A synthetic cell-free pathway for biocatalytic upgrading of one-carbon substrates.

Biotechnological processes hold tremendous potential for the efficient and sustainable conversion of one-carbon (C1) substrates into complex multi-carbon products. However, the development of robust and versatile biocatalytic systems for this purpose remains a significant challenge. In this study, we report a hybrid electrochemical-biochemical cell-free system for the conversion of C1 substrates into the universal biological building block acetyl-CoA.

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.

Intersubunit Coupling Enables Fast CO-Fixation by Reductive Carboxylases.

Enoyl-CoA carboxylases/reductases (ECRs) are some of the most efficient CO-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 .

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

Carbon-negative synthesis of biochemical products has the potential to mitigate global CO emissions. An attractive route to do this is the reverse β-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.

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.

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.

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 CO.

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.

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 -often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as , are industrially proven and have desirable metabolic features but have several hurdles to mainstream use.

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.

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.

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.

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.

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

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO 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.

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.

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 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 CO fixation, sequesters its reactive intermediate acrylyl-CoA.

'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'.

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

The enoyl-thioester reductase InhA catalyzes an essential step in fatty acid biosynthesis of and is a key target of antituberculosis drugs to combat multidrug-resistant 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.

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.

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).

Engineered Production of Short-Chain Acyl-Coenzyme A Esters in Saccharomyces cerevisiae.

Short-chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and the production of polyketides, biopolymers and other value-added chemicals. S. cerevisiae is a model organism that has been utilized for the biosynthesis of such biologically and economically valuable compounds.

A conserved threonine prevents self-intoxication of enoyl-thioester reductases.

Enzymes are highly specific biocatalysts, yet they can promote unwanted side reactions. Here we investigated the factors that direct catalysis in the enoyl-thioester reductase Etr1p. We show that a single conserved threonine is essential to suppress the formation of a side product that would otherwise act as a high-affinity inhibitor of the enzyme.

A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters.

Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies.

The use of ene adducts to study and engineer enoyl-thioester reductases.

An improved understanding of enzymes' catalytic proficiency and stereoselectivity would further enable applications in chemistry, biocatalysis and industrial biotechnology. We use a chemical probe to dissect individual catalytic steps of enoyl-thioester reductases (Etrs), validating an active site tyrosine as the cryptic proton donor and explaining how it had eluded definitive identification. This information enabled the rational redesign of Etr, yielding mutants that create products with inverted stereochemistry at wild type-like turnover frequency.