Search Results (30)

Heparosan, a microbially synthesized capsular polysaccharide, possesses a polysaccharide backbone structurally analogous to heparin. Its biosynthesis holds significant importance for achieving the chemoenzymatic synthesis of heparin. Here, we developed a systematic metabolic engineering strategy in Escherichia coli Nissle 1917 to establish an efficient heparosan production platform. Through the systematic engineering of the glycolytic pathway involving the targeted knockout of zwf, pfkAB, pgi, and fruA (or alternatively fbaA) genes, we generated recombinant strains that lost the capacity to utilize glucose or fructose as sole carbon sources in a minimal medium. This metabolic reprogramming established glycerol as the exclusive carbon source for cell growth, thereby creating a tripartite carbon allocation system, including glycerol for biomass, glucose for UDP-glucuronic acid, and fructose for UDP-N-acetylglucosamine. Therefore, heparosan production was significantly improved from 137.68 mg/L in the wild type to 414.40 mg/L in the recombinant strain. Building upon this foundation, the overexpression of glmM, pgm, and galU genes in the biosynthetic pathway enabled a heparosan titer of 773.78 mg/L in shake-flask cultures. Temporal induction optimization further enhanced titers to 1049.96 mg/L, representing a 7.60-fold enhancement compared to the wild-type strain. This study establishes a triple-carbon-source co-utilization strategy, which holds promising implications for the biosynthesis of heparosan-like microbial polysaccharides. Full article

Marine organisms represent a source of unique chemical entities with valuable biomedical potentialities, broad diversity, and complexity. It is essential to ensure a reliable and sustainable supply of marine natural products (MNPs) for their translation into commercial drugs and other valuable products. From a structural point of view and with few exceptions, MNPs of pharmaceutical importance derive from the so-called secondary metabolism of marine organisms. When production strategies rely on marine macroorganisms, harvesting or culturing coupled with extraction procedures frequently remain the only alternative to producing these compounds on an industrial scale. Their supply can often be implemented with laboratory scale cultures for bacterial, fungal, or microalgal sources. However, a diverse approach, combining traditional methods with modern synthetic biology and biosynthesis strategies, must be considered for invertebrate MNPs, as they are usually naturally accumulated in only very small quantities. This review offers a comprehensive examination of various production strategies for MNPs, addressing the challenges related to supply, synthesis, and scalability. It also underscores recent biotechnological advancements that are likely to transform the current industrial-scale manufacturing methods for pharmaceuticals derived from marine sources. Full article

Natural cyclic peptides, a diverse class of bioactive compounds, have been isolated from various natural sources and are renowned for their extensive structural variability and broad spectrum of medicinal properties. Over 40 cyclic peptides or their derivatives are currently approved as medicines, underscoring their significant therapeutic potential. These compounds are employed in diverse roles, including antibiotics, antifungals, antiparasitics, immune modulators, and anti-inflammatory agents. Their unique ability to combine high specificity with desirable pharmacokinetic properties makes them valuable tools in addressing unmet medical needs, such as combating drug-resistant pathogens and targeting challenging biological pathways. Due to the typically low concentrations of cyclic peptides in nature, effective synthetic strategies are indispensable for their acquisition, characterization, and biological evaluation. Cyclization, a critical step in their synthesis, enhances metabolic stability, bioavailability, and receptor binding affinity. Advances in synthetic methodologies—such as solid-phase peptide synthesis (SPPS), chemoenzymatic approaches, and orthogonal protection strategies—have transformed cyclic peptide production, enabling greater structural complexity and precision. This review compiles recent progress in the total synthesis and biological evaluation of natural cyclic peptides from 2017 onward, categorized by cyclization strategies: head-to-tail; head-to-side-chain; tail-to-side-chain; and side-chain-to-side-chain strategies. Each account includes retrosynthetic analyses, synthetic advancements, and biological data to illustrate their therapeutic relevance and innovative methodologies. Looking ahead, the future of cyclic peptides in drug discovery is bright. Emerging trends, including integrating computational tools for rational design, novel cyclization techniques to improve pharmacokinetic profiles, and interdisciplinary collaboration among chemists, biologists, and computational scientists, promise to expand the scope of cyclic peptide-based therapeutics. These advancements can potentially address complex diseases and advance the broader field of biological drug development. Full article

The presence and the level of antibodies in human sera against bacterial glycans are indications of prior encounters with similar antigens and/or the bacteria that express them by the immune system. An increasing number of pathogenic bacteria that cause human diseases have been shown to express polysaccharides containing a bacterial nonulosonic acid called 5,7-di-N-acetyllegionaminic acid (Leg5,7Ac₂). To investigate the immune recognition of Leg5,7Ac₂, which is critical for the fight against bacterial infections, a highly effective chemoenzymatic synthon strategy was applied to construct a library of α2–3/6-linked Leg5,7Ac₂-glycans via their diazido-derivatives (Leg5,7diN₃-glycans) formed by efficient one-pot three-enzyme (OP3E) synthetic systems from a diazido-derivative of a six-carbon monosaccharide precursor. Glycan microarray studies using this synthetic library of a Leg5,7Ac₂-capped collection of diverse underlying glycan carriers and their matched sialoside counterparts revealed specific recognition of Leg5,7Ac₂ by human IgG antibodies pooled from thousands of healthy donors (IVIG), suggesting prior human encounters with Leg5,7Ac₂-expressing pathogenic bacteria at the population level. These biologically relevant Leg5,7Ac₂-glycans and their immune recognition assays are important tools to begin elucidating their biological roles, particularly in the context of infection and host–pathogen interactions. Full article

Currently, intense interest is focused on the discovery and application of new multisubunit cage proteins and spherical virus capsids to the fields of bionanotechnology, drug delivery, and diagnostic imaging as their internal cavities can serve as hosts for fluorophores or bioactive molecular cargo. Bacterioferritin is unusual in the ferritin protein superfamily of iron-storage cage proteins in that it contains twelve heme cofactors and is homomeric. The goal of the present study is to expand the capabilities of ferritins by developing new approaches to molecular cargo encapsulation employing bacterioferritin. Two strategies were explored to control the encapsulation of a diverse range of molecular guests compared to random entrapment, a predominant strategy employed in this area. The first was the inclusion of histidine-tag peptide fusion sequences within the internal cavity of bacterioferritin. This approach allowed for the successful and controlled encapsulation of a fluorescent dye, a protein (fluorescently labeled streptavidin), or a 5 nm gold nanoparticle. The second strategy, termed the heme-dependent cassette strategy, involved the substitution of the native heme with heme analogs attached to (i) fluorescent dyes or (ii) nickel-nitrilotriacetate (NTA) groups (which allowed for controllable encapsulation of a histidine-tagged green fluorescent protein). An in silico docking approach identified several small molecules able to replace the heme and capable of controlling the quaternary structure of the protein. A transglutaminase-based chemoenzymatic approach to surface modification of this cage protein was also accomplished, allowing for future nanoparticle targeting. This research presents novel strategies to control a diverse set of molecular encapsulations and adds a further level of sophistication to internal protein cavity engineering. Full article

The increasing concentration of atmospheric CO₂ due to human activities has resulted in serious environmental issues such as global warming and calls for efficient ways to reduce CO₂ from the environment. The conversion of CO₂ into value-added compounds such as methane, formic acid, and methanol has emerged as a promising strategy for CO₂ utilization. Among the different techniques, the enzymatic approach based on the CO₂ metabolic process in cells presents a powerful and eco-friendly method for effective CO₂ conversion and upcycling. This review discusses the catalytic conversion of CO₂ using single and multienzyme systems, followed by various chemo-enzymatic processes to produce bicarbonates, bulk chemicals, synthetic organic fuel and synthetic polymer. We also highlight the challenges and prospects for future progress in CO₂ conversion via chemo-enzymatic processes for a sustainable solution to reduce the global carbon footprint. Full article

The aim of this study was to produce epoxidized monoalkyl esters (EMAE), a valuable class of oleochemicals used in a wide range of products and industries, from used soybean cooking oil (USCO) and fusel oil via a three-step chemoenzymatic process. This process consists of a first enzymatic hydrolysis of USCO to produce free fatty acids (FFA). Here, five microbial lipases with different specificities were tested as biocatalysts. Full hydrolysis of USCO was obtained after a 180 min reaction time under vigorous stirring (1500 rpm) using a non-specific lipase from Candida rugosa (CRL). Then, monoalkyl esters (MAE) were produced via the esterification of FFA and fusel oil in a solvent-free system using the lipase Eversa^® Transform 2.0 (ET2.0) immobilized via physical adsorption on poly(styrenene-divinylbenzene) (PSty-DVB) beads as a biocatalyst. Different water removal strategies (closed and open reactors in the presence or absence of molecular sieves at 5% m.m⁻¹) on the reaction were evaluated. Maximum FFA conversions of 64.3 ± 2.3% (open reactor after a 30 min reaction time) and 73.5 ± 0.4% (closed reactor after a 45 min reaction time) were observed at 40 °C, using a stoichiometric FFA:fusel oil molar ratio (1:1), without molecular sieves, and 5 mg of immobilized protein per gram of reaction mixture. Under these conditions, maximum FFA conversion was only 30.2 ± 2.7% after a 210 min reaction time in a closed reactor using soluble lipase. Reusability tests showed better retention of the original activity of immobilized ET2.0 (around 82%) after eight successive batches of esterification reactions conducted in an open reactor. Finally, the produced MAE was epoxidized via the Prilezhaev reaction, a classical chemical epoxidation process, using hydrogen peroxide and formic acid as a homogeneous catalyst. The products were characterized by standard methods and identified using proton nuclear magnetic resonance (¹H NMR). Maximum unsaturated bond conversions into epoxy groups were at approximately 33%, with the experimental epoxy oxygen content (OOC_exp.) at 1.75–1.78%, and selectivity (S) at 0.81, using both MAEs produced (open or closed reactors). These results show that this new process is a promising approach for value-added oleochemical production from low-cost and renewable raw materials. Full article

As a versatile and valuable intermediate, furfuryl alcohol (FOL) is widely utilized in manufacturing vitamin C, perfume, fruit acid, lubricant, lysine, plasticizer, dispersing agent, resin, fuel additive, and biofuel. This study aimed at the establishment of a cascade catalysis of biomass to FOL via a hybrid approach in a deep eutectic solvent medium. The catalysis of corn stover (75 g/L) with solid acid AT-Sn-WLS (1.2 wt%) produced 110.5 mM FAL in a ChCl:PEG10000–water (20:80, wt/wt) system at 170 °C for 30 min, and then the formed FAL was biologically transformed into FOL with recombinant E. coli SF harboring aldehyde reductase at pH 7.0 and 35 °C. This established hybrid strategy could efficiently valorize corn stover into FOL, with the productivity of 0.41 g FOL per g xylan in corn stover. Consequently, one combination of chemocatalytic and biocatalytic reactions leading to a one-pot catalytic process was shown as an attractive approach in the valorization of lignocellulose into valuable biobased chemicals. Full article

Valorization of the abundant renewable lignocellulose through an efficient chemoenzymatic strategy to produce the furan-based platform compounds has raised great interest in recent years. In this work, a newly prepared sulfonated tin-loaded rice husk-based heterogeneous chemocatalyst UST-Sn-RH was utilized to transform corncob (75.0 g/L) into furfural (72.1 mM) at 170 °C for 30 min in an aqueous system. To upgrade furfural into furfuryl alcohol, whole cells of recombinant E. coli KPADH harboring alcohol dehydrogenase were employed to transform corncob-derived furfural into furfuryl alcohol at 30 °C and pH 7.5. In the established chemoenzymatic cascade process, corncob was efficiently transformed to furfuryl alcohol with a productivity of 0.304 g furfuryl alcohol/(g xylan in corncob). In general, biomass could be efficiently valorized into valuable furan-based chemicals in this tandem reaction with the chemocatalyst (bio-based UST-Sn-RH) and the biocatalyst (KPADH cell) in an aqueous system, which has potential application. Full article

Chondroitin sulfate (CS) is widely used across the world as a nutraceutical and pharmaceutical. Its high demand and potential limitations in current methods of extraction call for an alternative method of production. This review highlights glycosaminoglycan’s structure, its medical significance, animal extraction source, and the disadvantages of the extraction process. We cover alternative production strategies for CS and its precursor, chondroitin. We highlight chemical synthesis, chemoenzymatic synthesis, and extensively discuss how strains have been successfully metabolically engineered to synthesize chondroitin and chondroitin sulfate. We present microbial engineering as the best option for modern chondroitin and CS production. We also explore the biosynthetic pathway for chondroitin production in multiple microbes such as Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum. Lastly, we outline how the manipulation of pathway genes has led to the biosynthesis of chondroitin derivatives. Full article

Alkaloids are a large and structurally diverse group of marine-derived natural products. Most marine-derived alkaloids are biologically active and show promising applications in modern (agro)chemical, pharmaceutical, and fine chemical industries. Different approaches have been established to access these marine-derived alkaloids. Among these employed methods, biotechnological approaches, namely, (chemo)enzymatic synthesis, have significant potential for playing a central role in alkaloid production on an industrial scale. In this review, we discuss research progress on marine-derived alkaloid synthesis via enzymatic reactions and note the advantages and disadvantages of their applications for industrial production, as well as green chemistry for marine natural product research. Full article

In the last few years, nanomaterials based on fullerene have begun to be considered promising tools in the development of efficient adjuvant/delivery systems for vaccination, thanks to their several advantages such as biocompatibility, size, and easy preparation and modification. In this work we reported the chemoenzymatic synthesis of natural polymannan analogues (di- and tri-mannan oligosaccharides characterized by α1,6man and/or α1,2man motifs) endowed with an anomeric propargyl group. These sugar derivatives were submitted to 1,3 Huisgen dipolar cycloaddition with a malondiamide-based chain equipped with two azido terminal groups. The obtained sugar-modified malondiamide derivatives were used to functionalize the surface of Buckminster fullerene (C₆₀) in a highly controlled fashion, and yields (11–41%) higher than those so far reported by employing analogue linkers. The same strategy has been exploited to obtain C₆₀ endowed with natural and unnatural amino acid derivatives. Finally, the first double functionalization of fullerene with both sugar- and amino acid-modified malondiamide chains was successfully performed, paving the way to the possible derivatization of fullerenes with immunogenic sugars and more complex antigenic peptides. Full article

Bispecific antibodies (bsAbs) were first developed in the 1960s and are now emerging as a leading class of immunotherapies for cancer treatment with the potential to further improve clinical efficacy and safety. Many different formats of bsAbs have been established in the last few years, mainly generated genetically. Here we report on a novel, flexible, and fast chemo–enzymatic, as well as purely enzymatic strategies, for generating bispecific antibody fragments by covalent fusion of two functional antibody Fab fragments (Fabs). For the chemo–enzymatic approach, we first modified the single Fabs site-specifically with click anchors using an enhanced Trypsiligase variant (eTl) and afterward converted the modified Fabs into the final heterodimers via click chemistry. Regarding the latter, we used the strain-promoted alkyne-azide cycloaddition (SPAAC) and inverse electron-demand Diels–Alder reaction (IEDDA) click approaches well known for their fast reaction kinetics and fewer side reactions. For applications where the non-natural linkages or hydrophobic click chemistry products might interfere, we developed two purely enzymatic alternatives enabling C- to C- and C- to N-terminal coupling of the two Fabs via a native peptide bond. This simple system could be expanded into a modular system, eliminating the need for extensive genetic engineering. The bispecific Fab fragments (bsFabs) produced here to bind the growth factors ErbB2 and ErbB3 with similar K_D values, such as the sole Fabs. Tested in breast cancer cell lines, we obtained biologically active bsFabs with improved properties compared to its single Fab counterparts. Full article

In this work, the feasibility of chemoenzymatically transforming biomass-derived D-xylose to furfuryl alcohol was demonstrated in a tandem reaction with SO₄²⁻/SnO₂-CS chemocatalyst and reductase biocatalyst in the deep eutectic solvent (DES)–water media. The high furfural yield (44.6%) was obtained by catalyzing biomass-derived D-xylose (75.0 g/L) in 20 min at 185 °C with SO₄²⁻/SnO₂-CS (1.2 wt%) in DES ChCl:EG–water (5:95, v/v). Subsequently, recombinant E.coli CF cells harboring reductases transformed D-xylose-derived furfural (200.0 mM) to furfuryl alcohol in the yield of 35.7% (based on D-xylose) at 35 °C and pH 7.5 using HCOONa as cosubstrate in ChCl:EG–water. This chemoenzymatic cascade catalysis strategy could be employed for the sustainable production of value-added furan-based chemical from renewable bioresource. Full article

Glycans have been shown to play a key role in many biological processes, such as signal transduction, immunogenicity, and disease progression. Among the various glycosylation modifications found on cell surfaces and in biomolecules, sialylation is especially important, because sialic acids are typically found at the terminus of glycans and have unique negatively charged moieties associated with cellular and molecular interactions. Sialic acids are also crucial for glycosylated biopharmaceutics, where they promote stability and activity. In this regard, heterogenous sialylation may produce variability in efficacy and limit therapeutic applications. Homogenous sialylation may be achieved through cellular and molecular engineering, both of which have gained traction in recent years. In this paper, we describe the engineering of intracellular glycosylation pathways through targeted disruption and the introduction of carbohydrate active enzyme genes. The focus of this review is on sialic acid-related genes and efforts to achieve homogenous, humanlike sialylation in model hosts. We also discuss the molecular engineering of sialyltransferases and their application in chemoenzymatic sialylation and sialic acid visualization on cell surfaces. The integration of these complementary engineering strategies will be useful for glycoscience to explore the biological significance of sialic acids on cell surfaces as well as the future development of advanced biopharmaceuticals. Full article