Expanding the Enzymatic Polymerization Landscape by Lipid Mesophase Soft Nanoconfinement.

Soft nanoconfinement can increase chemical reactivity in nature and has therefore led to considerable interest in transferring this universal feature to artificial biological systems. However, little is known about the underlying principles of soft nanoconfinement responsible for the enhancement of biochemical reactions. Herein we demonstrate how enzymatic polymerization can be expanded, optimized, and engineered when carried out under soft nanoconfinement mediated by lipidic mesophases. By systematically varying the water content in the mesophase and thus the diameter of the confined water nanochannels, we show higher efficiency, turnover rate, and degrees of polymerization as compared to the bulk aqueous solution, all controlled by soft nanoconfinement effects. Furthermore, we exploit the unique properties of unfreezing soft nanoconfined water to perform the first enzymatic polymerization at -20 °C in pure aqueous media. These results underpin lipidic mesophases as a versatile host system for chemical reactions and promote them as an original and unexplored platform for enzymatic polymerization.

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.

A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation.

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.

Convergent Chemoenzymatic Strategy to Deliver a Diversity of Shigella flexneri Serotype-Specific O-Antigen Segments from a Unique Lightly Protected Tetrasaccharide Core.

Progress in glycoscience is strongly dependent on the availability of broadly diverse tailor-made, well-defined, and often complex oligosaccharides. Herein, going beyond natural resources and aiming to circumvent chemical boundaries in glycochemistry, we tackle the development of an in vitro chemoenzymatic strategy holding great potential to answer the need for molecular diversity characterizing microbial cell-surface carbohydrates. The concept is exemplified in the context of Shigella flexneri, a major cause of diarrhoeal disease. Aiming at a broad serotype coverage S. flexneri glycoconjugate vaccine, a non-natural lightly protected tetrasaccharide was designed for compatibility with (i) serotype-specific glucosylations and O-acetylations defining S. flexneri O-antigens, (ii) recognition by suitable α-transglucosylases, and (iii) programmed oligomerization following enzymatic α-d-glucosylation. The tetrasaccharide core was chemically synthesized from two crystalline monosaccharide precursors. Six α-transglucosylases found in the glycoside hydrolase family 70 were shown to transfer glucosyl residues on the non-natural acceptor. The successful proof of concept is achieved for a pentasaccharide featuring the glucosylation pattern from the S. flexneri type IV O-antigen. It demonstrates the potential of appropriately planned chemoenzymatic pathways involving non-natural acceptors and low-cost donor/transglucosylase systems to achieve the demanding regioselective α-d-glucosylation of large substrates, paving the way to microbial oligosaccharides of vaccinal interest.

Futile Encounter Engineering of the DSR-M Dextransucrase Modifies the Resulting Polymer Length.

The factors that define the resulting polymer length of distributive polymerases are poorly understood. Here, starting from the crystal structure of the dextransucrase DSR-M in complex with an isomaltotetraose, we define different anchoring points for the incoming acceptor. Mutation of one of these, Trp624, decreases the catalytic rate of the enzyme but equally skews the size distribution of the resulting dextran chains toward shorter chains. Nuclear magnetic resonance analysis shows that this mutation influences both the dynamics of the active site and the water accessibility. Monte Carlo simulation of the elongation process allows interpretation of these results in terms of enhanced futile encounters, whereby the less effective binding increases the pool of effective seeds for the dextran chains and thereby directly determines the length distribution of the final polymers.

The stability of an mRNA is influenced by its concentration: a potential physical mechanism to regulate gene expression.

Changing mRNA stability is a major post-transcriptional way of controlling gene expression, particularly in newly encountered conditions. As the concentration of mRNA is the result of an equilibrium between transcription and degradation, it is generally assumed that at constant transcription, any change in mRNA concentration is the consequence of mRNA stabilization or destabilization. However, the literature reports many cases of opposite variations in mRNA concentration and stability in bacteria. Here, we analyzed the causal link between the concentration and stability of mRNA in two phylogenetically distant bacteria Escherichia coli and Lactococcus lactis. Using reporter mRNAs, we showed that modifying the stability of an mRNA had unpredictable effects, either higher or lower, on its concentration, whereas increasing its concentration systematically reduced stability. This inverse relationship between the concentration and stability of mRNA was generalized to native genes at the genome scale in both bacteria. Higher mRNA turnover in the case of higher concentrations appears to be a simple physical mechanism to regulate gene expression in the bacterial kingdom. The consequences for bacterial adaptation of this control of the stability of an mRNA by its concentration are discussed.

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.

GH13 amylosucrases and GH70 branching sucrases, atypical enzymes in their respective families.

Amylosucrases and branching sucrases are α-retaining transglucosylases found in the glycoside-hydrolase families 13 and 70, respectively, of the clan GH-H. These enzymes display unique activities in their respective families. Using sucrose as substrate and without mediation of nucleotide-activated sugars, amylosucrase catalyzes the formation of an α-(1 â†’ 4) linked glucan that resembles amylose. In contrast, the recently discovered branching sucrases are unable to catalyze polymerization of glucosyl units as they are rather specific for dextran branching through α-(1 â†’ 2) or α-(1 â†’ 3) branching linkages depending on the enzyme regiospecificity. In addition, GH13 amylosucrases and GH70 branching sucrases are naturally promiscuous and can glucosylate different types of acceptor molecules including sugars, polyols, or flavonoids. Amylosucrases have been the most investigated glucansucrases, in particular to control product profiles or to successfully develop tailored α-transglucosylases able to glucosylate various molecules of interest, for example, chemically protected carbohydrates that are planned to enter in chemoenzymatic pathways. The structural traits of these atypical enzymes will be described and compared, and an overview of the potential of natural or engineered enzymes for glycodiversification and chemoenzymatic synthesis will be highlighted.

Evaluation of dough rheological properties and bread texture of pearl millet-wheat flour mix.

This study was undertaken with the objective of formulating composite bread using pearl millet (Pennisetum glaucum) and wheat (Triticum aestivum) flours . Rheological and bread making properties of composite flours were evaluated. Mixolab results revealed torque increased and dough stability time decreased upon incorporation of pearl millet flour in wheat flour. The incorporation of millet flour at optimum level (5 %) led to an increase of the dough strength (W) and the elasticity-to-extensibility ratio (P/L) by 31 % and 65 % respectively. The bread texture and volume were also improved. These findings indicated the potentiality of using millet flour in bread making.

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.

A chemo-enzymatic pathway to expand cellooligosaccharide chemical space through amine bond introduction.

Enzymatic functionalization of oligosaccharides is a useful and environmentally friendly way to expand their structural chemical space and access to a wider range of applications in the health, food, feed, cosmetics and other sectors. In this work, we first tested the laccase/TEMPO system to generate oxidized forms of cellobiose and methyl ß-D-cellobiose, and obtained high yields of novel anionic disaccharides (>60 %) at pH 6.0. Laccase/TEMPO system was then applied to a mix of cellooligosaccharides and to pure D-cellopentaose. The occurrence of carbonyl and carboxyl groups in the oxidation products was shown by LC-HRMS, MALDI-TOF and reductive amination of the carbonyl groups was attempted with p-toluidine a low molar mass amine to form the Schiff base, then reduced by 2-picoline borane to generate a more stable amine bond. The new grafted products were characterized by LC-HRMS, LC-UV-MS/MS and covalent grafting was evidenced. Next, the same procedure was adopted to successfully graft a dye, the rhodamine 123, larger in size than toluidine. This two-step chemo-enzymatic approach, never reported before, for functionalization of oligosaccharides, offers attractive opportunities to anionic cellooligosaccharides and derived glucoconjugates of interest for biomedical or neutraceutical applications. It also paves the way for more environmentally-friendly cellulose fabric staining procedures.

Characterization of a novel dextransucrase from Weissella confusa isolated from sourdough.

Weissella confusa and Weissella cibaria isolated from wheat sourdoughs produce, from sucrose, linear dextrans due to a single soluble dextransucrase. In this study, the first complete gene sequence encoding dextransucrase from a W. confusa strain (LBAE C39-2) along with the one from a W. cibaria strain (LBAE K39) were reported. Corresponding gene cloning was achieved using specific primers designed on the basis of the draft genome sequence of these species. Deduced amino acid sequence of W. confusa and W. cibaria dextransucrase revealed common structural features of the glycoside hydrolase family 70. Notably, the regions located in the vicinity of the catalytic triad (D, E, D) are highly conserved. However, comparison analysis also revealed that Weissella dextransucrases form a distinct phylogenetic group within glucansucrases of other lactic acid bacteria. We then cloned the W. confusa C39-2 dextransucrase gene and successfully expressed the mature corresponding enzyme in Escherichia coli. The purified recombinant enzyme rDSRC39-2 catalyzed dextran synthesis from sucrose with a K m of 8.6 mM and a V max of 20 µmol/mg/min. According to (1)H and (13)C NMR analysis, the polymer is a linear class 1 dextran with 97.2 % α-(1→6) linkages and 2.8 % α-(1→3) branch linkages, similar to the one produced by W. confusa C39-2 strain. The enzyme exhibited optimum catalytic activity for temperatures ranging from 35 to 40 °C and a pH of 5.4 in 20 mM sodium acetate buffer. This novel dextransucrase is responsible for production of dextran with predominant α-(1→6) linkages that could find applications as food hydrocolloids.

Phosphorylated polysaccharides: Applications, natural abundance, and new-to-nature structures generated by chemical and enzymatic functionalisation.

Polysaccharides are foreseen as serious candidates for the future generation of polymers, as they are biosourced and biodegradable materials. Their functionalisation is an attractive way to modify their properties, thereby increasing their range of applications. Introduction of phosphate groups in polysaccharide chains for the stimulation of the immune system was first described in the nineteen seventies. Since then, the use of phosphorylated polysaccharides has been proposed in various domains, such as healthcare, water treatment, cosmetic, biomaterials, etc. These alternative usages capitalize on newly acquired physico-chemical or biological properties, leading to materials as diverse as flame-resistant agents or drug delivery systems. Phosphorylated polysaccharides are found in Nature and need to be extracted to assess their biological potential. However, they are not abundant, often present complex backbones hard to characterize, and most of them have a low phosphate content. These drawbacks have pushed forward the development of chemical phosphorylation employing a wide variety of phosphorylating agents to obtain polysaccharides with a large range of phosphate content. Chemical phosphorylation requires the use of harsh conditions and toxic, petroleum-based solvents, which hinders their exploitation in the food and health industry. Over the last 20 years, although enzymes are regiospecific catalysts that work in aqueous and mild conditions, enzymatic phosphorylation has been little investigated. To date, only three families of enzymes have been used for the in vitro phosphorylation of polysaccharides. Considering the number of unresolved metabolic pathways leading to phosphorylated polysaccharides, the huge diversity of kinase sequences, and the recent progress in protein engineering one can envision native and engineered kinases as promising tools for polysaccharide phosphorylation.

Genome sequence of Weissella confusa LBAE C39-2, isolated from a wheat sourdough.

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.

Genome sequences of three Leuconostoc citreum strains, LBAE C10, LBAE C11, and LBAE E16, isolated from wheat sourdoughs.

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.

Applying pairwise combinations of amino acid mutations for sorting out highly efficient glucosylation tools for chemo-enzymatic synthesis of bacterial oligosaccharides.

Iterative saturation mutagenesis and combinatorial active site saturation focused on vicinal amino acids were used to alter the acceptor specificity of amylosucrase from Neisseria polysaccharea , a sucrose-utilizing α-transglucosidase, and sort out improved variants. From the screening of three semirational sublibraries accounting in total for 20,000 variants, we report here the isolation of three double mutants of N. polysaccharea amylosucrase displaying a spectacular specificity enhancement toward both sucrose, the donor substrate, and the allyl 2-acetamido-2-deoxy-α-D-glucopyranoside acceptor as compared to the wild-type enzyme. Such levels of activity improvement have never been reported before for this class of carbohydrate-active enzymes. X-ray structure of the best performing enzymes supported by molecular dynamics simulations showed local rigidity of the -1 subsite as well as flexibility of loops involved in active site topology, which both account for the enhanced catalytic performances of the mutants. The study well illustrates the importance of taking into account the local conformation of catalytic residues as well as protein dynamics during the catalytic process, when designing enzyme libraries.

Structure and property engineering of α-D-glucans synthesized by dextransucrase mutants.

Seven dextran types, displaying from 3 to 20% α(1→3) glycosidic linkages, were synthesized in vitro from sucrose by mutants of dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, obtained by combinatorial engineering. The structural and physicochemical properties of these original biopolymers were characterized. When asymmetrical flow field flow fractionation coupled with multiangle laser light scattering was used, it was determined that weight average molar masses and radii of gyration ranged from 0.76 to 6.02 × 10(8) g·mol(-1) and from 55 to 206 nm, respectively. The ν(G) values reveal that dextrans Gcn6 and Gcn7, which contain 15 and 20% α(1→3) linkages, are highly branched and contain long ramifications, while Gcn1 is rather linear with only 3% α(1→3) linkages. Others display intermediate molecular structures. Rheological investigation shows that all of these polymers present a classical non-Newtonian pseudoplastic behavior. However, Gcn_DvΔ4N, Gcn2, Gcn3, and Gcn7 form weak gels, while others display a viscoelastic behavior that is typical of entangled polymer solutions. Finally, glass transition temperature T(g) was measured by differential scanning calorimetry. Interestingly, the T(g) of Gcn1 and Gcn5 are equal to 19.0 and 29.8 °C, respectively. Because of this low T(g), these two original dextrans are able to form rubber and flexible films at ambient temperature without any plasticizer addition. The mechanical parameters determined for Gcn1 films from tensile tests are very promising in comparison to the films obtained with other polysaccharides extracted from plants, algae or microbial fermentation. These results lead the way to using these dextrans as innovative biosourced materials.

NMR-based structural glycomics for high-throughput screening of carbohydrate-active enzyme specificity.

We report here the development of a straightforward, sensitive, and quantitative NMR-based method for high-throughput characterization of carbohydrate structure and screening of carbohydrate active enzyme (CAZyme) specificity. Automated assays starting from gene library expression to carbohydrate structure determination directly from crude reaction media have been established and successfully used to screen a library of 4032 CAZymes obtained by combinatorial engineering, at a rate of 480 enzyme variants per day. This allowed one to accurately discriminate 303 enzyme variants with altered specificity. The results demonstrate the potential of high-throughput NMR technology in glycomics, to mine artificial and natural enzyme diversity for novel biocatalysts.

Isolation of a gene from Leuconostoc citreum B/110-1-2 encoding a novel dextransucrase enzyme.

The amplicon encoding dextransucrase DSR-F from Leuconostoc citreum B/110-1-2, a novel sucrose glucosyltransferase (GTF)-specific for α-1,6 and α-1,3 glucosidic bond synthesis, with α-1,4 branching was cloned, sequenced, and expressed into Escherichia coli JM109. Recombinant enzyme catalyzed oligosaccharides synthesis from sucrose as donor and maltose acceptor. The dsrF gene encodes for a protein (DSR-F) of 1,528 amino acids, with a theoretical molecular mass of 170447.72 Da (~170 kDa). From amino acid sequence comparison, it appears that DSR-F possesses the same domains as those described for GTFs. However, the variable region is longer than in other GTFs (by 100 amino acids) and two APY repeats (a 79 residue long motif with a high number of conserved glycine and aromatic residues, characterized by the presence of the three consecutive residues Ala, Pro, and Tyr) were identified in the glucan binding domain. The DSR-F catalytic domain possesses the catalytic triad involved in the glucosyl enzyme formation. The amino acid sequence of this domain shares a 56% identity with catalytic domain of the alternansucrase ASR from L. citreum NRRL B-1355 and with the catalytic domain of a putative alternansucrase sequence found in the genome of L. citreum KM20. A truncated active variant DSR-F-∆SP-∆GBD of 1,251 amino acids, with a molecular mass of 145 544 Da (~145 kDa), was obtained.

A novel dextransucrase is produced by Leuconostoc citreum strain B/110-1-2: an isolate used for the industrial production of dextran and dextran derivatives.

The industrial Leuconostoc strain B/110-1-2 producing dextran and dextran derivatives was taxonomically identified by 16S rRNA as L. citreum. Its dextransucrase enzymes were characterized according to their cellular location and reaction specificity. In the presence of sucrose, the strain B/110-1-2 produced two cell-associated dextransucrases (31.54% of the total glucosyltransferase activity) with molecular weights of 160 and 240 kDa and a soluble dextransucrase (68.46%) at 160-180 kDa. Two open reading frames (ORF) coding for L. citreum strain B/110-1-2 dextransucrases were identified. One of them shared a 52% identity with the alternansucrase ASR of L. citreum NRRL B-1355 and with a putative annotated alternansucrase sequence found in the genome of L. citreum KM20. The structural analysis (HPAEC-PAD, HPSEC, and (13)C-NMR) of the polymer and oligodextrans produced by the B/110-1-2 dextransucrases suggest this novel glucansucrase has specificity similar to a dextransucrase but not to an alternansucrase, producing a soluble linear dextran with glucose molecules linked mainly in α-1,6 and α-1,3 with α-1,4 branches. These results enhance the understanding of this industrially significant strain and will aid in distinguishing between physiologically similar Leuconostoc spp. strains.