•Recent insights into TEMPO chemistry applied on polysaccharides are reviewed.•This chemistry gives birth to polyuronides with various biological applications.•Oxidized cellulose derivatives have ...mechanical and physico-chemical properties.•The biodegradability potential of oxidized polysaccharides is of first interest.
The oxidation of natural polysaccharides by TEMPO has become by now an “old chemical reaction” which led to numerous studies mainly conducted on cellulose. This regioselective oxidation of primary alcohol groups of neutral polysaccharides has generated a new class of polyuronides not identified before in nature, even if the discovery of enzymes promoting an analogous oxidation has been more recently reported. Around the same time, the scientific community discovered the surprising biological and techno-functional properties of these anionic macromolecules with a high potential of application in numerous industrial fields. The objective of this review is to establish the state of the art of TEMPO chemistry applied to polysaccharide oxidation, its history, the resulting products, their applications and the associated modifying enzymes.
The Cover Feature shows the carrier‐free immobilization of self‐assembled multi‐enzyme complexes formed on the basis of polypeptide interactions and enzyme oligomerization. Such supermolecular and ...water‐insoluble enzyme complexes feature a high reaction rate and good recycling capability for the biological multi‐enzyme biocatalytic conversion of starch into inositol. The proposed strategy for the carrier‐free immobilization of the enzyme complex sheds light on improving the catalytic efficiency of in vitro synthetic enzymatic biosystems. More information can be found in the Research Article by M. Liu et al.
Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi‐enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a ...novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi‐enzyme systems. Two SCAB systems are developed, one employing head‐to‐tail interactions with reversible covalent disulfide bonds, the other relying on non‐covalent metal‐driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non‐reducing environment or by metal presence. A proof‐of‐concept with formate dehydrogenase (FDH) and L‐alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6‐fold compared to free enzymes, with the covalent stapling outperforming the metal‐driven assembly. This enhancement likely stems from higher‐order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial‐organizing tools, for more efficient enzymatic cascade reactions.
Highly efficient cell-free enzymatic systems are typically difficult to achieve in traditional immobilized enzyme systems because of the lack of optimal spatial control of enzyme localization, ...substrate and product diffusion, and enzyme and coenzyme accessibility. Here, we report a strategy for expanding the pore apertures (from 3.3 to 6.7 nm) of a series of Zr-based metallic-organic frameworks (MOFs) (termed NU-100x, x = 3, 4, 5, 6, 7) with interconnected hierarchical pores by maintaining precise control of torsional angles associated with the linkers. As a proof of concept, we use the expanded NU-100x MOF structures to encapsulate lactate dehydrogenase (LDH) and demonstrate the use of the captured protein in a cell-free biosynthetic catalytic system. Remarkably, LDH immobilized in the large pores of the MOF is accessible to nicotinamide adenine dinucleotide coenzymes (NAD and NADH), allowing for in situ coenzyme regeneration leading to higher activity than that of the free enzyme.
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•A strategy for expanding csq-net Zr-based metal-organic frameworks (MOFs)•Isoreticular NU-100x series features interconnected hierarchical mesopores (3.3–6.7 nm)•Coenzymes regeneration is realized by enzymes immobilized in NU-100x•Potential application for biofuel production and biomass conversion
Cell-free synthetic biology is an emerging field of biotechnology that is expanding the capabilities of natural biological systems, as demonstrated by the rapid development of various systems for biofuel and biochemical production. To achieve large-scale industrial production of cell-free enzymatic systems, efficient immobilization strategies are needed to overcome challenges such as high production costs of enzymes and coenzymes. Here, we create a series of ideal enzyme carriers based on metal-organic frameworks (MOFs) with interconnected hierarchical mesoporous channels. These MOF enzyme carriers show water stability, high enzyme-loading capacity, and coenzyme accessibility and demonstrate the use of the captured protein in a cell-free biosynthetic catalytic system. This work provides a strategy for constructing novel immobilized cell-free enzymatic systems for industrially relevant applications.
Farha and colleagues have developed a strategy for expanding the pore apertures of csq-net Zr-based MOFs to obtain an isoreticular series of MOF structures with pore apertures ranging from 3.3 to 6.7 nm. Enzymes immobilized in the MOF are accessible to coenzymes and show higher activity than that of the free enzymes.
•Cell-free synthesis of lipid-linked mannopentaose-di-(N-acetylglucosamine) from a lipid-linked precursor, polyphosphate, mannose and GDP.•In-vitro N-glycosylation of peptides using purified ...oligosaccharyltransferase T. brucei STT3A.•GDP-mannose synthesis and regeneration cascade coupled to three mannosyltransferases.•Liquid chromatography - mass spectrometry (LC–MS) analysis of in-vitro N-glycosylated peptides.
A wide range of glycoproteins can be recombinantly expressed in aglycosylated forms in bacterial and cell-free production systems. To investigate the effect of glycosylation of these proteins on receptor binding, stability, efficacy as drugs, pharmacodynamics and pharmacokinetics, an efficient glycosylation platform is required. Here, we present a cell-free synthetic platform for the in vitro N-glycosylation of peptides mimicking the endoplasmic reticulum (ER) glycosylation machinery of eukaryotes. The one-pot, two compartment multi-enzyme cascade consisting of eight recombinant enzymes including the three Leloir glycosyltransferases, Alg1, Alg2 and Alg11, expressed in E. coli and S. cerevisiae, respectively, has been engineered to produce the core lipid-linked (LL) oligosaccharide mannopentaose-di-(N-acetylglucosamine) (LL-Man5). Pythanol (C20H42O), a readily available alcohol consisting of regular isoprenoid units, was utilized as the lipid anchor. As part of the cascade, GDP-mannose was de novo produced from the inexpensive substrates ADP, polyphosphate and mannose. To prevent enzyme inhibition, the nucleotide sugar cascade and the glycosyltransferase were segregated into two compartments by a cellulose ester membrane with 3.5 kDa cut-off allowing for the effective diffusion of GDP-mannose across compartments. Finally, as a proof-of-principle, pythanyl-linked Man5 and the single-subunit oligosaccharyltransferase Trypanosoma brucei STT3A expressed in Sf9 insect cells were used to in vitro N-glycosylate a synthetic peptide of ten amino acids bearing the eukaryotic consensus motif N-X-S/T.
Saccharides have recently attracted considerable attention because of their biological functions and potential applications in the pharmaceutical, cosmetic and food industries. Over the decades, a ...large amount of enzymes involved in saccharide synthesis have been discovered and characterised with the aid of available genome sequences. The advancement of metabolic engineering and synthetic biology strategies facilitated the artificial pathway design and construction for production of multiple sugars in vitro and in vivo based on those characterized enzymes. This review presented a panoramic view of enzymes related to saccharide synthesis and gave the detailed information. Furthermore, we provide an extensive overview of the recent advances in the construction of cell-free reaction systems and engineering of microbial cells for the production of natural or unnatural saccharides. In addition, the future trends in the synthesis of sugars with high structural diversity through the combination of multiple pathways are presented and evaluated.
In vitro biosynthetic pathways that condense and reduce molecules through coenzyme A (CoASH) activation demand energy and redox power in the form of ATP and NAD(P)H, respectively. These coenzymes ...must be orthogonally recycled by ancillary reactions that consume chemicals, electricity, or light, impacting the atom economy and/or the energy consumption of the biosystem. In this work, we have exploited vinyl esters as dual acyl and electron donor substrates to synthesize β‐hydroxy acids through a non‐decarboxylating Claisen condensation, reduction and hydrolysis stepwise cascade, including a NADH recycling step, catalyzed by a total of 4 enzymes. Herein, the chemical energy to activate the acyl group with CoASH and the redox power for the reduction are embedded into the vinyl esters. Upon optimization, this self‐sustaining cascade reached a titer of (S)‐3‐hydroxy butyrate of 24 mM without requiring ATP and simultaneously recycling CoASH and NADH. This work illustrates the potential of in vitro biocatalysis to transform simple molecules into multi‐functional ones.
A 4‐enzyme cell‐free system transforms vinyl esters into β‐hydroxy acids in the absence of ATP and ancillary electron donors through concurrent abiotic thiolysis, non‐decarboxylating Claisen condensation, ketone reduction and hydrolysis steps. The same substrate provides the energy to activate the acyl group and the redox power to reduce the condensed ketone.
A method is developed for carrier‐free immobilization of multi‐enzyme complexes with more than four enzymes by utilization of polypeptide interactions (SpyCatcher‐SpyTag and dockerin‐cohesin) and ...enzyme component self‐oligomerization. Two pairs of scaffoldins with different arrangements of SpyCatcher‐SpyTag and cohesins are prepared to recruit the four dockerin‐containing cascade enzymes (i. e., alpha‐glucan phosphorylase, phosphoglucomutase, inositol 1‐phosphate synthase, and inositol 1‐phosphatase) that can convert starch into inositol, forming multi‐enzyme complexes. These self‐assembled enzyme complexes show higher initial reaction rates than the four‐enzyme cocktail. Moreover, water‐insoluble self‐assembled multi‐enzyme complexes are observed, being the carrier‐free immobilized multi‐enzyme complex aggregates. These immobilized enzyme complexes can be recycled easily by simple centrifuging followed by resuspension for another round of reaction. Not only can these immobilized enzyme complexes be obtained by mixing the purified enzyme components, but also by the mixing of crude cell extracts. Therefore, the strategy for the carrier‐free immobilization of enzyme complex sheds light on improving the catalytic capability of in vitro synthetic enzymatic biosystems.
So many enzymes: With a view to potential application of multi‐enzyme complexes in biological multi‐enzyme biocatalysis, a strategy for the carrier‐free immobilization based on protein scaffold is developed by taking the protein‐protein interactions (SpyCatcher‐SpyTag and dockerin‐cohesin) and enzyme oligomerization into account, whereby higher reaction rates and better enzyme recycling are realized.
The activity orchestration of an unprecedented cell‐free enzyme system with self‐sufficient cofactor recycling enables the stepwise transformation of aliphatic diols into ω‐hydroxy acids at the ...expense of molecular oxygen as electron acceptor. The efficiency of the biosynthetic route was maximized when two compatible alcohol dehydrogenases were selected as specialist biocatalysts for each one of the oxidative steps required for the oxidative lactonization of diols. The cell‐free system reached up to 100 % conversion using 100 mM of linear C5 diols and performed the desymmetrization of prochiral branched diols into the corresponding ω‐hydroxy acids with an exquisite enantioselectivity (ee>99 %). Green metrics demonstrate superior sustainability of this system compared to traditional metal catalysts and even to whole cells for the synthesis of 5‐hydroxypetanoic acid. Finally, the cell‐free system was assembled into a consortium of heterogeneous biocatalysts that allowed the enzyme reutilization. This cascade illustrates the potential of systems biocatalysis to access new heterofunctional molecules such as ω‐hydroxy acids.
Who needs cells? We describe the cell‐free biosynthesis of short ω‐hydroxy acids from bio‐based 1,ω‐diols by a multi‐enzyme system combining two specialized alcohol dehydrogenases. This system is proven to transform a wide scope of diols. Finally, we intensify this cell‐free biotransformation by increasing the substrate concentration to 100 mM and immobilizing the system to recycle a consortium of heterogeneous biocatalysts in consecutive operational cycles.
Multi-enzyme complexes have the potential to achieve high catalytic efficiency for sequence reactions due to their advantages in eliminating product inhibition, facilitating intermediate transfer and ...in situ regenerating cofactors. Constructing functional multi-enzyme systems to mimic natural multi-enzyme complexes is of great interest for multi-enzymatic biosynthesis and cell-free synthetic biotransformation, but with many challenges. Currently, various assembly strategies have been developed based on the interaction of biomacromolecules such as DNA, peptide and scaffolding protein. On the other hand, chemical-induced assembly is based on the affinity of enzymes with small molecules including inhibitors, cofactors and metal ions has the advantage of simplicity, site-to-site oriented structure control and economy. This review summarizes advances and progresses employing these strategies. Furthermore, challenges and perspectives in designing multi-enzyme systems are highlighted.
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