oenococcus, Dextrans, biology.organism_classification, Recombinant Proteins, 0104 chemical sciences, 030104 developmental biology, Dextran, [CHIM.POLY]Chemical Sciences/Polymers, Biochemistry, Glucosyltransferases, Oenococcus kitaharae, dextran, polysaccharide, biology.protein, Biocatalysis, saccharase, Oenococcus
Abstract

A gene encoding a novel dextransucrase was identified in the genome of Oenococcus kitaharae DSM17330 and cloned into E. coli . With a kcat of 691 s −1 and a half-life time of 111 h 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 × 10 9 g·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.

Published
2018
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4. Investigations on the Determinants Responsible for Low Molar Mass Dextran Formation by DSR-M Dextransucrase

Author

Marion Claverie, Guy Lippens, Pierre Roblin, Magali Remaud-Simeon, Nelly Monties, Marlène Vuillemin, Claire Moulis, Gianluca Cioci, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Laboratoire de Génie Chimique (LGC), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées, French Ministry of Higher Education and Research, Agence Nationale de la Recherche (ANR), Centre National de la Recherche Scientifique - CNRS (FRANCE), Institut National Polytechnique de Toulouse - Toulouse INP (FRANCE), Institut National de la Recherche Agronomique - INRA (FRANCE), Institut National des Sciences Appliquées de Toulouse - INSA (FRANCE), Université Toulouse III - Paul Sabatier - UT3 (FRANCE), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), and Université de Toulouse (UT)

Subjects
0301 basic medicine, crystal structure, Sucrose, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, carbohydrate-binding protein, Population, medicine.disease_cause, Catalysis, Dextransucrase, sucrose 6-glucosyltransferase, 03 medical and health sciences, chemistry.chemical_compound, [CHIM.GENI]Chemical Sciences/Chemical engineering, polymère, Leuconostoc citreum, medicine, Génie chimique, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, education, Génie des procédés, chemistry.chemical_classification, dextran, education.field_of_study, Molar mass, Chemistry, glucan-binding domain, dextransucrase, Substrate (chemistry), General Chemistry, glucansucrases, dextransucrases, 030104 developmental biology, Enzyme, Dextran, Biochemistry, nonprocessive polymerization, GH70 family
Abstract

International audience; Certain enzymes of the GH70 family dextransucrases synthesize very high molar mass dextran polymers, whereas others produce a mixed population of very high and low molar mass products directly from sucrose substrate. Identifying the determinants dictating polymer elongation would allow the tight control of dextran size. To explore this central question, we focus on the recently discovered DSR-M enzyme from Leuconostoc citreum NRRL B-1299, which is the sole enzyme that naturally, exclusively, and very efficiently produces only low molar mass dextrans from sucrose. Extensive biochemical and structural characterization of a truncated form of DSR-M (DSR-MΔ2, displaying the same biochemical behavior as the parental enzyme) and X-ray structural analysis of complexes with sucrose and isomaltotetraose molecules together with accurate monitoring of the resulting polymer formation reveal that DSR-MΔ2 adopts a nonprocessive mechanism attributed to (i) a high propensity to recognize sucrose as a preferred acceptor at the initial stage of catalysis, (ii) an ability to elongate oligodextrans irrespective of their size, and (iii) the presence of a domain V showing a weak ability to bind to the growing dextran chains. In this study, we present the 3D structure with the largest defined domain V reported to date in the GH70 family and map sugar binding pockets on the basis of the structure of the complex obtained with isomaltotetraose. Altogether, these findings give insights into the interplay between the domain V and the catalytic site during polymerization. They open promising strategies for GH70 enzyme engineering aiming at modulating glucan size.

Published
2017
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5. Characterization of the First α-(1→3) Branching Sucrases of the GH70 Family

Author

Sandrine Morel, Marlène Vuillemin, Etienne Séverac, Yoann Brison, Claire Moulis, Marion Claverie, Pauline Bondy, Magali Remaud-Simeon, Pierre Monsan, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), ANR-12-CDII-0005, Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), ANR-12-CDII-0005,ENGEL,Ingénierie d'une Plateforme Microbienne pour la Production de Biopolymères et Glycoconjugués(2012), Moulis, Claire, Remaud Simeon, Magali, and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], 030106 microbiology, gène codant, Biology, medicine.disease_cause, Biochemistry, law.invention, Sucrase, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, law, Leuconostoc citreum, Glucansucrase, medicine, Leuconostoc, Glycoside hydrolase, Molecular Biology, Gene, analyse phylogénique, bactérie, Cell Biology, biology.organism_classification, bacterium, Molecular biology, 030104 developmental biology, Dextran, chemistry, Enzymology, biology.protein, Recombinant DNA
Abstract

Leuconostoc citreum NRRL B-742 has been known for years to produce a highly alpha-(1 -> 3)-branched dextran for which the synthesis had never been elucidated. In this work a gene coding for a putative alpha-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 alpha-(1 -> 6) dextrans through the specific formation of alpha-(1 -> 3) linkages. To date, BRS-B is the first reported alpha-(1 -> 3) branching sucrase. Using a suitable sucrose/dextran ratio, a comb-like dextran with 50% of alpha-(1 -> 3) branching was synthesized, suggesting that BRS-B is likely involved in the comb-like dextran produced by L. citreum NRRL 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 of Leuconostoc fallax KCTC3537 and Lactobacillus kunkeei EFB6. Biochemical characterization of the corresponding recombinant enzymes confirmed their branching specificity, revealing that branching sucrases are not only found in L. citreum species. According to phylogenetic analyses, these enzymes are proposed to constitute a new subgroup of the GH70 family.

Published
2016
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6. Inventory of the GH70 enzymes encoded by Leuconostoc citreum NRRL B-1299 - identification of three novel -transglucosylases

Author

Catherine Fontagné-Faucher, Pierre Monsan, Delphine Passerini, Valentin Loux, Claire Moulis, Magali Remaud-Simeon, Marlène Vuillemin, Sandrine Morel, Lisa Ufarté, Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Institut National Polytechnique (Toulouse) (Toulouse INP), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Mathématiques et Informatique Appliquées du Génome à l'Environnement [Jouy-En-Josas] (MaIAGE), Institut National de la Recherche Agronomique (INRA), Laboratoire de Biotechnologies Agroalimentaire et Environnementale (LBAE), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut Universitaire de Technologie - Paul Sabatier (IUT Paul Sabatier), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), Région Midi-Pyrénées (France), ANR-10-ALIA-0003,OENOPOLYS,Les polysaccharides de la bactérie lactique Oenococcus oeni, de l'élucidation de leurs structures et voies de biosynthèse à leur valorisation technologique(2010), ANR-12-CDII-0005,ENGEL,Ingénierie d'une Plateforme Microbienne pour la Production de Biopolymères et Glycoconjugués(2012), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT), Université de Toulouse (UT), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut Universitaire de Technologie - Paul Sabatier (IUT Paul Sabatier), and Université de Toulouse (UT)-Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3)

Subjects
[SDV]Life Sciences [q-bio], Molecular Sequence Data, glucansucrase, Biology, medicine.disease_cause, Biochemistry, Genome, branching sucrase, Substrate Specificity, Sucrase, Bacterial Proteins, Leuconostoc citreum, [SDV.IDA]Life Sciences [q-bio]/Food engineering, Glucansucrase, medicine, Consensus sequence, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, [INFO]Computer Science [cs], Amino Acid Sequence, [MATH]Mathematics [math], Molecular Biology, Gene, Prophage, transglucosylase, dextransucrase, Molecular Sequence Annotation, Cell Biology, Hydrogen-Ion Concentration, Kinetics, Leuconostoccitreum NRRL B-1299, Glucosyltransferases, Chromosomal region, biology.protein, Leuconostoc
Abstract

International audience; Leuconostoccitreum NRRL B-1299 has long been known to produce -glucans containing both -(16) and -(12) linkages, which are synthesized by -transglucosylases of the GH70 family. We sequenced the genome of Leuconostoccitreum 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 sucraseA (BRS-A) grafts linear -(16) dextran with -(12)-linked glucosyl units, and is probably involved in the -(12) branching of L.citreum NRRL B-1299 dextran. This is the first report of a naturally occurring -(12) branching sucrase. DSR-M and DSR-DP are dextransucrases that are specific for -(16) linkage synthesis and mainly produce oligomers or short dextrans with molar masses between 580 and 27000gmol(-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.

Published
2015
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7. Complete Genome Sequence of Leuconostoc citreum Strain NRRL B-742

Author

Hervé Robert, Delphine Passerini, Valérie Gabriel, Valentin Loux, Pierre Monsan, Bruno Gabriel, Marlène Vuillemin, Sandrine Laguerre, Magali Remaud-Simeon, Claire Moulis, Sandrine Morel, Myriam Amari, Catherine Fontagné-Faucher, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biotechnologies Agroalimentaire et Environnementale (LBAE), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut Universitaire de Technologie - Paul Sabatier (IUT Paul Sabatier), Université de Toulouse (UT)-Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT), Unité Mathématique Informatique et Génome (MIG), Institut National de la Recherche Agronomique (INRA), ANR-12-CDII-0005,ENGEL,Ingénierie d'une Plateforme Microbienne pour la Production de Biopolymères et Glycoconjugués(2012), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Fédérale Toulouse Midi-Pyrénées, Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut Universitaire de Technologie - Paul Sabatier (IUT Paul Sabatier), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), and Toulouse White Biotechnology (TWB)

Subjects
microbial, [SDV]Life Sciences [q-bio], medicine.disease_cause, Genome, Microbiology, 03 medical and health sciences, chemistry.chemical_compound, Leuconostoc citreum, [SDV.IDA]Life Sciences [q-bio]/Food engineering, Genetics, medicine, genome, genomics, [INFO]Computer Science [cs], [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Prokaryotes, [MATH]Mathematics [math], Molecular Biology, Fermentation in food processing, 030304 developmental biology, Whole genome sequencing, 0303 health sciences, biology, Strain (chemistry), 030306 microbiology, food and beverages, biology.organism_classification, Lactic acid, carbohydrates (lipids), chemistry, bacteria, Bacteria
Abstract

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.

Published
2014
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8. Optimizing the production of an α-(1→2) branching sucrase in [i]Escherichia coli[/i] using statistical design

Author

Claire Moulis, Magali Remaud-Simeon, Marlène Vuillemin, Yannick Malbert, Sandrine Laguerre, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), French National Research Agency [ANR-10-ALIA-0003], Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA)

Subjects
0106 biological sciences, Time Factors, [SDV]Life Sciences [q-bio], Glucansucrase, Biology, Biostatistics, medicine.disease_cause, 01 natural sciences, Applied Microbiology and Biotechnology, law.invention, Sucrase, 03 medical and health sciences, chemistry.chemical_compound, Erlenmeyer flask, Response surface methodology, law, 010608 biotechnology, medicine, Escherichia coli, Inducer, Lactose, E. coli heterologous protein production, 030304 developmental biology, 0303 health sciences, Expression vector, Protein expression optimization, Temperature, General Medicine, Culture Media, chemistry, Biochemistry, biology.protein, Box-Behnken design, Biotechnology
Abstract

Experimental design and Response Surface Methodology (RSM) were used to optimize the production of a dagger N-123-GBD-CD2, an alpha-(1 -> aEuro parts per thousand 2) branching sucrase previously reported as mainly produced in inclusion bodies. The a dagger N-123-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.

Published
2014
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9. Genome Sequences of Three Leuconostoc citreum Strains, LBAE C10, LBAE C11, and LBAE E16, Isolated from Wheat Sourdoughs

Author

Sandrine Laguerre, Sandrine Morel, Magali Remaud-Simeon, Valérie Gabriel, Catherine Fontagné-Faucher, Christophe Klopp, Valentin Loux, Hervé Robert, Bruno Gabriel, Claire Moulis, Marlène Vuillemin, Myriam Amari, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biotechnologies Agroalimentaire et Environnementale (LBAE), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-IUT 'A' Paul Sabatier, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Laboratoire de Biotechnologies Agroalimentaire et Environnementale, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Institut Universitaire de Technologie, Unité Mathématique, Informatique et Génome (MIG), Institut National de la Recherche Agronomique (INRA), Unité de Biométrie et Intelligence Artificielle (UBIA), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut Universitaire de Technologie - Paul Sabatier (IUT Paul Sabatier), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut Universitaire de Technologie - Paul Sabatier (IUT Paul Sabatier), Université de Toulouse (UT)-Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT), Unité Mathématique Informatique et Génome (MIG), and Unité de Biométrie et Intelligence Artificielle (ancêtre de MIAT) (UBIA)

Subjects
DNA, Bacterial, Sequence analysis, Molecular Sequence Data, comparative genomics, medicine.disease_cause, Microbiology, Genome, génomique comparée, génomique, 03 medical and health sciences, Leuconostoc citreum, medicine, genomics, Leuconostoc, Food microbiology, bacteria, Molecular Biology, Fermentation in food processing, Triticum, 030304 developmental biology, 2. Zero hunger, Genetics, Whole genome sequencing, Comparative genomics, bacterie, 0303 health sciences, biology, 030306 microbiology, food and beverages, Sequence Analysis, DNA, biology.organism_classification, GENOMIQUE, Genome Announcements, carbohydrates (lipids), [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Food Microbiology, Genome, Bacterial
Abstract

Leuconostoc citreum is a key microorganism in fermented foods of plant origin. Here we report the draft genome sequence for three strains of Leuconostoc citreum , LBAE C10, LBAE C11, and LBAE E16, which have been isolated from traditional French wheat sourdoughs.

Published
2012
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10. Genome sequence of weissella confusa LBAE C39-2, isolated from a wheat sourdough

Author

Bruno Gabriel, Sandrine Morel, Sandrine Laguerre, Valentin Loux, Myriam Amari, Magali Remaud-Simeon, Hervé Robert, Valérie Gabriel, Marlène Vuillemin, Catherine Fontagné-Faucher, Christophe Klopp, Claire Moulis, Laboratoire de Biotechnologies Agroalimentaire et Environnementale (LBAE), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-IUT 'A' Paul Sabatier, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Unité Mathématique, Informatique et Génome (MIG), Institut National de la Recherche Agronomique (INRA), Unité de Biométrie et Intelligence Artificielle (UBIA), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut Universitaire de Technologie - Paul Sabatier (IUT Paul Sabatier), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut Universitaire de Technologie - Paul Sabatier (IUT Paul Sabatier), Université de Toulouse (UT)-Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Unité Mathématique Informatique et Génome (MIG), and Unité de Biométrie et Intelligence Artificielle (ancêtre de MIAT) (UBIA)

Subjects
DNA, Bacterial, Weissella, Sequence analysis, Molecular Sequence Data, Microbiology, Genome, 03 medical and health sciences, Food microbiology, Food science, microbiologie, bacteria, Molecular Biology, genome, Triticum, 030304 developmental biology, Whole genome sequencing, bactérie, 0303 health sciences, biology, 030306 microbiology, Strain (biology), Microbiology and Parasitology, food and beverages, Sequence Analysis, DNA, biology.organism_classification, génome, GENOMIQUE, 6. Clean water, Microbiologie et Parasitologie, Genome Announcements, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Food Microbiology, Leuconostocaceae, Bacteria, Genome, Bacterial
Abstract

Weissella confusa is a rod-shaped heterofermentative lactic acid bacterium from the family of Leuconostocaceae . Here we report the draft genome sequence of the strain W. confusa LBAE C39-2 isolated from a traditional French wheat sourdough.

Published
2012
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11. The Endo-α(1,4) Specific Fucoidanase Fhf2 From Formosa haliotis Releases Highly Sulfated Fucoidan Oligosaccharides

Author

Vo Thi Dieu Trang, Maria Dalgaard Mikkelsen, Marlene Vuillemin, Sebastian Meier, Hang Thi Thuy Cao, Jan Muschiol, Valentina Perna, Thuan Thi Nguyen, Vy Ha Nguyen Tran, Jesper Holck, Tran Thi Thanh Van, Huynh Hoang Nhu Khanh, and Anne S. Meyer

Subjects
FTIR, Fucus evanescens, Sargassum mcclurei, calcium dependency, T9SS, sulfation, Plant culture, SB1-1110
Abstract

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.

Published
2022
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12. Exopolysaccharide (EPS) Synthesis by Oenococcus oeni: From Genes to Phenotypes

Author

Maryline Roques, Marion Favier, Virginie Moine, Marguerite Dols-Lafargue, Hugo Campbell-Sills, Magali Remaud-Simeon, Thierry Doco, Melina Petrel, Cécile Miot-Sertier, Marlène Vuillemin, Pascale Williams, Joana Coulon, Patricia Ballestra, Claire Moulis, Maria Dimopoulou, Etienne Gontier, Patrick Lucas, Unité de Recherche Oenologie [Villenave d'Ornon], Institut National de la Recherche Agronomique (INRA)-Université de Bordeaux (UB)-Institut des Sciences de la Vigne et du Vin (ISVV), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), USC 1366 Oenologie, Institut National de la Recherche Agronomique (INRA), Sciences Pour l'Oenologie (SPO), Institut National de la Recherche Agronomique (INRA)-Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut de Recherche pour le Développement (IRD [Nouvelle-Calédonie])-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Université Sciences et Technologies - Bordeaux 1, Université Montpellier 1 (UM1)-Institut National de la Recherche Agronomique (INRA)-Université de Montpellier (UM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Unité de Recherche Oenologie [Villenave d'Ornon] (OENO), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Université Sciences et Technologies - Bordeaux 1 (UB)

Subjects
Genotype, bactérie lactique, [SDV]Life Sciences [q-bio], lcsh:Medicine, Biochemistry, Microbiology, oenococcus oeni, Homopolysaccharide, chemistry.chemical_compound, [SDV.IDA]Life Sciences [q-bio]/Food engineering, Gene Order, vin, Genetics, Malolactic fermentation, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, lcsh:Science, Gene, Oenococcus, Phylogeny, Oenococcus oeni, 2. Zero hunger, fermentation alcoolique, Multidisciplinary, biology, lcsh:R, Polysaccharides, Bacterial, Glycosyltransferase Gene, Chromosome Mapping, Biology and Life Sciences, Genomics, Chromosomes, Bacterial, biology.organism_classification, Phenotype, Biosynthetic Pathways, oenologie, chemistry, Genes, Bacterial, Genetic Loci, Multigene Family, Galactose, exopolysaccharide, lcsh:Q, expression des gènes, Research Article, Biotechnology
Abstract

International audience; 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 β-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 β-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.

Published
2014
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13. Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering

Author

Marlene Vuillemin, Jesper Holck, Martin Matwiejuk, Eduardo S. Moreno Prieto, Jan Muschiol, Dora Molnar-Gabor, Anne S. Meyer, and Birgitte Zeuner

Subjects
lacto-N-biosidase, GH20, transglycosylation, protein engineering, lacto-N-tetraose, Technology, Engineering (General). Civil engineering (General), TA1-2040, Biology (General), QH301-705.5, Physics, QC1-999, Chemistry, QD1-999
Abstract

The lacto-N-biosidase LnbB from Bifidobacterium bifidum JCM 1254 was engineered to improve its negligible transglycosylation efficiency with the purpose of enzymatically synthesizing lacto-N-tetraose (LNT; Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc) in one enzymatic step. LNT is a prebiotic human milk oligosaccharide in itself and constitutes the structural core of a range of more complex human milk oligosaccharides as well. Thirteen different LnbB variants were expressed and screened for transglycosylation activity by monitoring transglycosylation product formation using lacto-N-biose 1,2-oxazoline as donor substrate and lactose as acceptor substrate. LNT was the major reaction product, yet careful reaction analysis revealed the formation of three additional LNT isomers, which we identified to have a β1,2-linkage, a β1,6-linkage, and a 1,1-linkage, respectively, between lacto-N-biose (Gal-β1,3-GlcNAc) and lactose. Considering both maximal transglycosylation yield and regioselectivity as well as minimal product hydrolysis, the best variant was LnbB W394H, closely followed by W465H and Y419N. A high transglycosylation yield was also obtained with W394F, yet the substitution of W394 and W465 of the subsite −1 hydrophobic platform in the enzyme with His dramatically impaired the undesirable product hydrolysis as compared to substitution with Phe; the effect was most pronounced for W465. Using p-nitrophenyl-β-lacto-N-bioside as donor substrate manifested W394 as an important target position. The optimization of the substrate concentrations confirmed that high initial substrate concentration and high acceptor-to-donor ratio both favor transglycosylation.

Published
2021
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14. Specificities and Synergistic Actions of Novel PL8 and PL7 Alginate Lyases from the Marine Fungus Paradendryphiella salina

Author

Bo Pilgaard, Marlene Vuillemin, Jesper Holck, Casper Wilkens, and Anne S. Meyer

Subjects
alginate lyase, polysaccharide lyase, marine fungi, enzyme characterization, alginate, polymannuronic acid, Biology (General), QH301-705.5
Abstract

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.

Published
2021
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15. Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum

Author

Birgitte Zeuner, Marlene Vuillemin, Jesper Holck, Jan Muschiol, and Anne S. Meyer

Subjects
fucosidase, xyloglucan, 2′-fucosyllactose, human milk oligosaccharides, protein engineering, GH29, Biology (General), QH301-705.5
Abstract

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.

Published
2020
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16. Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis

Author

Marlene Vuillemin, Artem S. Silchenko, Hang Thi Thuy Cao, Maxim S. Kokoulin, Vo Thi Dieu Trang, Jesper Holck, Svetlana P. Ermakova, Anne S. Meyer, and Maria Dalgaard Mikkelsen

Subjects
fucoidan, fucoidanase, Fucus evanescens, GH107, fuco-oligosaccharides, Biology (General), QH301-705.5
Abstract

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.

Published
2020
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