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Human carbonic anhydrase II (hCAII) naturally catalyzes the reaction between two achiral molecules - water and carbon dioxide - to yield the achiral product carbonic acid through a zinc hydroxide intermediate. We have previously shown that a zinc hydride, instead of a hydroxide, can be generated in this enzyme to create a catalyst for the reduction of aryl ketones. Dialkyl ketones are more challenging to reduce, and the enantioselective reduction of dialkyl ketones with two alkyl groups that are similar in size and electronic properties, is a particularly challenging transformation to achieve with high activity and selectivity. Here, we show that hCAII, as well as a double variant of it, catalyzes the enantioselective reduction of dialkyl ketones with high yields and enantioselectivities, even when the two alkyl groups are similar in size. We also show that variants of hCAII catalyze the site-selective reduction of one ketone over the other in an unsymmetrical aliphatic diketone. Computational docking of a dialkyl ketone to the double variant containing the zinc hydride provides insights into the origins of the reactivity of various substrates and the high enantioselectivity of the transformations and show how a confined environment can control the enantioselectivity of an abiological intermediate.
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Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii, self-assembles into filaments in vitro, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve â¼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M. jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.
Assuntos
Methanocaldococcus , Chaperonas Moleculares , Chaperonas Moleculares/química , Proteínas Arqueais/química , Proteínas Arqueais/ultraestrutura , Microscopia de Fluorescência/métodos , Imageamento Tridimensional/métodosRESUMO
Electronically conductive protein-based materials can enable the creation of bioelectronic components and devices from sustainable and nontoxic materials, while also being well-suited to interface with biological systems, such as living cells, for biosensor applications. However, as proteins are generally electrical insulators, the ability to render protein assemblies electroactive in a tailorable manner can usher in a plethora of useful materials. Here, an approach to fabricate electronically conductive protein nanowires is presented by aligning heme molecules in proximity along protein filaments, with these nanowires also possessing charge transfer abilities that enable energy harvesting from ambient humidity. The heme-incorporated protein nanowires demonstrate electron transfer over micrometer distances, with conductive atomic force microscopy showing individual nanowires having comparable conductance to other previously characterized heme-based bacterial nanowires. Exposure of multilayer nanowire films to humidity produces an electrical current, presumably through water molecules ionizing carboxyl groups in the filament and creating an unbalanced total charge distribution that is enhanced by the heme. Incorporation of heme and potentially other metal-center porphyrin molecules into protein nanostructures could pave the way for structurally- and electrically-defined protein-based bioelectronic devices.
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Condutividade Elétrica , Heme , Nanofios , Nanofios/química , Heme/química , Microscopia de Força Atômica , Proteínas/químicaRESUMO
Electromicrobial production (EMP), where electrochemically generated substrates (e.g., H2) are used as energy sources for microbial processes, has garnered significant interest as a method of producing fuels and other value-added chemicals from CO2. Combining these processes with direct air capture (DAC) has the potential to enable a truly circular carbon economy. Here, we analyze the economics of a hypothetical system that combines adsorbent-based DAC with EMP to produce n-butanol, a potential replacement for fossil fuels. First-principles-based modeling is used to predict the performance of the DAC and bioprocess components. A process model is then developed to map material and energy flows, and a techno-economic assessment is performed to determine the minimum fuel selling price. Beyond assessing a specific set of conditions, this analytical framework provides a tool to reveal potential pathways toward the economic viability of this process. We show that an EMP system utilizing an engineered knallgas bacterium can achieve butanol production costs of <$6/gal ($1.58/L) if a set of optimistic assumptions can be realized.
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1-Butanol , Dióxido de CarbonoRESUMO
Sustainable fertilizer production is a pressing challenge due to a growing human population. The manufacture of synthetic nitrogen fertilizer involves intensive emissions of greenhouse gases. The synthetic nitrogen that ends up in biowaste such as animal waste perturbs the nitrogen cycle through significant nitrogen losses in the form of ammonia volatilization, a major human health and environmental hazard. Low-temperature air-plasma treatment of animal waste holds promise for sustainable fertilizer production on farmlands by enabling nitrogen fixation via ionization, forming nitrogen oxyacids. Although the formation of nitrogen oxyacids in plasma treatment of water is well-established, the extent of nitrogen oxyanion enrichment in animal waste and its downstream effects on acidifying the waste remain elusive because many compounds found in complex biowaste media may interfere with absorbed NOx species. This work aims to establish that plasma treatment of dairy manure can suppress ammonia loss by volatilization via acidification of animal waste while enriching the waste in total nitrogen due to nitrogen retained in ammonia as well as adding nitrogen oxyacids by reacting NOx with the aqueous slurry. To this end, air-plasma effluent containing NOx is bubbled through dairy manure, which is then analyzed for changes in the nitrogen oxyanion content and pH. Increasing the plasma treatment time results in more acidic manure, reduced ammonium content in the downstream acid trap, and increased nitrogen oxyanion content, where the yield of nitrogen oxyanion from absorbed NOx species is approximately 100%. Increased plasma treatment also led to an increase in the total Kjeldahl nitrogen and the total nitrogen. These results indicate that plasma treatment of animal waste can significantly suppress ammonia pollution from animal husbandry facilities such as dairy farms while upcycling animal waste as a rich organic source of nitrogen.
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Alkylidene cyclopropanes (ACPs) are valuable synthetic intermediates because of their constrained structure and opportunities for further diversification. Although routes to ACPs are known, preparations of ACPs with control of both the configuration of the cyclopropyl (R vs S) group and the geometry of the alkene (E vs Z) are unknown. We describe enzymatic cyclopropanation of allenes with ethyl diazoacetate (EDA) catalyzed by an iridium-containing cytochrome (Ir(Me)-CYP119) that controls both stereochemical elements. Two mutants of Ir(Me)-CYP119 identified by 6-codon (6c, VILAFG) saturation mutagenesis catalyze the formation of (E)-ACPs with -93% to >99% ee and >99:1 E/Z ratio with just three rounds of 96 mutants. By four additional rounds of mutagenesis, an enzyme variant was identified that forms (Z)-ACPs with up to 94% ee and a 28:72 E/Z ratio. Computational studies show that the orientation of the carbene unit dictated by the mutated positions accounts for the stereoselectivity.
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Alcadienos , Irídio , Catálise , Alcenos/químicaRESUMO
Nature often uses dynamically assembling multienzymatic complexes called metabolons to achieve spatiotemporal control of complex metabolic reactions. Researchers are aiming to mimic this strategy of organizing enzymes to enhance the performance of artificial biocatalytic systems. Biomolecular condensates formed through liquid-liquid phase separation (LLPS) can serve as a powerful tool to drive controlled assembly of enzymes. Diverse enzymatic pathways have been reconstituted within catalytic condensates in vitro as well as synthetic membraneless organelles in living cells. Furthermore, in vivo condensates have been engineered to regulate metabolic pathways by selectively sequestering enzymes. Thus, harnessing LLPS for controlled organization of enzymes provides an opportunity to dynamically regulate biocatalytic processes.
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Células Artificiais , Condensados Biomoleculares , Biocatálise , Catálise , Separação de FasesRESUMO
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 candidates for bioplastic production. The native synthesis 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 enable increased PHB productivity in balanced, nonstress growth conditions. We observe average increases in PHB productivity of 56% and 41% relative to the wildtype parent strain upon deleting each gene 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, nonstress 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. Due to uncertainty in the two-component system's input signal and regulon, the mechanism by which these genes impart this phenotype remains unclear. Such strains may allow for the use of single-stage, continuous bioreactor systems, which are far simpler than many PHB bioproduction schemes used previously, given a similar product yield to batch systems in such a configuration. 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 expands our understanding of the regulation of PHB accumulation in Cupriavidus, in particular the initiation of this process upon transition into unbalanced growth regimes.
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Cupriavidus necator , Cupriavidus , Hidroxibutiratos/metabolismo , Cupriavidus/genética , Reatores Biológicos , Nitrogênio/metabolismo , Poliésteres/metabolismoRESUMO
Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.
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Azasserina , Azasserina/biossíntese , Azasserina/química , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Família Multigênica/genética , Estireno/química , Ciclopropanos/química , Coenzimas/química , Coenzimas/metabolismo , Biocatálise , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismoRESUMO
BACKGROUND: Intracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis. RESULTS: To overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended in distilled water, this engineered strain experienced 75% cell lysis, although decreases in cell growth rate due to higher salt concentrations were observed. CONCLUSIONS: Our strategy is shown to be a simple and effective way to lyse cells for the purification of intracellular biomacromolecules and may be applicable in many bacteria used for bioproduction.
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Cupriavidus necator , Proteínas de Escherichia coli , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Canais Iônicos/genética , Cupriavidus necator/metabolismo , Cloreto de Sódio/farmacologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Bactérias/metabolismo , Água , Mamíferos/metabolismoRESUMO
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.
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Hidrogéis , Seda , Seda/química , Hidrogéis/química , Peroxidase do Rábano Silvestre/metabolismo , Materiais Biocompatíveis/química , CatáliseRESUMO
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.
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Algoritmos , Dinâmica não Linear , Incerteza , Reatores Biológicos , Modelos BiológicosRESUMO
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.
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Metaloproteínas , Metaloproteínas/metabolismo , Terpenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Vias BiossintéticasRESUMO
Electromicrobial production (EMP) 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 EMP enabled by direct electron uptake. We also identify potential electroautotrophic organisms and metabolic engineering strategies to enable electroautotrophy in organisms lacking the native capability. Systematic model comparisons of microbial respiration and carbon fixation strategies revealed that, under aerobic conditions, the CO2 fixation rate is limited to < 6 µmol/cm2/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, predicted multiple probable electroautotrophic organisms and a significant number of genetically tractable strains that require heterologous expression of < 5 proteins to gain electroautotrophic function. The model and analysis presented here will guide microbial engineering and reactor design for practical EMP systems.
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Dióxido de Carbono , Elétrons , Dióxido de Carbono/metabolismo , FilogeniaRESUMO
The potential applications afforded by the generation and reactivity of artificial metalloenzymes (ArMs) in microorganisms are vast. We show that a non-pathogenic E. coli strain, Nissle 1917 (EcN), is a suitable host for the creation of ArMs from cytochrome P450s and artificial heme cofactors. An outer-membrane receptor in EcN transports an iridium porphyrin into the cell, and the Ir-CYP119 (CYP119 containing iridium porphyrin) assembled in vivo catalyzes carbene insertions into benzylic C-H bonds enantioselectively and site-selectively. The application of EcN as a whole-cell screening platform eliminates the need for laborious processing procedures, drastically increases the ease and throughput of screening, and accelerates the development of Ir-CYP119 with improved catalytic properties. Studies to identify the transport machinery suggest that a transporter different from the previously assumed ChuA receptor serves to usher the iridium porphyrin into the cytoplasm.
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Escherichia coli/metabolismo , Evolução Molecular , Metaloproteínas/metabolismo , Carbono/química , Catálise , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hidrogênio/química , Irídio/química , Metaloproteínas/química , Metaloproteínas/genética , Metano/análogos & derivados , Metano/química , Mutagênese Sítio-Dirigida , Porfirinas/química , EstereoisomerismoRESUMO
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 α-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.
Assuntos
MetaloproteínasRESUMO
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.
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Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Terpenos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Irídio/química , Mesoporfirinas/química , Engenharia Metabólica , Estereoisomerismo , Sulfolobus solfataricus/enzimologia , Terpenos/químicaRESUMO
Providing life-support materials to crewed space exploration missions is pivotal for mission success. However, as missions become more distant and extensive, obtaining these materials from in situ resource utilization is paramount. The combination of microorganisms with electrochemical technologies offers a platform for the production of critical chemicals and materials from CO2 and H2O, two compounds accessible on a target destination like Mars. One such potential commodity is poly(3-hydroxybutyrate) (PHB), a common biopolyester targeted for additive manufacturing of durable goods. Here, we present an integrated two-module process for the production of PHB from CO2. An autotrophic Sporomusa ovata (S. ovata) process converts CO2 to acetate which is then directly used as the primary carbon source for aerobic PHB production by Cupriavidus basilensis (C. basilensis). The S. ovata uses H2 as a reducing equivalent to be generated through electrocatalytic solar-driven H2O reduction. Conserving and recycling media components is critical, therefore we have designed and optimized our process to require no purification or filtering of the cell culture media between microbial production steps which could result in up to 98% weight savings. By inspecting cell population dynamics during culturing we determined that C. basilensis suitably proliferates in the presence of inactive S. ovata. During the bioprocess 10.4 mmol acetate L -1 day-1 were generated from CO2 by S. ovata in the optimized media. Subsequently, 12.54 mg PHB L-1 hour-1 were produced by C. basilensis in the unprocessed media with an overall carbon yield of 11.06% from acetate. In order to illustrate a pathway to increase overall productivity and enable scaling of our bench-top process, we developed a model indicating key process parameters to optimize.
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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.
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Hidrogéis/química , Methanocaldococcus/química , Chaperonas Moleculares/química , Reagentes de Ligações Cruzadas/química , Temperatura Alta , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estabilidade ProteicaRESUMO
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.