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Cell-free biocatalysis is gaining momentum in producing value-added chemicals, particularly in stepwise reaction cascades. However, the stability of enzyme cascades in industrial settings is often compromised when free enzymes are involved. In this study, we have developed a stable multifunctional heterogeneous biocatalyst coimmobilizing five enzymes on microparticles to transform 1,ω-diols into 1,ω-hydroxy acids. We improved the operational efficiency and stability of the heterogeneous biocatalyst by fine-tuning the enzyme loading and spatial organization. Stability issues are overcome through postimmobilization polymer coating. The general applicability of this heterogeneous biocatalyst is demonstrated by its scale-up in both batch and packed bed reactors, allowing a product yield of >80%. The continuous process is fed with H2O2 as the oxygen source, reaching a space-time yield (STY) of 0.76 g·L-1·h-1, maintained for the first 12 h. Finally, this flow system is telescoped with a second plug-flow reactor packed with a different heterogeneous biocatalyst integrating an additional transaminase. As a result, this 6-enzyme 2-reactor system sequentially transforms 1,ω-diols into 1,ω-amino acids while in situ recycling NAD+, depleting H2O2, and generating O2.

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Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi-enzyme system is extremely difficult and relies on a combinatorial trial-and-error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96-well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python-based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four-enzyme system to transform vinyl acetate into (S)-3-hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi-enzyme pathways for chemical manufacturing.

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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.

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In this work, the adsorption of Candida antarctica B (CALB) and Rhizomucor miehei (RML) lipases into hydrophobic wrinkled silica nanoparticles (WSNs) is investigated. WSNs are hydrophobized by chemical vapor deposition. Both proteins are homogeneously distributed inside the pores of the nanoparticles, as confirmed by Transmission Electron Microscopy and Energy Dispersive X-ray measurements. The maximum enzyme load of CALB is twice that obtained for RML. Fourier Transform Infrared Spectroscopy confirms the preservation of the enzyme secondary structure after immobilization for both enzymes. Adsorption isotherms fit to a Langmuir model, resulting in a binding constant (KL) for RML 4.5-fold higher than that for CALB, indicating stronger binding for the former. Kinetic analysis reveals a positive correlation between enzyme load and RML activity unlike CALB where activity decreases along the enzyme load increases. Immobilization allows for enhancing the thermal stability of both lipases. Finally, CALB outperforms RML in the hydrolysis of ethyl-3-hydroxybutyrate. However, immobilized CALB yielded 20 % less 3-HBA than free lipase, while immobilized RML increases 3-fold the 3-HBA yield when compared with the free enzyme. The improved performance of immobilized RML can be explained due to the interfacial hyperactivation undergone by this lipase when immobilized on the superhydrophobic surface of WSNs.

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Heterogeneous biocatalysis is highly relevant in biotechnology as it offers several benefits and practical uses. To leverage the full potential of heterogeneous biocatalysts, the establishment of well-crafted protocols, and a deeper comprehension of enzyme immobilization on solid substrates are essential. These endeavors seek to optimize immobilized biocatalysts, ensuring maximal enzyme performance within confined spaces. For this aim, multidimensional characterization of heterogeneous biocatalysts is required. In this context, spectroscopic and microscopic methodologies conducted at different space and temporal scales can inform about the intraparticle enzyme kinetics, the enzyme spatial distribution, and the mass transport issues. In this Minireview, we identify enzyme immobilization, enzyme catalysis, and enzyme inactivation as the three main processes for which advanced characterization tools unveil fundamental information. Recent advances in operando characterization of immobilized enzymes at the single-particle (SP) and single-molecule (SM) levels inform about their functional properties, unlocking the full potential of heterogeneous biocatalysis toward biotechnological applications.

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Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

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The in vitro synthesis of Coenzyme A (CoA)-thioester intermediates opens new avenues to transform simple molecules into more complex and multifunctional ones by assembling cell-free biosynthetic cascades. In this review, we have systematically cataloged known CoA-dependent enzyme reactions that have been successfully implemented in vitro. To faciliate their identification, we provide their UniProt ID when available. Based on this catalog, we have organized enzymes into three modules: activation, modification, and removal. i) The activation module includes enzymes capable of fusing CoA with organic molecules. ii) The modification module includes enzymes capable of catalyzing chemical modifications in the structure of acyl-CoA intermediates. And iii) the removal module includes enzymes able to remove the CoA and release an organic molecule different from the one activated in the upstream. Based on these reactions, we constructed a reaction network that summarizes the most relevant CoA-dependent biosynthetic pathways reported until today. From the information available in the articles, we have plotted the total turnover number of CoA as a function of the product titer, observing a positive correlation between both parameters. Therefore, the success of a CoA-dependent in vitro pathway depends on its ability to regenerate CoA, but also to regenerate other cofactors such as NAD(P)H and ATP.

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Enzyme immobilization is a key enabling technology for a myriad of industrial applications, yet immobilization science is still too empirical to reach highly active and robust heterogeneous biocatalysts through a general approach. Conventional protein immobilization methods lack control over how enzymes are oriented on solid carriers, resulting in negative conformational changes that drive enzyme deactivation. Site-selective enzyme immobilization through peptide tags and protein domains addresses the orientation issue, but this approach limits the possible orientations to the N- and C-termini of the target enzyme. In this work, we engineer the surface of two model dehydrogenases to introduce histidine clusters into flexible regions not involved in catalysis, through which immobilization is driven. By varying the position and the histidine density of the clusters, we create a small library of enzyme variants to be immobilized on different carriers functionalized with different densities of various metal chelates (Co2+, Cu2+, Ni2+, and Fe3+). We first demonstrate that His-clusters can be as efficient as the conventional His-tags in immobilizing enzymes, recovering even more activity and gaining stability against some denaturing agents. Furthermore, we find that the enzyme orientation as well as the type and density of the metal chelates affect the immobilization parameters (immobilization yield and recovered activity) and the stability of the immobilized enzymes. According to proteomic studies, His-clusters enable a different enzyme orientation as compared to His-tag. Finally, these oriented heterogeneous biocatalysts are implemented in batch reactions, demonstrating that the stability achieved by an optimized orientation translates into increased operational stability.

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Cell-free systems for the in vitro production of proteins have revolutionized the synthetic biology field. In the last decade, this technology is gaining momentum in molecular biology, biotechnology, biomedicine and even education. Materials science has burst into the field of in vitro protein synthesis to empower the value of existing tools and expand its applications. In this sense, the combination of solid materials (normally functionalized with different biomacromolecules) together with cell-free components has made this technology more versatile and robust. In this chapter, we discuss the combination of solid materials with DNA and transcription-translation machinery to synthesize proteins within compartments, to immobilize and purify in situ the nascent protein, to transcribe and transduce DNAs immobilized on solid surfaces, and the combination of all or some of these strategies.

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Biocatalysis is a key technology enabling plastic recycling. However, despite advances done in the development of plastic-degrading enzymes, the molecular mechanisms that govern their catalytic performance are poorly understood, hampering the engineering of more efficient enzyme-based technologies. In this work, we study the hydrolysis of PET-derived diesters and PET trimers catalyzed by the highly promiscuous lipase B from Candida antarctica (CALB) through QM/MM molecular dynamics simulations supported by experimental Michaelis-Menten kinetics. The computational studies reveal the role of the pH on the CALB regioselectivity toward the hydrolysis of bis-(hydroxyethyl) terephthalate (BHET). We exploit this insight to perform a pH-controlled biotransformation that selectively hydrolyzes BHET to either its corresponding diacid or monoesters using both soluble and immobilized CALB. The discoveries presented here can be exploited for the valorization of BHET resulting from the organocatalytic depolymerization of PET.

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Invited for the cover of this issue are the groups of Gonzalo Jiménez-Osés and Fernando López-Gallego at CIC bioGUNE and CIC biomaGUNE, respectively. The image depicts the substrate scope of an engineered acyl transferases for the synthesis of statin derivatives. Read the full text of the article at 10.1002/chem.202300911.

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This study identifies new acyl donors for manufacturing statin analogues through the acylation of monacolin J acid by the laboratory evolved acyltransferase LovD9. Vinyl and p-nitrophenyl esters have emerged as alternate substrates for LovD9-catalyzed acylation. While vinyl esters can reach product yields as high as the ones obtained by α-dimethyl butyryl-S-methyl-3-mercaptopropionate (DMB-SMMP), the thioester for which LovD9 was evolved, p-nitrophenyl esters display a reactivity even higher than DMB-SMMP for the first acylation step yet the acylation product yield is lower. The reaction mechanisms were elucidated through quantum mechanics (QM) calculations.

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Multi-enzymatic cascades with enzymes arranged in close-proximity through a protein scaffold can trigger a substrate channeling effect, allowing for efficient cofactor reuse with industrial potential. However, precise nanometric organization of enzymes challenges the design of scaffolds. In this study, we create a nanometrically organized multi-enzymatic system exploiting engineered Tetrapeptide Repeat Affinity Proteins (TRAPs) as scaffolding for biocatalysis. We genetically fuse TRAP domains and program them to selectively and orthogonally recognize peptide-tags fused to enzymes, which upon binding form spatially organized metabolomes. In addition, the scaffold encodes binding sites to selectively and reversibly sequester reaction intermediates like cofactors via electrostatic interactions, increasing their local concentration and, consequently, the catalytic efficiency. This concept is demonstrated for the biosynthesis of amino acids and amines using up to three enzymes. Scaffolded multi-enzyme systems present up to 5-fold higher specific productivity than the non-scaffolded ones. In-depth analysis suggests that channeling of NADH cofactor between the assembled enzymes enhances the overall cascade throughput and the product yield. Moreover, we immobilize this biomolecular scaffold on solid supports, creating reusable heterogeneous multi-functional biocatalysts for consecutive operational batch cycles. Our results demonstrate the potential of TRAP-scaffolding systems as spatial-organizing tools to increase the efficiency of cell-free biosynthetic pathways.

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Biocatalysis can improve current bioprocesses by identifying or improving enzymes that withstand harsh and unnatural operating conditions. Immobilized Biocatalyst Engineering (IBE) is a novel strategy integrating protein engineering and enzyme immobilization as a single workflow. Using IBE, it is possible to obtain immobilized biocatalysts whose soluble performance would not be selected. In this work, Bacillus subtilis lipase A (BSLA) variants obtained through IBE were characterized as soluble and immobilized biocatalysts, and how the interactions with the support affect their structure and catalytic performance were analyzed using intrinsic protein fluorescence. Variant P5G3 (Asn89Asp, Gln121Arg) showed a 2.6-fold increased residual activity after incubation at 76 °C compared to immobilized wild-type (wt) BSLA. On the other hand, variant P6C2 (Val149Ile) showed 4.4 times higher activity after incubation in 75 % isopropyl alcohol (36 °C) compared to Wt_BSLA. Furthermore, we studied the advancement of the IBE platform by performing synthesis and immobilizing the BSLA variants using a cell-free protein synthesis (CFPS) approach. The observed differences in immobilization performance, high temperature, and solvent resistance between the in vivo-produced variants and Wt_BSLA were confirmed for the in vitro synthesized enzymes. These results open the door for designing strategies integrating IBE and CFPS to generate and screen improved immobilized enzymes from genetic diversity libraries. Furthermore, it was confirmed that IBE is a platform that can be used to obtain improved biocatalysts, especially those with an unremarkable performance as soluble biocatalysts, which wouldn't be selected for immobilization and further development for specific applications.

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Immobilized multienzyme systems are gaining momentum in applied biocatalysis; however, the coimmobilization of several enzymes on one carrier is still challenging. In this work, we exploited a heterofunctional support activated with three different chemical functionalities to immobilize a wide variety of different enzymes. This support is based on agarose microbeads activated with aldehyde, amino, and cobalt chelate moieties that allow a fast and irreversible immobilization of enzymes, enhancing the thermostability of most of the heterogeneous biocatalysts (up to 21-fold higher than the soluble one). Furthermore, this trifunctional support serves to efficiently coimmobilize a multienzyme system composed of an alcohol dehydrogenase, a reduced nicotinamide adenine dinucleotide (NADH) oxidase, and a catalase. The confined multienzymatic system demonstrates higher performance than its free counterpart, achieving a total turnover number (TTN) of 1 × 105 during five batch consecutive cycles. We envision this solid material as a platform for coimmobilizing multienzyme systems with enhanced properties to catalyze stepwise biotransformations.

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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 ß-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.

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ß-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose, a substrate for fermentation to produce the carbon-neutral fuel bioethanol. Enzyme thermal stability and reusability can be improved through immobilization onto insoluble supports. Moreover, nanoscaled matrixes allow for preserving high reaction rates. In this work, BG was physically immobilized onto wrinkled SiO2 nanoparticles (WSNs). The adsorption procedure was tuned by varying the BG:WSNs weight ratio to achieve the maximum controllability and maximize the yield of immobilization, while different times of immobilization were monitored. Results show that a BG:WSNs ratio equal to 1:6 wt/wt provides for the highest colloidal stability, whereas an immobilization time of 24 h results in the highest enzyme loading (135 mg/g of support) corresponding to 80% yield of immobilization. An enzyme corona is formed in 2 h, which gradually disappears as the protein diffuses within the pores. The adsorption into the silica structure causes little change in the protein secondary structure. Furthermore, supported enzyme exhibits a remarkable gain in thermal stability, retaining complete folding up to 90 °C. Catalytic tests assessed that immobilized BG achieves 100% cellobiose conversion. The improved adsorption protocol provides simultaneously high glucose production, enhanced yield of immobilization, and good reusability, resulting in considerable reduction of enzyme waste in the immobilization stage.

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The development of methods to engineer and immobilize amine transaminases (ATAs) to improve their functionality and operational stability is gaining momentum. The quest for robust, fast, and easy-to-use methods to screen the activity of large collections of transaminases, is essential. This work presents a novel and multiplex fluorescence-based kinetic assay to assess ATA activity using 4-dimethylamino-1-naphthaldehyde as an amine acceptor. The developed assay allowed us to screen a battery of amine donors using free and immobilized ATAs from different microbial sources as biocatalysts. As a result, using chromatographic methods, 4-hydroxybenzylamine was identified as the best amine donor for the amination of 5-(hydroxymethyl)furfural. Finally, we adapted this method to determine the apparent Michaelis-Menten parameters of a model immobilized ATA at the microscopic (single-particle) level. Our studies promote the use of this multiplex, multidimensional assay to screen ATAs for further improvement.

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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.

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Scalability, process control, and modularity are some of the advantages that make flow biocatalysis a key-enabling technology for green and sustainable chemistry. In this context, rigid porous solid membranes hold the promise to expand the toolbox of flow biocatalysis due to their chemical stability and inertness. Yttrium-stabilized zirconia (YSZ) fulfills these properties; however, it has been scarcely exploited as a carrier for enzymes. Here, we discovered an unprecedented interaction between YSZ materials and His-tagged enzymes that enables the fabrication of multifunctional biocatalytic membranes for bioredox cascades. X-ray photoelectron spectroscopy suggests that enzyme immobilization is driven by coordination interactions between the imidazole groups of His-tags and both Zr and Y atoms. As model enzymes, we coimmobilized in-flow a thermophilic hydroxybutyryl-CoA dehydrogenase (TtHBDH-His) and a formate dehydrogenase (His-CbFDH) for the continuous asymmetric reduction of ethyl acetoacetate with in situ redox cofactor recycling. Fluorescence confocal microscopy deciphered the spatial organization of the two coimmobilized enzymes, pointing out the importance of the coimmobilization sequence. Finally, the coimmobilized system succeeded in situ, recycling the redox cofactor, maintaining the specific productivity using only 0.05 mM NADH, and accumulating a total enzyme turnover number of 4000 in 24 h. This work presents YSZ materials as ready-to-use carriers for the site-directed enzyme in-flow immobilization and the application of the resulting heterogeneous biocatalysts for continuous biomanufacturing.

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