A rapid cell-free expression and screening platform for antibody discovery.

Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly.

Intersubunit Coupling Enables Fast CO2-Fixation by Reductive Carboxylases.

Enoyl-CoA carboxylases/reductases (ECRs) are some of the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying the extraordinary catalytic activity of ECRs on the level of the protein assembly remain elusive. Here we used a combination of ambient-temperature X-ray free electron laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. The K. setae ECR is a homotetramer that differentiates into a pair of dimers of open- and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in the K. setae ECR across the pair of dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intrasubunit communications of this remarkably efficient CO2-fixing enzyme during catalysis.

Cell-free prototyping enables implementation of optimized reverse ß-oxidation pathways in heterotrophic and autotrophic bacteria.

Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse ß-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimize and implement r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generates designer strains for the selective production of butanoic acid (4.9 ± 0.1 gL-1), as well as hexanoic acid (3.06 ± 0.03 gL-1) and 1-hexanol (1.0 ± 0.1 gL-1) at the best performance reported to date in this bacterium. We also generate Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL-1 in a 1.5 L continuous fermentation. Our strategy enables optimization of r-BOX derived products for biomanufacturing and industrial biotechnology.

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

Cell-Free Protein Synthesis for High-Throughput Biosynthetic Pathway Prototyping.

Biological systems provide a sustainable and complimentary approach to synthesizing useful chemical products. Metabolic engineers seeking to establish economically viable biosynthesis platforms strive to increase product titers, rates, and yields. Despite continued advances in genetic tools and metabolic engineering techniques, cellular workflows remain limited in throughput. It may take months to test dozens of unique pathway designs even in a robust model organism, such as Escherichia coli. In contrast, cell-free protein synthesis enables the rapid generation of enzyme libraries that can be combined to reconstitute metabolic pathways in vitro for biochemical synthesis in days rather than weeks. Cell-free reactions thereby enable comparison of hundreds to thousands of unique combinations of enzyme homologs and concentrations, which can quickly identify the most productive pathway variants to test in vivo or further characterize in vitro. This cell-free pathway prototyping strategy provides a complementary approach to accelerate cellular metabolic engineering efforts toward highly productive strains for metabolite production.

Benzylmalonyl-CoA dehydrogenase, an enzyme involved in bacterial auxin degradation.

A novel acyl-CoA dehydrogenase involved in degradation of the auxin indoleacetate by Aromatoleum aromaticum was identified as a decarboxylating benzylmalonyl-CoA dehydrogenase (IaaF). It is encoded within the iaa operon coding for enzymes of indoleacetate catabolism. Using enzymatically produced benzylmalonyl-CoA, the reaction was characterized as simultaneous oxidation and decarboxylation of benzylmalonyl-CoA to cinnamoyl-CoA and CO2. Oxygen served as electron acceptor and was reduced to H2O2, whereas electron transfer flavoprotein or artificial dyes serving as electron acceptors for other acyl-CoA dehydrogenases were not used. The enzyme is homotetrameric, contains an FAD cofactor and is enantiospecific in benzylmalonyl-CoA turnover. It shows high catalytic efficiency and strong substrate inhibition with benzylmalonyl-CoA, but otherwise accepts only a few medium-chain alkylmalonyl-CoA compounds as alternative substrates with low activities. Its reactivity of oxidizing 2-carboxyacyl-CoA with simultaneous decarboxylation is unprecedented and indicates a modified reaction mechanism for acyl-CoA dehydrogenases, where elimination of the 2-carboxy group replaces proton abstraction from C2.

Toward sustainable, cell-free biomanufacturing.

Industrial biotechnology is an attractive approach to address the need for low-cost fuels and products from sustainable resources. Unfortunately, cells impose inherent limitations on the effective synthesis and release of target products. One key constraint is that cellular survival objectives often work against the production objectives of biochemical engineers. Additionally, industrial strains release CO2 and struggle to utilize sustainable, potentially profitable feedstocks. Cell-free biotechnology, which uses biological machinery harvested from cells, can address these challenges with advantages including: (i) shorter development times, (ii) higher volumetric production rates, and (iii) tolerance to otherwise toxic molecules. In this review, we highlight recent advances in cell-free technologies toward the production of non-protein products beyond lab-scale demonstrations and describe guiding principles for designing cell-free systems. Specifically, we discuss carbon and energy sources, reaction homeostasis, and scale-up. Expanding the scope of cell-free biomanufacturing practice could enable innovative approaches for the industrial production of green chemicals.

Cell-Free Exploration of the Natural Product Chemical Space.

Natural products and secondary metabolites comprise an indispensable resource from living organisms that have transformed areas of medicine, agriculture, and biotechnology. Recent advances in high-throughput DNA sequencing and computational analysis suggest that the vast majority of natural products remain undiscovered. To accelerate the natural product discovery pipeline, cell-free metabolic engineering approaches used to develop robust catalytic networks are being repurposed to access new chemical scaffolds, and new enzymes capable of performing diverse chemistries. Such enzymes could serve as flexible biocatalytic tools to further expand the unique chemical space of natural products and secondary metabolites, and provide a more sustainable route to manufacture these molecules. Herein, we highlight select examples of natural product biosynthesis using cell-free systems and propose how cell-free technologies could facilitate our ability to access and modify these structures to transform synthetic and chemical biology.

Modular cell-free expression plasmids to accelerate biological design in cells.

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms-organisms that are easy to use like Escherichia coli-often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have desirable metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes, and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has matured as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute's DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of functional enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for Clostridium autoethanogenum expression with efficiencies ranging from 68% to 90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

Tuning the Cell-Free Protein Synthesis System for Biomanufacturing of Monomeric Human Filaggrin.

The modern cell-free protein synthesis (CFPS) system is expanding the opportunity of cell-free biomanufacturing as a versatile platform for synthesizing various therapeutic proteins. However, synthesizing human protein in the bacterial CFPS system remains challenging due to the low expression level, protein misfolding, inactivity, and more. These challenges limit the use of a bacterial CFPS system for human therapeutic protein synthesis. In this study, we demonstrated the improved performance of a customized CFPS platform for human therapeutic protein production by investigating the factors that limit cell-free transcription-translation. The improvement of the CFPS platform has been made in three ways. First, the cell extract was prepared from the rare tRNA expressed host strain, and CFPS was performed with a codon-optimized gene for Escherichia coli codon usage bias. The soluble protein yield was 15.2 times greater with the rare tRNA overexpressing host strain as cell extract and codon-optimized gene in the CFPS system. Next, we identify and prioritize the critical biomanufacturing factors for highly active crude cell lysate for human protein synthesis. Lastly, we engineer the CFPS reaction conditions to enhance protein yield. In this model, the therapeutic protein filaggrin expression was significantly improved by up to 23-fold, presenting 28 ± 5 µM of soluble protein yield. The customized CFPS system for filaggrin biomanufacturing described here demonstrates the potential of the CFPS system to be adapted for studying therapeutic proteins.

Awakening a latent carbon fixation cycle in Escherichia coli.

Carbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. We identify the GED (Gnd-Entner-Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner-Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.

A common approach for absolute quantification of short chain CoA thioesters in prokaryotic and eukaryotic microbes.

BACKGROUND: Thioesters of coenzyme A participate in 5% of all enzymatic reactions. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5-10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance of these metabolites explains the high interest to trace and quantify them in microbial cells. RESULTS: Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach assessed intracellular CoA thioesters down to the picomolar level and exhibited high precision and reproducibility for all microbes, as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA metabolism. A succinyl-CoA synthase defective mutant of C. glutamicum exhibited an unaffected level of succinyl-CoA that indicated a complete compensation by the L-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the actinomycete S. albus. The microbe revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum. CONCLUSIONS: The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use one single protocol promises to facilitate automatized efforts, which rely on standardized workflows.

A critical comparison of cellular and cell-free bioproduction systems.

Conversion of biological feedstocks into value-added chemicals is mostly performed via microbial fermentation. An emerging alternative approach is the use of cell-free systems, consisting of purified enzymes and cofactors. Unfortunately, the in vivo and in vitro research communities rarely interact, which leads to oversimplifications and exaggerations that do not permit fair comparison of the two strategies and impede synergistic interactions. Here, we provide a comprehensive account for the advantages and drawbacks associated with each strategy, and further discuss recent research efforts that aim to breach the limits of cellular and cell-free production. We also explore emerging hybrid solutions that integrate the benefits of both worlds and could expand the boundaries of biosynthesis.

Four amino acids define the CO2 binding pocket of enoyl-CoA carboxylases/reductases.

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO2) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO2 molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO2 within the active site of Ecr from Kitasatospora setae Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO2-fixing enzyme. Together, these 4 residues anchor and position the CO2 molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO2 fixation. Altogether, our study reveals unprecedented molecular details of selective CO2 binding and C-C-bond formation during the catalytic cycle of nature's most efficient CO2-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO2 in biology and chemistry.

Crystal structure of archaeal HMG-CoA reductase: insights into structural changes of the C-terminal helix of the class-I enzyme.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyses the last step in mevalonate biosynthesis. HMGR is the target of statin inhibitors that regulate cholesterol concentration in human blood. Here, we report the properties and structures of HMGR from an archaeon Methanothermococcus thermolithotrophicus (mHMGR). The structures of the apoenzyme and the NADPH complex are highly similar to those of human HMGR. A notable exception is C-terminal helix (Lα10-11) that is straight in both mHMGR structures. This helix is kinked and closes the active site in the human enzyme ternary complex, pointing to a substrate-induced structural rearrangement of C-terminal in class-I HMGRs during the catalytic cycle.

'Negative' and 'positive catalysis': complementary principles that shape the catalytic landscape of enzymes.

Our understanding of enzyme catalysis is dominated by transition state theory. According to this concept, an enzymatic reaction is guided along a desired reaction coordinate through the stabilization of favorable transition state. But how much is the outcome of an enzyme reaction controlled by the destabilization of unwanted transition states? Here, we revive and critically review the hypothesis that the active site of enzymes also features elements of 'negative catalysis'. We provide examples that show that enzyme catalysis can be achieved by the combined action of positive and negative constraints at the active site of an enzyme. This integrated view of enzyme catalysis has direct consequences for our studies on the catalytic landscape of enzymes, as well as current efforts in enzyme engineering and the de novo-design of enzymes.

The multicatalytic compartment of propionyl-CoA synthase sequesters a toxic metabolite.

Cells must cope with toxic or reactive intermediates formed during metabolism. One coping strategy is to sequester reactions that produce such intermediates within specialized compartments or tunnels connecting different active sites. Here, we show that propionyl-CoA synthase (PCS), an ∼ 400-kDa homodimer, three-domain fusion protein and the key enzyme of the 3-hydroxypropionate bi-cycle for CO2 fixation, sequesters its reactive intermediate acrylyl-CoA. Structural analysis showed that PCS forms a multicatalytic reaction chamber. Kinetic analysis suggested that access to the reaction chamber and catalysis are synchronized by interdomain communication. The reaction chamber of PCS features three active sites and has a volume of only 33 nm3. As one of the smallest multireaction chambers described in biology, PCS may inspire the engineering of a new class of dynamically regulated nanoreactors.

InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis.

The enoyl-thioester reductase InhA catalyzes an essential step in fatty acid biosynthesis of Mycobacterium tuberculosis and is a key target of antituberculosis drugs to combat multidrug-resistant M. tuberculosis strains. This has prompted intense interest in the mechanism and intermediates of the InhA reaction. Here, using enzyme mutagenesis, NMR, stopped-flow spectroscopy, and LC-MS, we found that the NADH cofactor and the CoA thioester substrate form a covalent adduct during the InhA catalytic cycle. We used the isolated adduct as a molecular probe to directly access the second half-reaction of the catalytic cycle of InhA (i.e. the proton transfer), independently of the first half-reaction (i.e. the initial hydride transfer) and to assign functions to two conserved active-site residues, Tyr-158 and Thr-196. We found that Tyr-158 is required for the stereospecificity of protonation and that Thr-196 is partially involved in hydride transfer and protonation. The natural tendency of InhA to form a covalent C2-ene adduct calls for a careful reconsideration of the enzyme's reaction mechanism. It also provides the basis for the development of effective tools to study, manipulate, and inhibit the catalytic cycle of InhA and related enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily. In summary, our work has uncovered the formation of a covalent adduct during the InhA catalytic cycle and identified critical residues required for catalysis, providing further insights into the InhA reaction mechanism important for the development of antituberculosis drugs.

Combining Promiscuous Acyl-CoA Oxidase and Enoyl-CoA Carboxylase/Reductases for Atypical Polyketide Extender Unit Biosynthesis.

The incorporation of different extender units generates structural diversity in polyketides. There is significant interest in engineering substrate specificity of polyketide synthases (PKSs) to change their chemical structure. Efforts to change extender unit selectivity are hindered by the lack of simple screening methods and easily available atypical extender units. Here, we present a chemo-biosynthetic strategy that employs biocatalytic proofreading and allows access to a large variety of extender units. First, saturated acids are chemically coupled to free coenzyme A (CoA). The corresponding acyl-CoAs are then converted to alkylmalonyl-CoAs in a "one-pot" reaction through the combined action of an acyl-CoA oxidase and enoyl-CoA carboxylase/reductase. We synthesized six different extender units and used them in in vitro competition screens to investigate active site residues conferring extender unit selectivity. Our results show the importance of an uncharacterized glutamine in extender unit selectivity and open the possibility for comprehensive studies on extender incorporation in PKSs.

Archaeal acetoacetyl-CoA thiolase/HMG-CoA synthase complex channels the intermediate via a fused CoA-binding site.

Many reactions within a cell are thermodynamically unfavorable. To efficiently run some of those endergonic reactions, nature evolved intermediate-channeling enzyme complexes, in which the products of the first endergonic reactions are immediately consumed by the second exergonic reactions. Based on this concept, we studied how archaea overcome the unfavorable first reaction of isoprenoid biosynthesis-the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA catalyzed by acetoacetyl-CoA thiolases (thiolases). We natively isolated an enzyme complex comprising the thiolase and 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) from a fast-growing methanogenic archaeon, Methanothermococcus thermolithotrophicus HMGCS catalyzes the second reaction in the mevalonate pathway-the exergonic condensation of acetoacetyl-CoA and acetyl-CoA to HMG-CoA. The 380-kDa crystal structure revealed that both enzymes are held together by a third protein (DUF35) with so-far-unknown function. The active-site clefts of thiolase and HMGCS form a fused CoA-binding site, which allows for efficient coupling of the endergonic thiolase reaction with the exergonic HMGCS reaction. The tripartite complex is found in almost all archaeal genomes and in some bacterial ones. In addition, the DUF35 proteins are also important for polyhydroxyalkanoate (PHA) biosynthesis, most probably by functioning as a scaffold protein that connects thiolase with 3-ketoacyl-CoA reductase. This natural and highly conserved enzyme complex offers great potential to improve isoprenoid and PHA biosynthesis in biotechnologically relevant organisms.