Selective microbial production of lacto-N-fucopentaose I in Escherichia coli using engineered α-1,2-fucosyltransferases.

Lacto-N-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2'-fucosyllactose (2'-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B Streptococcus and antiviral effects against Enterovirus and Norovirus. Microbial production of HMOs by engineered Escherichia coli is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the N-acetylglucosamine moiety of the lacto-N-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2'-FL. In this study, we constructed LNFP I-producing strains of E. coli with various α1,2-fucTs, and observed undesired 2'-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2'-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2'-FL production. Therefore, to decrease the formation of byproduct 2'-FL, we designed 15 variants of FsFucT from Francisella sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2'-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154-171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the E. coli strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154-171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2'-FL, while the E. coli strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2'-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2'-FL by combining protein engineering of α1,2-FucT designed through in silico structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.

Peculiarities of Phase Formation in Mn-Based Na SuperIonic Conductor (NaSICon) Systems: The Case of Na1+2x Mn x Ti2-x (PO4)3 (0.0 ≤ x ≤ 1.5).

NAtrium SuperIonic CONductor (NASICON) structured phosphate framework compounds are attracting a great deal of interest as suitable electrode materials for "rocking chair" type batteries. Manganese-based electrode materials are among the most favored due to their superior stability, resource non-criticality, and high electrode potentials. Although a large share of research was devoted to Mn-based oxides for Li- and Na-ion batteries, the understanding of thermodynamics and phase formation in Mn-rich polyanions is still generally lacking. In this study, we investigate a bifunctional Na-ion battery electrode system based on NASICON-structured Na1+2x Mn x Ti2-x (PO4)3 (0.0 ≤ x ≤ 1.5). In order to analyze the thermodynamic and phase formation properties, we construct a composition-temperature phase diagram using a computational sampling by density functional theory, cluster expansion, and semi-grand canonical Monte Carlo methods. The results indicate finite thermodynamic limits of possible Mn concentrations in this system, which are primarily determined by the phase separation into stoichiometric Na3MnTi(PO4)3 (x = 1.0) and NaTi2(PO4)3 for x < 1.0 or NaMnPO4 for x > 1.0. The theoretical predictions are corroborated by experiments obtained using X-ray diffraction and Raman spectroscopy on solid-state and sol-gel prepared samples. The results confirm that this system does not show a solid solution type behavior but phase-separates into thermodynamically more stable sodium ordered monoclinic α-Na3MnTi(PO4)3 (space group C2) and other phases. In addition to sodium ordering, the anti-bonding character of the Mn-O bond as compared to Ti-O is suggested as another important factor governing the stability of Mn-based NASICONs. We believe that these results will not only clarify some important questions regarding the thermodynamic properties of NASICON frameworks but will also be helpful for a more general understanding of polyanionic systems.

An Enolate-Structure-Enabled Anionic Cascade Cyclization Reaction: Easy Access to Complex Scaffolds with Contiguous Six-, Five-, and Four-Membered Rings.

Catalyst-free addition of ketone enolate to non-activated multiple C-C bonds involves non-complementary reaction partners and typically requires super-basic conditions. On the other hand, highly aggregated or solvated enolates are not reactive enough to undergo direct addition to alkenes or alkynes. Herein, we report a new anionic cascade reaction for one-step assembly of intriguing molecular scaffolds possessing contiguous six-, five-, and four-membered rings, representing a formal [2+2] enol-allene cycloaddition. Reaction proceeds under very mild conditions and with excellent diastereoselectivity. Deeper mechanistic and computational studies revealed unusually slow proton transfer phenomenon in cyclic ketone intermediate and explained peculiar stereochemical outcome.

Site-Selective Modification of a Porpholactone-Selective Synthesis of 12,13- and 17,18-Dihydroporpholactones.

The reaction of meso-tetrakis(pentafluorophenyl)porpholactone with azomethine ylides and nitrones affords pyrrolidine-fused and isoxazolidine-fused dihydroporpholactones that display, respectively, isobacteriochlorin- and chlorin-type UV-Vis spectra. These reactions are site-selective, yielding, respectively, 17,18- or 12,13-dihydroporpholactones. The crystal and molecular features of pyrrolidine-fused and isoxazolidine-fused dihydroporpholactones were unveiled from single-crystal X-ray diffraction studies.

Selective Formation of S4- and T-Symmetric Supramolecular Tetrahedral Cages and Helicates in Polar Media Assembled via Cooperative Action of Coordination and Hydrogen Bonds.

We report on the synthesis and self-assembly study of novel supramolecular monomers encompassing quadruple hydrogen-bonding motifs and metal-coordinating 2,2'-bipyridine units. When mixed with metal ions such as Fe2+ or Zn2+, the tetrahedron cage complexes are formed in quantitative yields and full diastereoselectivity, even in highly polar acetonitrile or methanol solvents. The symmetry of the complexes obtained has been shown to depend critically on the flexibility of the ligand. Restriction of the rotation of the hydrogen-bonding unit with respect to the metal-coordinating site results in a T-symmetric cage, whereas introducing flexibility either through a methylene linker or rotating benzene ring allows the formation of S4-symmetric cages with self-filled interior. In addition, the possibility to select between tetrahedral cages or helicates and to control the dimensions of the aggregate has been demonstrated with a three-component assembly using external hydrogen-bonding molecular inserts or by varying the radius of the metal ion (Hg2+ vs Fe2+). Self-sorting studies of individual Fe2+ complexes with ligands of different sizes revealed their inertness toward ligand scrambling.