Synthetic Biology: Bottom-Up Assembly of Molecular Systems.

The bottom-up assembly of biological and chemical components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their composition and can thus provide specifically optimized environments for synthetic biological processes. This review aims to inspire future endeavors by providing a diverse toolbox of molecular modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important technical and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technological achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biology. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biological systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.

Pnictogen-Bonding Enzymes.

The objective of this study was to create artificial enzymes that capitalize on pnictogen bonding, a s-hole interaction that is essentially absent in biocatalysis.  For this purpose, stibine catalysts were equipped with a biotin derivative and combined with streptavidin mutants to identify an efficient transfer hydrogenation catalyst for the reduction of a fluorogenic quinoline substrate.  Increased catalytic activity from wild-type streptavidin to the best mutants coincides with the depth of the s hole on the Sb(V) center, and the emergence of saturation kinetic behavior.  Michaelis-Menten analysis reveals transition-state recognition in the low micromolar range, more than three orders of magnitude stronger than the millimolar substrate recognition.  Carboxylates preferred by the best mutants contribute to transition-state recognition by hydrogen-bonded ion pairing and anion-π interactions with the emerging pyridinium product.  The emergence of challenging stereoselectivity in aqueous systems further emphasizes compatibility of pnictogen bonding with higher order systems catalysis.

Binding Interactions and Inhibition Mechanisms of Gold Complexes in Thiamine Diphosphate-Dependent Enzymes.

Thiamine diphosphate (ThDP)-dependent enzymes possess the unique ability to generate a carbene within their active site. In this study, we sought to harness this carbene to produce a Au(I) N-heterocyclic complex directly in the active site of ThDP enzymes, thereby establishing a novel platform for artificial metalloenzymes. Because direct metalation of ThDP proved challenging, we synthesized a ThDP mimic that acts as a competitive inhibitor with a high affinity (Ki = 1.5 µM). Upon metalation with Au(I), we observed that the complex became a more potent inhibitor (Ki = 0.7 µM). However, detailed analysis of the inhibition mode, native mass spectrometry, and size exclusion experiments revealed that the complex does not bind specifically to the active site of ThDP enzymes. Instead, it exhibits unspecific binding and exceeds the 1:1 stoichiometry. Similar binding patterns were observed for other Au(I) species. These findings prompt an important question regarding the inherent propensity of ThDP enzymes to bind strongly to Au. If this phenomenon holds true, it could pave the way for the development of Au-based drugs targeting these enzymes.

An Artificial [Fe4S4]-Containing Metalloenzyme for the Reduction of CO2 to Hydrocarbons.

Iron-sulfur clusters have been reported to catalyze various redox transformations, including the multielectron reduction of CO2 to hydrocarbons. Herein, we report the design and assembly of an artificial [Fe4S4]-containing Fischer-Tropschase relying on the biotin-streptavidin technology. For this purpose, we synthesized a bis-biotinylated [Fe4S4] cofactor with marked aqueous stability and incorporated it in streptavidin. The effect of the second coordination sphere provided by the protein environment was scrutinized by cyclic voltammetry, highlighting the accessibility of the doubly reduced [Fe4S4] cluster. The Fischer-Tropschase activity was improved by chemo-genetic means for the reduction of CO2 to hydrocarbons with up to 14 turnovers.

Artificial Metalloenzyme-Catalyzed Enantioselective Amidation via Nitrene Insertion in Unactivated C(sp3)-H Bonds.

Enantioselective C-H amidation offers attractive means to assemble C-N bonds to synthesize high-added value, nitrogen-containing molecules. In recent decades, complementary enzymatic and homogeneous-catalytic strategies for C-H amidation have been reported. Herein, we report on an artificial metalloenzyme (ArM) resulting from anchoring a biotinylated Ir-complex within streptavidin (Sav). The resulting ArM catalyzes the enantioselective amidation of unactivated C(sp3)-H bonds. Chemogenetic optimization of the Ir cofactor and Sav led to significant improvement in both the activity and enantioselectivity. Up to >700 TON and 92% ee for the amidation of unactivated C(sp3)-H bonds was achieved. The single crystal X-ray analysis of the artificial nitrene insertase (ANIase) combined with quantum mechanics-molecular mechanics (QM-MM) calculations sheds light on critical second coordination sphere contacts leading to improved catalytic performance.

Water-Soluble Tris(cyclometalated) Iridium(III) Complexes for Aqueous Electron and Energy Transfer Photochemistry.

Cyclometalated iridium(III) complexes are frequently employed in organic light emitting diodes, and they are popular photocatalysts for solar energy conversion and synthetic organic chemistry. They luminesce from redox-active excited states that can have high triplet energies and long lifetimes, making them well suited for energy transfer and photoredox catalysis. Homoleptic tris(cyclometalated) iridium(III) complexes are typically very hydrophobic and do not dissolve well in polar solvents, somewhat limiting their application scope. We developed a family of water-soluble sulfonate-decorated variants with tailored redox potentials and excited-state energies to address several key challenges in aqueous photochemistry.First, we aimed at combining enzyme with photoredox catalysis to synthesize enantioenriched products in a cyclic reaction network. Since the employed biocatalyst operates best in aqueous solution, a water-soluble photocatalyst was needed. A new tris(cyclometalated) iridium(III) complex provided enough reducing power for the photochemical reduction of imines to racemic mixtures of amines and furthermore was compatible with monoamine oxidase (MAO-N-9), which deracemized this mixture through a kinetic resolution of the racemic amine via oxidation to the corresponding imine. This process led to the accumulation of the unreactive amine enantiomer over time. In subsequent studies, we discovered that the same iridium(III) complex photoionizes under intense irradiation to give hydrated electrons as a result of consecutive two-photon excitation. With visible light as energy input, hydrated electrons become available in a catalytic fashion, thereby allowing the comparatively mild reduction of substrates that would typically only be reactive under harsher conditions. Finally, we became interested in photochemical upconversion in aqueous solution, for which it was desirable to obtain water-soluble iridium(III) compounds with very high triplet excited-state energies. This goal was achieved through improved ligand design and ultimately enabled sensitized triplet-triplet annihilation upconversion unusually far into the ultraviolet spectral range.Studies of photoredox catalysis, energy transfer catalysis, and photochemical upconversion typically rely on the use of organic solvents. Water could potentially be an attractive alternative in many cases, but photocatalyst development lags somewhat behind for aqueous solution compared to organic solvent. The purpose of this Account is to provide an overview of the breadth of new research perspectives that emerged from the development of water-soluble fac-[Ir(ppy)]3 complexes (ppy = 2-phenylpyridine) with sulfonated ligands. We hope to inspire the use of some of these or related coordination compounds in aqueous photochemistry and to stimulate further conceptual developments at the interfaces of coordination chemistry, photophysics, biocatalysis, and sustainable chemistry.

Spiers Memorial Lecture: Shielding the active site: a streptavidin superoxide-dismutase chimera as a host protein for asymmetric transfer hydrogenation.

By anchoring a metal cofactor within a host protein, so-called artificial metalloenzymes can be generated. Such hybrid catalysts combine the versatility of transition metals in catalyzing new-to-nature reactions with the power of genetic-engineering to evolve proteins. With the aim of gaining better control over second coordination-sphere interactions between a streptavidin host-protein (Sav) and a biotinylated cofactor, we engineered a hydrophobic dimerization domain, borrowed from superoxide dismutase C (SOD), on Sav's biotin-binding vestibule. The influence of the SOD dimerization domain (DD) on the performance of an asymmetric transfer hydrogenase (ATHase) resulting from anchoring a biotinylated Cp*Ir-cofactor - [Cp*Ir(biot-p-L)Cl] (1-Cl) - within Sav-SOD is reported herein. We show that, depending on the nature of the residue at position Sav S112, the introduction of the SOD DD on the biotin-binding vestibule leads to an inversion of configuration of the reduction product, as well as a fivefold increase in catalytic efficiency. The findings are rationalized by QM/MM calculations, combined with X-ray crystallography.

Manganese Transfer Hydrogenases Based on the Biotin-Streptavidin Technology.

Artificial (transfer) hydrogenases have been developed for organic synthesis, but they rely on precious metals. Native hydrogenases use Earth-abundant metals, but these cannot be applied for organic synthesis due, in part, to their substrate specificity. Herein, we report the design and development of manganese transfer hydrogenases based on the biotin-streptavidin technology. By incorporating bio-mimetic Mn(I) complexes into the binding cavity of streptavidin, and through chemo-genetic optimization, we have obtained artificial enzymes that hydrogenate ketones with nearly quantitative yield and up to 98 % enantiomeric excess (ee). These enzymes exhibit broad substrate scope and high functional-group tolerance. According to QM/MM calculations and X-ray crystallography, the S112Y mutation, combined with the appropriate chemical structure of the Mn cofactor plays a critical role in the reactivity and enantioselectivity of the artificial metalloenzyme (ArMs). Our work highlights the potential of ArMs incorporating base-meal cofactors for enantioselective organic synthesis.

An Artificial Metalloenzyme Based on a Copper Heteroscorpionate Enables sp3 C-H Functionalization via Intramolecular Carbene Insertion.

The selective functionalization of sp3 C-H bonds is a versatile tool for the diversification of organic compounds. Combining attractive features of homogeneous and enzymatic catalysts, artificial metalloenzymes offer an ideal means to selectively modify these inert motifs. Herein, we report on a copper(I) heteroscorpionate complex embedded within streptavidin that catalyzes the intramolecular insertion of a carbene into sp3 C-H bonds. Target residues for genetic optimization of the artificial metalloenzyme were identified by quantum mechanics/molecular mechanics simulations. Double-saturation mutagenesis yielded detailed insight on the contribution of individual amino acids on the activity and the selectivity of the artificial metalloenzyme. Mutagenesis at a third position afforded a set of artificial metalloenzymes that catalyze the enantio- and regioselective formation of ß- and γ-lactams with high turnovers and promising enantioselectivities.

Directed evolution of artificial metalloenzymes for in vivo metathesis.

The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions. For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist: repurposing natural metalloenzymes for abiotic transformations; in silico metalloenzyme (re-)design; and incorporation of abiotic cofactors into proteins. The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein. This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction without equivalent in nature. Building on previous work on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the 'metathase' because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.

Ultrahigh-Throughput Screening of an Artificial Metalloenzyme using Double Emulsions.

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols.

HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridium Dye.

HaloTag is a small self-labeling protein that is frequently used for creating fluorescent reporters in living cells. The small-molecule dyes used with HaloTag are almost exclusively based on rhodamine scaffolds, which are often expensive and challenging to synthesize. Herein, we report the engineering of HaloTag for use with a chemically accessible, inexpensive fluorophore based on the dimethylamino-styrylpyridium dye. Through directed evolution, the maximum fluorogenicity and the apparent second-order bioconjugation rate constants could be improved up to 4-fold and 42-fold, respectively. One of the top variants, HT-SP5, enabled reliable imaging in mammalian cells, with a 113-fold fluorescence enhancement over the parent protein. Additionally, crystallographic characterization of selected mutants suggests the chemical origin of the fluorescent enhancement. The improved dye system offers a valuable tool for imaging and illustrates the viability of engineering self-labeling proteins for alternative fluorophores.

Expanding the Potential of the Solvent-Assisted Method to Create Bio-Interfaces from Amphiphilic Block Copolymers.

Artificial membranes, as materials with biomimetic properties, can be applied in various fields, such as drug screening or bio-sensing. The solvent-assisted method (SA) represents a straightforward method to prepare lipid solid-supported membranes. It overcomes the main limitations of established membrane preparation methods, such as Langmuir-Blodgett (LB) or vesicle fusion. However, it has not yet been applied to create artificial membranes based on amphiphilic block copolymers, despite their enhanced mechanical stability compared to lipid-based membranes and bio-compatible properties. Here, we applied the SA method on different amphiphilic di- and triblock poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) copolymers and optimized the conditions to prepare artificial membranes on a solid support. The real-time membrane formation, the morphology, and the mechanical properties have been evaluated by a combination of atomic force microscopy and quartz crystal microbalance. Then, selected biomolecules including complementary DNA strands and an artificial deallylase metalloenzyme (ADAse) were incorporated into these membranes relying on the biotin-streptavidin technology. DNA strands served to establish the capability of these synthetic membranes to interact with biomolecules by preserving their correct conformation. The catalytic activity of the ADAse following its membrane anchoring induced the functionality of the biomimetic platform. Polymer membranes on solid support as prepared by the SA method open new opportunities for the creation of artificial membranes with tailored biomimetic properties and functionality.

Bioorthogonal strategies for the in vivo synthesis or release of drugs.

The site-specific delivery of antitumor agents is a rapidly developing field that relies on prodrug activation and uncaging strategies. For this purpose, a wide range of homogeneous and heterogeneous biocompatible activators/catalysts have been developed to convert caged drugs with low toxicity and high stability in physiological settings into active substances in a bioorthogonal manner. The current methods allow for the site-specific delivery of activators and prodrugs to organelles, target cells, or tumors in living organisms. Here, we present an overview of the latest advances in catalytic drugs, highlighting the expanding toolbox of bioorthogonal activation strategies made possible by transition metals acting as activators or catalysts.

Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey.

Evolution is essential to the generation of complexity and ultimately life. It relies on the propagation of the properties, traits, and characteristics that allow an organism to survive in a challenging environment. It is evolution that shaped our world over about four billion years by slow and iterative adaptation. While natural evolution based on selection is slow and gradual, directed evolution allows the fast and streamlined optimization of a phenotype under selective conditions. The potential of directed evolution for the discovery and optimization of enzymes is mostly limited by the throughput of the tools and methods available for screening. Over the past twenty years, versatile tools based on droplet microfluidics have been developed to address the need for higher throughput. In this Review, we provide a chronological overview of the intertwined development of microfluidics droplet-based compartmentalization methods and in vivo directed evolution of enzymes.

Enantioselective Hydroxylation of Benzylic C(sp3)-H Bonds by an Artificial Iron Hydroxylase Based on the Biotin-Streptavidin Technology.

The selective hydroxylation of C-H bonds is of great interest to the synthetic community. Both homogeneous catalysts and enzymes offer complementary means to tackle this challenge. Herein, we show that biotinylated Fe(TAML)-complexes (TAML = Tetra Amido Macrocyclic Ligand) can be used as cofactors for incorporation into streptavidin to assemble artificial hydroxylases. Chemo-genetic optimization of both cofactor and streptavidin allowed optimizing the performance of the hydroxylase. Using H2O2 as oxidant, up to ∼300 turnovers for the oxidation of benzylic C-H bonds were obtained. Upgrading the ee was achieved by kinetic resolution of the resulting benzylic alcohol to afford up to >98% ee for (R)-tetralol. X-ray analysis of artificial hydroxylases highlights critical details of the second coordination sphere around the Fe(TAML) cofactor.

Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Enzymatic Cascades and Directed Evolution.

Artificial metalloenzymes (ArMs) result from anchoring a metal-containing moiety within a macromolecular scaffold (protein or oligonucleotide). The resulting hybrid catalyst combines attractive features of both homogeneous catalysts and enzymes. This strategy includes the possibility of optimizing the reaction by both chemical (catalyst design) and genetic means leading to achievement of a novel degree of (enantio)selectivity, broadening of the substrate scope, or increased activity, among others. In the past 20 years, the Ward group has exploited, among others, the biotin-(strept)avidin technology to localize a catalytic moiety within a well-defined protein environment. Streptavidin has proven versatile for the implementation of ArMs as it offers the following features: (i) it is an extremely robust protein scaffold, amenable to extensive genetic manipulation and mishandling, (ii) it can be expressed in E. coli to very high titers (up to >8 g·L-1 in fed-batch cultures), and (iii) the cavity surrounding the biotinylated cofactor is commensurate with the size of a typical metal-catalyzed transition state. Relying on a chemogenetic optimization strategy, varying the orientation and the nature of the biotinylated cofactor within genetically engineered streptavidin, 12 reactions have been reported by the Ward group thus far. Recent efforts within our group have focused on extending the ArM technology to create complex systems for integration into biological cascade reactions and in vivo. With the long-term goal of complementing in vivo natural enzymes with ArMs, we summarize herein three complementary research lines: (i) With the aim of mimicking complex cross-regulation mechanisms prevalent in metabolism, we have engineered enzyme cascades, including cross-regulated reactions, that rely on ArMs. These efforts highlight the remarkable (bio)compatibility and complementarity of ArMs with natural enzymes. (ii) Additionally, multiple-turnover catalysis in the cytoplasm of aerobic organisms was achieved with ArMs that are compatible with a glutathione-rich environment. This feat is demonstrated in HEK-293T cells that are engineered with a gene switch that is upregulated by an ArM equipped with a cell-penetrating module. (iii) Finally, ArMs offer the fascinating prospect of "endowing organometallic chemistry with a genetic memory." With this goal in mind, we have identified E. coli's periplasmic space and surface display to compartmentalize an ArM, while maintaining the critical phenotype-genotype linkage. This strategy offers a straightforward means to optimize by directed evolution the catalytic performance of ArMs. Five reactions have been optimized following these compartmentalization strategies: ruthenium-catalyzed olefin metathesis, ruthenium-catalyzed deallylation, iridium-catalyzed transfer hydrogenation, dirhodium-catalyzed cyclopropanation and carbene insertion in C-H bonds. Importantly, >100 turnovers were achieved with ArMs in E. coli whole cells, highlighting the multiple turnover catalytic nature of these systems.

Artificial Iron Proteins: Modeling the Active Sites in Non-Heme Dioxygenases.

An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S112E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic FeII complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S112E. Two aqua ligands complete the primary coordination sphere of the FeII center with both involved in hydrogen bond networks within Sav. The corresponding FeIII protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The FeIII protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.

Artificial Metalloenzymes: Reaction Scope and Optimization Strategies.

The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970's. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000's. Perhaps the most intriguing of potential ArM properties is their ability to endow homogeneous catalysts with a genetic memory. Indeed, incorporating a homogeneous catalyst into a genetically encoded scaffold offers the opportunity to improve ArM performance by directed evolution. This capability could, in turn, lead to improvements in ArM efficiency similar to those obtained for natural enzymes, providing systems suitable for practical applications and greater insight into the role of second coordination sphere interactions in organometallic catalysis. Since its renaissance in the early 2000's, different aspects of artificial metalloenzymes have been extensively reviewed and highlighted. Our intent is to provide a comprehensive overview of all work in the field up to December 2016, organized according to reaction class. Because of the wide range of non-natural reactions catalyzed by ArMs, this was done using a functional-group transformation classification. The review begins with a summary of the proteins and the anchoring strategies used to date for the creation of ArMs, followed by a historical perspective. Then follows a summary of the reactions catalyzed by ArMs and a concluding critical outlook. This analysis allows for comparison of similar reactions catalyzed by ArMs constructed using different metallocofactor anchoring strategies, cofactors, protein scaffolds, and mutagenesis strategies. These data will be used to construct a searchable Web site on ArMs that will be updated regularly by the authors.