Coding Synthetic Chemistry Strategies for Furan Valorization into Bacterial Designer Cells

Following a synthetic chemistry blueprint for the valorization of lignocellulosic platform chemicals, this study showcases a so far unprecedented approach to implement non-natural enzyme modules in vivo. For the design of a novel functional whole cell tool, two purely abiotic transformations, a styrene monooxygenase-catalyzed Achmatowicz rearrangement and an alcohol dehydrogenase-mediated borrowing hydrogen redox isomerization, were incorporated into a recombinant bacterial host. Introducing this type of chemistry otherwise unknown in biosynthesis, the cellular factories were enabled to produce complex lactone building blocks in good yield from bio-based furan substrates. This whole cell system streamlined the synthetic cascade, eliminated isolation and purification steps, and provided a high degree of stereoselectivity that has so far been elusive in the chemical methodology.Peer reviewe

In silico characterization of bacterial chitinase : illuminating its relationship with archaeal and eukaryotic cousins

BackgroundChitin is one of the most abundant biopolymers on Earth, only trailing second after cellulose. The enzyme chitinase is responsible for the degradation of chitin. Chitinases are found to be produced by wide range of organisms ranging from archaea to higher plants. Though chitin is a major component of fungal cell walls and invertebrate exoskeletons, bacterial chitinase can be industrially generated at low cost, in facile downstream processes at high production rate. Microbial chitinases are more stable, active, and economically practicable compared to the plant- and animal-derived enzymes.ResultsIn the present study, computationally obtained results showed functional characteristics of chitinase with particular emphasis on bacterial chitinase which is fulfilling all the required qualities needed for commercial production. Sixty-two chitinase sequences from four different groups of organisms were collected from the RCSB Protein Data Bank. Considering one suitable exemplary sequence from each group is being compared with others. Primary, secondary, and tertiary structures are determined by in silico models. Different physical parameters, viz., pI, molecular weight, instability index, aliphatic index, GRAVY, and presence of functional motifs, are determined, and a phylogenetic tree has been constructed to elucidate relationships with other groups of organisms.ConclusionsThis study provides novel insights into distribution of chitinase among four groups and their characterization. The results represent valuable information toward bacterial chitinase in terms of the catalytic properties and structural features, can be exploited to produce a range of chitin-derived products.Peer reviewe

Enzymatic Bromocyclization of alpha- and gamma-Allenols by Chloroperoxidase from Curvularia inaequalis

Vanadate-dependent chloroperoxidase from Curvularia inaequalis catalyzes 5-endo-trig bromocyclizations of alpha-allenols to produce valuable halofunctionalized furans as versatile synthetic building blocks. In contrast to other haloperoxidases, also the more challenging 5-exo-trig halocyclizations of gamma-allenols succeed with this system even though the scope still remains more narrow. Benefitting from the vanadate chloroperoxidase's high resiliency towards oxidative conditions, cyclization-inducing reactive hypohalite species are generated in situ from bromide salts and hydrogen peroxide. Crucial requirements for high conversions are aqueous biphasic emulsions as reaction media, stabilized by either cationic or non-ionic surfactants.Peer reviewe

Synthesis of silver and gold nanoparticles-enzyme-polymer conjugate hybrids as dual-activity catalysts for chemoenzymatic cascade reactions

Novel hybrids containing silver or gold nanoparticles have been synthesized in aqueous media and at room temperature using enzymes or tailor-made enzyme-polymer conjugates, which directly induced the formation of inorganic silver or gold species. The choice of pH, protein, or bioconjugate strongly affected the final metallic nanoparticles hybrid formation. Using Candida antarctica lipase (CALB) in a solution, nanobiohybrids containing Ag2O nanoparticles of 9 nm average diameter were obtained. The use of tailor-made bioconjugates, for example, the CALB modified with dextran-aspartic acid polymer (Dext6kDa), resulted in a nanobiohybrid containing smaller Ag(0)/Ag2O nanoparticles. In the case of nanobiohybrids based on gold, Au(0) species were found in all cases. The Au-CALB hybrid contained spherical nanoparticles with 18 nm average diameter size, with a minor range of larger ones (>100 nm) while the AuNPs-CALB-Dext6kDa hybrid was formed by much smaller nanoparticles (9 nm, minor range of 22 nm), and also nanorods of 20-30/40-50 nm length. Using Thermomyces lanuginosus lipase (TLL), apart from the nanoparticle formation, nanoflowers with a diameter range of 100-200 nm were obtained. All nanobiohybrids maintained (dual) enzymatic and metallic activities. For instance, these nanobiohybrids exhibited exquisite dual-activity for hydrolysis/cycloisomerization cascades starting from allenic acetates. By merging the transition metal reactivity with the inherent lipase catalysis, allenic acetates directly converted to the corresponding O-heterocycles in enantiopure form catalysed by AgNPs-CALB-Dext6kDa, taking advantage of a kinetic resolution/cyclization pathway. These results showed the high applicability of these novel hybrids, offering new opportunities for the design of novel reaction cascades.Peer reviewe

Methanol-Driven Oxidative Rearrangement of Biogenic Furans - Enzyme Cascades vs. Photobiocatalysis

The oxidative ring expansion of bio-derived furfuryl alcohols to densely functionalized six-membered O-heterocycles represents an attractive strategy in the growing network of valorization routes to synthetic building blocks out of the lignocellulosic biorefinery feed. In this study, two scenarios for the biocatalytic Achmatowicz-type rearrangement using methanol as terminal sacrificial reagent have been evaluated, comparing multienzymatic cascade designs with a photo-bio-coupled activation pathway.Peer reviewe

Chemoenzymatic Hydrogen Production from Methanol through the Interplay of Metal Complexes and Biocatalysts

Microbial methylotrophic organisms can serve as great inspiration in the development of biomimetic strategies for the dehydrogenative conversion of C1 molecules under ambient conditions. In this Concept article, a concise personal perspective on the recent advancements in the field of biomimetic catalytic models for methanol and formaldehyde conversion, in the presence and absence of enzymes and co-factors, towards the formation of hydrogen under ambient conditions is given. In particular, formaldehyde dehydrogenase mimics have been introduced in stand-alone C1-interconversion networks. Recently, coupled systems with alcohol oxidase and dehydrogenase enzymes have been also developed for in situ formation and decomposition of formaldehyde and/or reduced/oxidized nicotinamide adenine dinucleotide (NADH/ NAD+). Although C1 molecules are already used in many industries for hydrogen production, these conceptual bioinspired low-temperature energy conversion processes may lead one day to more efficient energy storage systems enabling renewable and sustainable hydrogen generation for hydrogen fuel cells under ambient conditions using C1 molecules as fuels for mobile and miniaturized energy storage solutions in which harsh conditions like those in industrial plants are not applicable.Peer reviewe

Bioinduced Room-Temperature Methanol Reforming

Imitating nature's approach in nucleophile-activated formaldehyde dehydrogenation, air-stable ruthenium complexes proved to be exquisite catalysts for the dehydrogenation of formaldehyde hydrate as well as for the transfer hydrogenation to unsaturated organic substrates at loadings as low as 0.5 mol%. Concatenation of the chemical hydrogen-fixation route with an oxidase-mediated activation of methanol gives an artificial methylotrophic in vitro metabolism providing methanol-derived reduction equivalents for synthetic hydrogenation purposes. Moreover, for the first time methanol reforming at room temperature was achieved on the basis of this bioinduced dehydrogenation path delivering hydrogen gas from aqueous methanol

Chelated peptide enolates in palladium-catalyzed allylic substitutions

Die nachträgliche Manipulation physiologisch aktiver Peptide stellt sowohl für die Entwicklung und das Optimieren peptidbasierter Pharmazeutika als auch in der Aufklärung von Struktur-Aktivitäts-Beziehungen ein viel versprechendes, synthetisches Werkzeug dar. Daher sind flexible und selektive Konzepte zur Backbone- wie Seitenketten-Modifizierung von Peptiden gefragt. Im Rahmen dieser Arbeit konnten die Grundlagen für ein neuartiges Verfahren zur Backbone-Modifizierung kleiner Peptide gelegt werden, wobei die Möglichkeit der Anwendung chelatisierter Peptidenolate als Nukleophile in der Palladium-katalysierten allylischen Substitution intensiv untersucht wurde. Die entwickelte Methode konnte ihre außerordentliche Flexibilität dadurch unter Beweis stellen, dass sowohl eine große Bandbreite an Peptiden als auch verschiedenste Substitutionsmuster am Allylfragment toleriert wurden. Eine Vielzahl an Di- und Tripeptiden konnten auf diese Weise in durchweg sehr guten Ausbeuten und Stereoselektivitäten allyliert werden. Das Verfahren erlaubte den nachträglichen, stereoselektiven Aufbau unnatürlicher, alkyl-, aryl- und heterosubstituierter Aminosäuren. Weiterhin eröffnete die Einführung stannylierter Seitenketten einen Zugang zu metallierten Peptid-Derivaten, welche anschließend sehr effizient in Seitenketten-Modifikationen durch Palladium-katalysierte Kreuzkupplungen eingesetzt werden konnten.The subsequent manipulation of physiologically active peptides represents a promising synthetic tool both for the development and the optimization of peptide-based pharmaceuticals as well as for the elucidation of structure-activity-relationships. Therefore selective and flexible concepts for the modification of peptides are desired. This thesis deals with the evaluation of chelated peptide enolates as nucleophiles in the palladium-catalyzed allylic substitution, forming the fundament for a new methodology for the peptide backbone modification by introducing functionalized ã,ä-unsaturated side chains. During these investigations this approach could prove its extraordinary flexibility, as a broad range of peptides as well as different substitution patterns at the allylic fragment were tolerated. A variety of di- and tripeptides could be allylated in generally high yields and stereoselectivities. This procedure allowed the generation of unnatural, alkyl-, aryl- or heterosubstituted amino acid residues within a peptide. Furthermore the highly diastereoselective introduction of stannylated side chains opened up access to metalated peptide derivatives, which could be used very efficiently as precursors for subsequent side chain modifications via palladium-catalyzed cross-coupling reactions

Enzymatic C-N Bond Formation via Aerobic Nitroso Ene Reactions

A novel biocatalytic protocol enables the direct and selective introduction of nitrogen functionalities via activation of allylic C-H bonds. Utilizing an oxidase/peroxidase couple for the formal dehydrogenation of N-hydroxycarbamates and hydroxamic acids with air as terminal oxidant, acylnitroso species are generated under particularly mild aqueous conditions. The reactive intermediates undergo C-N bond formation through an ene-type mechanism and provide high yields both in intramolecular and intermolecular enzymatic aminations. Investigations on alternative reaction pathways and labelling studies provide more insights into this unprecedented biocatalytic promiscuity of classical oxidoreductases as catalysts for nitroso ene-based transformations.</p