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In this Perspective, we present progress, outstanding challenges, and opportunities for the incorporation of artificial metalloenzymes (ArMs) into biosynthetic pathways. We first explain discoveries within the field of ArMs that led to the potential inclusion of these enzymes in biosynthesis. We then describe the specific barriers that our laboratory, in collaboration with the laboratories of Keasling and Mukhopadhyay, addressed to establish a biosynthetic pathway containing an ArM. This biosynthesis produced an unnatural cyclopropyl terpenoid by combining heterologous production of the terpene with modification of its terminal alkene by an ArM built from a cytochrome P450. Finally, we describe the remaining challenges and opportunities related to the application of ArMs in synthetic biology.

Protein-based hydrogel biomaterials provide a platform for different biological applications, including the encapsulation and stabilization of different biomolecules. These hydrogel properties can be modulated by controlling the design parameters to match specific needs; thus, multicomponent hydrogels have distinct advantages over single-component hydrogels due to their enhanced versatility. Here, silk fibroin and γ-prefoldin chaperone protein based composite hydrogels were prepared and studied. Different ratios of the proteins were chosen, and the hydrogels were prepared by enzyme-assisted cross-linking. The secondary structure of the two proteins, dityrosine bond formation, and mechanical properties were assessed. The results obtained can be used as a platform for the rational design of composite thermostable hydrogel biomaterials to facilitate protection (due to hydrogel mechanics) and retention of bioactivity (e.g., of enzymes and other biomolecules) due to chaperone-like properties of γ-prefoldin.

Species of bacteria from the genus Cupriavidus are known, in part, for their ability to produce high amounts of poly-hydroxybutyrate (PHB) making them attractive candidate bioplastic producers. The native production of PHB occurs during periods of metabolic stress, and the process regulating the initiation of PHB accumulation in these organisms is not fully understood. Screening an RB-TnSeq transposon library of Cupriavidus basilensis 4G11 allowed us to identify two genes of an apparent, uncharacterized two component system which, when omitted from the genome, are capable of increased PHB productivity in balanced, non-stress growth conditions. We observe average increases in PHB productivity of 56% and 41% relative to the wildtype parent strain, upon deleting each of two genes individually from the genome. The increased PHB phenotype disappears, however, in nitrogen-free unbalanced growth conditions suggesting the phenotype is specific to fast-growing, replete, non-stress growth. Bioproduction modeling suggests this phenotype could be due to a decreased reliance on metabolic stress induced by nitrogen limitation to initiate PHB production in the mutant strains. Such strains may allow for the use of single stage, continuous bioreactor systems, which are far simpler than PHB bioproduction schemes used previously. Bioproductivity modeling suggests that omitting this regulation in the cells may increase PHB productivity up to 24% relative to the wildtype organism when using single stage continuous systems. This work furthermore expands our understanding of the regulation of PHB accumulation in Cupriavidus, in particular the initiation of this process upon transition into unbalanced growth regimes.

Addressing the three major stresses facing our planet, clean air, clean energy, and clean water, is within our reach. At present, new materials such as metal-organic frameworks and covalent organic frameworks, produced by reticular chemistry, are at the forefront of efforts to capture carbon dioxide from air and harvest water from air. We envision that the products of these two capture processes (carbon dioxide and water) can be fed into a conversion cycle in which they are used to produce fuels and chemicals via artificial photosynthesis. The use of air as a nonpolluting, cyclable, and sustainable resource for carbon and water can be powered by sunlight. We describe how the scientific basis for realizing this vision is either already achieved or being established, and that in the fullness of time this paradigm may lead to new global industries and a thriving “air economy.”

Computational models are increasingly used to investigate and predict the complex dynamics of biological and biochemical systems. Nevertheless, governing equations of a biochemical system may not be (fully) known, which would necessitate learning the system dynamics directly from, often limited and noisy, observed data. On the other hand, when expensive models are available, systematic and efficient quantification of the effects of model uncertainties on quantities of interest can be an arduous task. This paper leverages the notion of flow-map (de)compositions to present a framework that can address both of these challenges via learning data-driven models useful for capturing the dynamical behavior of biochemical systems. Data-driven flow-map models seek to directly learn the integration operators of the governing differential equations in a black-box manner, irrespective of structure of the underlying equations. As such, they can serve as a flexible approach for deriving fast-to-evaluate surrogates for expensive computational models of system dynamics, or, alternatively, for reconstructing the long-term system dynamics via experimental observations. We present a data-efficient approach to data-driven flow-map modeling based on polynomial chaos Kriging. The approach is demonstrated for discovery of the dynamics of various benchmark systems and a coculture bioreactor subject to external forcing, as well as for uncertainty quantification of a microbial electrosynthesis reactor. Such data-driven models and analyses of dynamical systems can be paramount in the design and optimization of bioprocesses and integrated biomanufacturing systems.

Combining the diverse chemical functionality of proteins with the predictable structural assembly of nucleic acids has enabled the creation of hybrid nanostructures for a range of biotechnology applications. Through the attachment of proteins onto or within nucleic acid nanostructures, materials with dynamic capabilities can be created that include switchable enzyme activity, targeted drug delivery, and multienzyme cascades for biocatalysis. Investigations of difficult-to-study biological mechanisms have also been aided by using DNA-protein assemblies that mimic natural processes in a controllable manner. Furthermore, advances that enable the recombinant production and intracellular assembly of hybrid nanostructures have the potential to overcome the significant manufacturing cost that has limited the use of DNA and RNA nanotechnology.

© 2020 Wiley Periodicals LLC Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on “The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes”. On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.

The transfer of electrons through protein complexes is central to cellular respiration. Exploiting proteins for charge transfer in a controllable fashion has the potential to revolutionize the integration of biological systems and electronic devices. Here we characterize the structure of an ultrastable protein filament and engineer the filament subunits to create electronically conductive nanowires under aqueous conditions. Cryoelectron microscopy was used to resolve the helical structure of gamma-prefoldin, a filamentous protein from a hyperthermophilic archaeon. Conjugation of tetra-heme c3-type cytochromes along the longitudinal axis of the filament created nanowires capable of long-range electron transfer. Electrochemical transport measurements indicated networks of the nanowires capable of conducting current between electrodes at the redox potential of the cytochromes. Functionalization of these highly engineerable nanowires with other molecules, such as redox enzymes, may be useful for bioelectronic applications.

Artificial metalloenzymes (ArMs), created by introducing synthetic cofactors into protein scaffolds, are an emerging class of catalyst for non-natural reactions. Although many classes of ArMs are known, in vitro reconstitution of cofactors and proteins has been a limiting step in the high-throughput screening and directed evolution of ArMs because purification of individual host proteins is time-consuming. We describe the application of a platform to combine mutants of the P450 enzyme CYP119 and the cofactor Ir(Me)MPIX in vivo, by coexpression of the CYP119 mutants with the heme transporter encoded by the hug operon, to the directed evolution of ArMs containing Ir(Me)MPIX in whole cells. We applied this platform to the development an ArMs catalyzing the insertion of the acyclic carbene from a-diazopropanoate esters (Me-EDA) into the N-H bonds of N -alkyl anilines, a combination of carbene and amine classes for which mutant enzymes of natural hemoproteins previously reacted with low enantioselectivity. The mutants of the artificial metalloenzyme Ir(Me)CYP119 identified by an evolution campaign involving more than 4000 mutants are shown to catalyze the reaction of Me-EDA with N -methyl anilines to form chiral chiral amino esters with high TON and good enantioselectivity, thereby demonstrating that the directed evolution of ArMs can rival that of natural enzymes in vivo. .

Electromicrobial production (EMP) systems can store renewable energy and CO

Transcription factors (TFs) are potent proteins that control gene expression and can thereby drive cell fate decisions. Fluorescent reporters have been broadly knocked into endogenous TF loci to investigate the biological roles of these factors; however, the sensitivity of such analyses in human pluripotent stem cells (hPSCs) is often compromised by low TF expression levels and/or reporter silencing. Complementarily, we report an inducible and quantitative reporter platform based on the Cre-LoxP recombination system that enables robust, quantifiable, and continuous monitoring of live hPSCs and their progeny to investigate the roles of TFs during human development and disease. Stem Cells 2019;37:1556–1566

The selective functionalization of one C-H bond over others in nearly identical steric and electronic environments can facilitate the construction of complex molecules. We report site-selective functionalizations of C-H bonds, differentiated solely by remote substituents, catalyzed by artificial metalloenzymes (ArMs) that are generated from the combination of an evolvable P450 scaffold and an iridium-porphyrin cofactor. The generated systems catalyze the insertion of carbenes into the C-H bonds of a range of phthalan derivatives containing substituents that render the two methylene positions in each phthalan inequivalent. These reactions occur with site-selectivity ratios of up to 17.8:1 and, in most cases, with pairs of enzyme mutants that preferentially form each of the two constitutional isomers. This study demonstrates the potential of abiotic reactions catalyzed by metalloenzymes to functionalize C-H bonds with site selectivity that is difficult to achieve with small-molecule catalysts.

We report a filamentous chaperone-based protein hydrogel capable of stabilizing enzymes against thermal inactivation. The hydrogel backbone consists of a thermostable chaperone protein, the gamma-prefoldin (γPFD) from Methanocaldococcus jannaschii, which self-assembles into a fibrous structure. Specific coiled-coil interactions engineered into the wildtype γPFD trigger the formation of a cross-linked network of protein filaments. The structure of the filamentous chaperone is preserved through the designed coiled-coil interactions. The resulting hydrogel enables entrapped enzymes to retain greater activity after exposure to high temperatures, presumably by virtue of the inherent chaperone activity of the γPFD.

Enzymatic reactions through mononuclear metal hydrides are unknown in nature, despite the prevalence of such intermediates in the reactions of synthetic transition-metal catalysts. If metalloenzymes would react through abiotic intermediates like these, then the scope of enzyme-catalyzed reactions would expand. Here we show that zinc-containing carbonic anhydrase enzymes catalyze hydride transfers from silanes to ketones with high enantioselectivity and report mechanistic data providing strong evidence that the process involves a mononuclear zinc hydride. This work shows that abiotic silanes can act as reducing equivalents in an enzyme-catalyzed process and that monomeric hydrides of electropositive metals, which are typically unstable in protic environments, can be catalytic intermediates in enzymatic processes. Overall, this work bridges a gap between the types of transformations in molecular catalysis and biocatalysis., Graphical Abstract

Human pluripotent stem cells harbor an unlimited capacity to generate therapeutically relevant cells for applications in regenerative medicine. However, to utilize these cells in the clinic, scalable culture systems that activate defined receptors and signaling pathways to sustain stem cell self-renewal are required; and synthetic materials offer considerable promise to meet these needs. De novo development of materials that target novel pathways has been stymied by a limited understanding of critical receptor interactions maintaining pluripotency. Here, we identify peptide agonists for the human pluripotent stem cell (hPSC) laminin receptor and pluripotency regulator, α6-integrin, through unbiased, library-based panning strategies. Biophysical characterization of adhesion suggests that identified peptides bind hPSCs through α6-integrin with sub-μM dissociation constants similar to laminin. By harnessing a high-throughput microculture platform, we developed predictive guidelines for presenting these integrin-targeting peptides alongside canonical binding motifs at optimal stoichiometries to generate nascent culture surfaces. Finally, when presented as self-assembled monolayers, predicted peptide combinations supported hPSC expansion, highlighting how unbiased screens can accelerate the discovery of targeted biomaterials.

Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium–porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature. [Figure not available: see fulltext.]

A central question surrounding possible human exploration of Mars is whether crewed missions can be supported by available technologies using in situ resources. Here, we show that photovoltaics-based power systems would be adequate and practical to sustain a crewed outpost for an extended period over a large fraction of the planet's surface. Climate data were integrated into a radiative transfer model to predict spectrally-resolved solar flux across the Martian surface. This informed detailed balance calculations for solar cell devices that identified optimal bandgap combinations for maximizing production capacity over a Martian year. We then quantified power systems, manufacturing, and agricultural demands for a six-person mission, which revealed that photovoltaics-based power generation would require, 5 pages, 4 figures, supplementary information

Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.

Microbial electrosynthesis (MES) systems can store renewable energy and CO2 in many-carbon molecules inaccessible to abiotic electrochemistry. Here, we develop a multiphysics model to investigate the fundamental and practical limits of MES enabled by direct electron uptake and we identify organisms in which this biotechnological CO2-fixation strategy can be realized. Systematic model comparisons of microbial respiration and carbon fixation strategies revealed that, under aerobic conditions, the CO2 fixation rate is limited to 2/hr by O2 mass transport despite efficient electron utilization. In contrast, anaerobic nitrate respiration enables CO2 fixation rates >50 μmol/cm2/hr for microbes using the reductive tricarboxylic acid cycle. Phylogenetic analysis, validated by recapitulating experimental demonstrations of electroautotrophy, uncovered multiple probable electroautotrophic organisms and a significant number of genetically tractable strains that require heterologous expression of

Mediated microbial electrosynthesis (MES) represents a promising strategy for the capture and conversion of CO2 into carbon-based products. We describe the development and application of a comprehensive multiphysics model to analyze a formate-mediated MES reactor. The model shows that this system can achieve a biomass productivity of ∼1.7 g L-1 h-1 but is limited by a competitive trade-off between O2 gas/liquid mass transfer and CO2 transport to the cathode. Synthetic metabolic strategies are evaluated for formatotrophic growth, which can enable an energy efficiency of ∼21 %, a 30 % improvement over the Calvin cycle. However, carbon utilization efficiency is only ∼10 % in the best cases due to a futile CO2 cycle, so gas recycling will be necessary for greater efficiency. Finally, separating electrochemical and microbial processes into separate reactors enables a higher biomass productivity of ∼2.4 g L-1 h-1 . The mediated MES model and analysis presented here can guide process design for conversion of CO2 into renewable chemical feedstocks.

Mediated microbial electrosynthesis (MES) represents a promising strategy for the capture and conversion of CO2 into carbon-based products. We describe the development and application of a comprehensive multiphysics model to analyze a formate-mediated MES reactor. The model shows that this system can achieve a biomass productivity of ∼1.7 g L-1 hr-1 but is limited by a competitive trade-off between O2 gas/liquid mass transfer and CO2 transport to the cathode. Synthetic metabolic strategies are evaluated for formatotrophic growth, which can enable an energy efficiency of ∼21%, a 30% improvement over the Calvin cycle. However, carbon utilization efficiency is only ∼10% in the best cases due to a futile CO2 cycle, so gas recycle will be necessary for greater efficiency. Finally, separating electrochemical and microbial processes into separate reactors enables a higher biomass productivity of ∼2.4 g L-1 hr-1. The mediated MES model and analysis presented here can guide process design for conversion of CO2 into renewable chemical feedstocks.

A high-throughput 3D hPSC differentiation screening strategy reveals basic and translational insight for CNS cell therapies., The emergence of several cell therapy candidates in the clinic is an encouraging sign for human diseases/disorders that currently have no effective treatment; however, scalable production of these cell therapies has become a bottleneck. To overcome this barrier, three-dimensional (3D) cell culture strategies have been considered for enhanced cell production. Here, we demonstrate a high-throughput 3D culture platform used to systematically screen 1200 culture conditions with varying doses, durations, dynamics, and combinations of signaling cues to derive oligodendrocyte progenitor cells and midbrain dopaminergic neurons from human pluripotent stem cells (hPSCs). Statistical models of the robust dataset reveal previously unidentified patterns about cell competence to Wnt, retinoic acid, and sonic hedgehog signals, and their interactions, which may offer insights into the combinatorial roles these signals play in human central nervous system development. These insights can be harnessed to optimize production of hPSC-derived cell replacement therapies for a range of neurological indications.

We describe a versatile CRISPR/Cas-based strategy to construct multi-enzyme complexes scaffolded on a DNA template in programmable patterns. Catalytically inactive dCas9 nuclease was used in combination with SpyCatcher-SpyTag chemistry to assemble enzymes in a highly modular fashion. Five enzymes comprising the violacein biosynthesis pathway were precisely organized in nanometer proximity; a notable increase in violacein production demonstrated the benefits of scaffolding.

Synthetic biology enables microbial hosts to produce complex molecules that areotherwise produced by organisms that are rare or difficult to cultivate, but the structures of thesemolecules are limited to chemical reactions catalyzed by natural enzymes. The integration ofartificial metalloenzymes (ArMs) that catalyze abiotic reactions into metabolic networks couldbroaden the cache of molecules produced biosynthetically by microorgansms. We report theassembly of an ArM containing an iridium-porphyrin complex in the cytoplasm of a terpeneproducing Escherichia coli by a heterologous heme transport machinery, and insertion of this ArMinto a natural biosynthetic pathway to produce an unnatural terpenoid. This work shows thatsynthetic biology and synthetic chemistry, incorporated together in whole cells, can producemolecules previously inaccessible to nature.

Advancing our knowledge of how neural stem cell (NSC) behavior in the adult hippocampus is regulated has implications for elucidating basic mechanisms of learning and memory as well as for neurodegenerative disease therapy. To date, numerous biochemical cues from the endogenous hippocampal NSC niche have been identified as modulators of NSC quiescence, proliferation, and differentiation; however, the complex repertoire of signaling factors within stem cell niches raises the question of how cues act in combination with one another to influence NSC physiology. To help overcome experimental bottlenecks in studying this question, we adapted a high-throughput microculture system, with over 500 distinct microenvironments, to conduct a systematic combinatorial screen of key signaling cues and collect high-content phenotype data on endpoint NSC populations. This novel application of the platform consumed only 0.2% of reagent volumes used in conventional 96-well plates, and resulted in the discovery of numerous statistically significant interactions among key endogenous signals. Antagonistic relationships between fibroblast growth factor 2, transforming growth factor β (TGF-β), and Wnt-3a were found to impact NSC proliferation and differentiation, whereas a synergistic relationship between Wnt-3a and Ephrin-B2 on neuronal differentiation and maturation was found. Furthermore, TGF-β and bone morphogenetic protein 4 combined with Wnt-3a and Ephrin-B2 resulted in a coordinated effect on neuronal differentiation and maturation. Overall, this study offers candidates for further elucidation of significant mechanisms guiding NSC fate choice and contributes strategies for enhancing control over stem cell-based therapies for neurodegenerative diseases.

Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell culture models have been used increasingly for in vitro cell culture because they may better model endogenous tissue environments. However, detailed studies of stem cell differentiation within 3D cultures remain limited, particularly with respect to high-throughput screening. Herein, we demonstrate the use of a microarray chip-based platform to screen, in high-throughput, individual and paired effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures. Dose-response analysis of selected combinations from the initial combinatorial screen revealed that the combined treatment of all-trans retinoic acid (RA) with the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) enhances neurogenesis while simultaneously decreases astrocyte differentiation, whereas the combined treatment of brain-derived neurotrophic factor and the small azide neuropathiazol enhances the differentiation into neurons and astrocytes. Subtype specification analysis of RA- and CHIR-differentiated cultures revealed that enhanced neurogenesis was not biased toward a specific neuronal subtype. Together, these results demonstrate a high-throughput screening platform for rapid evaluation of differentiation conditions in a 3D environment, which will aid the development and application of 3D stem cell culture models.

Two novel tandem catalysis approaches for the chiral synthesis of S-alcohols from reduction of their prochiral ketones with Horse Liver Alcohol Dehydrogenase (HLADH), and selective C-H oxidation reactions with protein engineered Cytochrome P450s, are presented. We utilized a co-factor regeneration procedure with three biomimetic NAD+ models that do not contain the pyrophosphate, nor the adenosine group, and either/or a ribose, N-1-benzylnicotinamide triflate, 1, N-4-methoxybenzylnicotinamide triflate, 2, and β-nicotinamide-5′-ribose methyl phosphate, 3, in conjunction with in situ formed [Cp*Rh(bpy)H]+ from [Cp*Rh(bpy)(H2O)]2+ (Cp* = η5-C5Me5, bpy = 2,2'-bipyridyl) and the hydride source, sodium formate, to regioselectively provide their 1,4-NADH analogs, N-benzyl-1,4-dihydronicotinamide, 4, N-4-methoxybenzyl-1,4-dihydronicotinamide, 5, and 1,4-dihydronicotinamide-5′-ribose methylphosphate, 6. Surprisingly, the 1,4-NADH biomimics, 4 and 6, were recognized, in the second tandem catalysis approach, by the natural 1,4-NADH dependent enzyme, HLADH, for catalyzed, highly enantioselective conversions of prochiral ketones to chiral S-alcohols. For example, with phenethylmethyl ketone and benzylmethyl ketone, the corresponding chiral alcohols were formed in >93% ee (S-enantiomer). Thus, 1,4-NADH biomimetic model recognition by HLADH does not significantly depend on the presence of the ribose, pyrophosphate, or adenosine groups to provide chiral products. We will also propose a plausible active site (HLADH)Zn-H intermediate, generated via a hydride transfer from bound 4/6 to Zn, for the enzymatic reduction of prochiral aryl/alkyl ketones to their chiral aryl/alkyl S-alcohols. Furthermore, the use of protein engineered cytochrome P450 enzymes provided improved molecular recognition of the above mentioned 1,4-NADH biomimetic co-factors, 4 and 5, for selective C-H oxidation reactions. For example, 1,4-NADH dependent mutants of natural 1,4-NAD(P)H dependent P450 BM-3 and 1,4-NADH dependent P450 CAM, with biomimetic co-factors 4 and 5, provided selective oxidation of p-nitrophenoxydecanoic acid to ω-oxydecanocarboxylic acid and p-nitrophenol, via C-H hydroxylation and β-hydrogen elimination, while oxidation of camphor provided hydroxycamphor, respectively. We will discuss the various parameters that effect molecular recognition of the biomimics, including protein engineering of both P450 BM-3 and P450 CAM enzymes, while determining the effect of the co-factor regeneration procedure on HLADH and P450 enzyme activity. These important observations have created new paradigms for the synthesis of organic compounds of interest, with the economically more favorable biomimics of NAD+, 1,4-NADH, and 1,4-NAD(P)H as co-factors, in tandem with the use of [Cp*Rh(bpy)(H)]+ as a regioselective catalytic reagent for co-factor regeneration.

Harnessing the ability of proteins to self-assemble into complex structures has enabled the creation of templates for applications in nanotechnology. Protein templates can be used to position functional molecules in regular patterns with nanometer precision over large surface areas. A difficult but successful approach to building customizable protein templates involves designing novel protein-protein interfaces to join protein building blocks into ordered arrangements. This approach was illustrated recently by engineering the protein interfaces of a molecular chaperone to produce filamentous templates composed of repeating subunits. In this chapter, we describe how these multicomponent protein templates can be produced recombinantly, assembled into filaments, and used as material templates. The templates enable the positioning and alignment of functional molecules at varying distances along the length of the filament, which can be demonstrated using a Förster resonance energy transfer (FRET) assay. In addition, we describe a method to quantify the chaperone ability of these filaments to stabilize and protect other proteins from thermal-induced aggregation-a useful property for bionanotechnology applications that involve molecular scaffolds for positioning and stabilizing enzymes.

Harnessing the ability of proteins to self-assemble into complex structures has enabled the creation of templates for applications in nanotechnology. Protein templates can be used to position functional molecules in regular patterns with nanometer precision over large surface areas. A difficult but successful approach to building customizable protein templates involves designing novel protein-protein interfaces to join protein building blocks into ordered arrangements. This approach was illustrated recently by engineering the protein interfaces of a molecular chaperone to produce filamentous templates composed of repeating subunits. In this chapter, we describe how these multicomponent protein templates can be produced recombinantly, assembled into filaments, and used as material templates. The templates enable the positioning and alignment of functional molecules at varying distances along the length of the filament, which can be demonstrated using a Forster resonance energy transfer (FRET) assay. In addition, we describe a method to quantify the chaperone ability of these filaments to stabilize and protect other proteins from thermal-induced aggregation-a useful property for bionanotechnology applications that involve molecular scaffolds for positioning and stabilizing enzymes.

Precisely organized enzyme complexes are often found in nature to support complex metabolic reactions in a highly efficient and specific manner. Scaffolding enzymes on artificial materials has thus gained attention as a promising biomimetic strategy to design biocatalytic systems with enhanced productivity. Herein, a versatile scaffolding platform that can immobilize enzymes on customizable nanofibers is reported. An ultrastable self-assembling filamentous protein, the gamma-prefoldin (γ-PFD), is genetically engineered to display an array of peptide tags, which can specifically and stably bind enzymes containing the counterpart domain through simple in vitro mixing. Successful immobilization of proteins along the filamentous template in tunable density is first verified using fluorescent proteins. Then, two different model enzymes, glucose oxidase and horseradish peroxidase, are used to demonstrate that scaffold attachment could enhance the intrinsic catalytic activity of the immobilized enzymes. Considering the previously reported ability of γ-PFD to bind and stabilize a broad range of proteins, the filament's interaction with the bound enzymes may have created a favorable microenvironment for catalysis. It is envisioned that the strategy described here may provide a generally applicable methodology for the scaffolded assembly of multienzymatic complexes for use in biocatalysis.

We demonstrate the synthesis of protein–polymer hybrid hydrogel that can be used as a platform for immobilizing functional proteins. Orthogonal chemistry was employed for cross-linking the hybrid network and conjugating proteins to the gel backbone, allowing for the convenient, one-pot formation of a functionalized hydrogel. The resulting hydrogel had tunable mechanical properties, was stable in solution, and biocompatible.

Summary A 3D cell culture chip was used for high-throughput screening of a human neural progenitor cell line. The differential toxicity of 24 compounds was determined on undifferentiated and differentiating NPCs. Five compounds led to significant differences in IC50 values between undifferentiated and differentiating cultures. This platform has potential use in phenotypic screening to elucidate molecular toxicology on human stem cells., Graphical Abstract, Highlights • Demonstrated chip platform for HTS of protein expression and toxicity of 3D cultures • Dose-response viability and proliferation of a 24-compound library on human NPC lines • Assessed differential toxicity between progenitors and differentiating progeny • Identified five compounds more toxic to undifferentiated progenitors, Dordick and colleagues demonstrate a 3D cell culture chip for high-throughput screening of immortalized human neural progenitor cells. The dose-response toxicity of 24 compounds was determined on undifferentiated and differentiating hNPCs. Five compounds led to significant differences in IC50 values between undifferentiated and differentiating cultures. This platform has potential use in phenotypic screening to elucidate molecular toxicology on human stem cells.