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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Mechanisms in the Synthesis of S -Alcohols with 1,4-NADH Biomimetic Co-factor N-Benzyl-1,4-dihydronicotinamide using Horse Liver Alcohol Dehydrogenase: A Hybrid Computational Study

The enantioselective reduction of prochiral ketones catalyzed by horse liver alcohol dehydrogenase (HLADH), was investigated via a hybrid computational approach, for molecular reactions involved in chiral synthesis of S-alcohols, when the natural co-factor, 1,4-dihyronicotinamide adenine dinucleotide, 1,4-NADH, was replaced with biomimetic co-factor, N-benzyl-1,4-dihydronicotinamide, 1. We surmised that different hydride and proton transfer mechanisms were involved using co-factor, 1. An alternative mechanism, where the hydride transfer step occurred, via an η 1 -keto-S-η 2 -5,6-1,4-dihydronicotinamide-Zn(II) complex, was previously investigated with a model of the HLADH-Zn(II) catalytic site (J. Organometal. Chem. 2021, 943, 121810). Presently, we studied canonical and alternative mechanisms compared to models of the entire enzyme structure. We disproved the η 2 -Zn(II) complex, and discovered a canonical hydride transfer from biomimetic 1,4-NADH, 1, to the Zn(II) bound prochiral ketone substrate, followed by a new proton relay, consisting of a water chain connecting His51 to Ser48 that accomplished the S-alkoxy anion's protonation to yield the final S-alcohol product. The HLADH catalysis, with biomimetic co-factor, 1, that replaced the ribose group, the 5'-diphosphate groups, and the adenine nucleotide with a N-benzyl group, has provided a new paradigm for the design of other structures of 1,4-NADH biomimetic co-factors, including their economic value in biocatalysis reactions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Characterization of Gas-Phase Native(-like) Proteins Using Structures for Lossless Ion Manipulations

High-resolution mobility-based ion separations in Structures for Lossless Ion Manipulations (SLIM) have been useful for ion mobility separations for a variety of molecular classes in the gas phase. Here, in this study, we present multipass SLIM separations for gas-phase proteins in their near-native state exhibiting charge-state-dependent arrival time distributions using carbonic anhydrase (29 kDa), alcohol dehydrogenase (148 kDa), and apo-transferrin (79 kDa). The experimental CCS values were obtained from calibration curves for the arrival times of Agilent Tune Mix ions. For multipass separations, the ATDs were converted to CCS values by deconvoluting the multipass arrival times into accurate single-pass values amenable to the single-pass calibration curves. Mass spectra of carbonic anhydrase (CA) showed three different charge states (z = 9+ to 11+). Their corresponding mobility peaks were baseline-separated by using 8-m single-pass separations. When compared to the corresponding drift tube ion mobility (DTIMS) measurements, the CCS values obtained from DTIMS and SLIM were in agreement within experimental error. Single-pass analysis of alcohol dehydrogenase (ADH) exhibits three predominant charge states (z = 23+ to 25+) with mobility overlap between adjacent charge states. The mobility peak resolution for ADH improved with multipass separations (up to 24-m path length). In addition, CCS distributions obtained for charge states z = 16+ to 18+ of apo-transferrin reveal a transition from a compact unimodal form (z = 18+ and 19+) to broader multimodal CCS distributions for z = 16+. For apo-transferrin, 40-m multipass separations were performed allowing for complete isolation of the selected mobility range corresponding to z = 17+, leading to selective isolation of a narrow arrival time window. The extended mobility separations provided minimal alterations to the structure of the proteins, and the experimentally derived CCS values showed minimal change as a function of the separation time or number of passes. Mobility-based ion separations for native-like proteins, using SLIM, open opportunities for native-IMS applications as well as other manipulations enabled by SLIM-like mobility-selective isolation and collection.

charge state distribution↗
DOE OSTI2025

The role of AdhE mutations in Thermoanaerobacterium saccharolyticum

ABSTRACT Thermoanaerobacterium saccharolyticum is a thermophilic anaerobic bacterium that natively ferments a variety of hemicellulose substrates to organic acids and alcohols. It has recently been engineered to produce ethanol at high yield and titer; however, it uses a unique metabolic pathway for ethanol production that is poorly characterized. One of the distinctive aspects of this pathway is the presence of acetyl-CoA as an intermediate metabolite. In this organism, acetyl-CoA is converted to ethanol by a bifunctional AdhE enzyme. This enzyme has been a frequent target for mutations, and in many cases, the function of these mutations was unknown. Using a combination of genetic modifications, enzyme assays, and computational analysis, we have developed a better understanding of how mutations in AdhE affect ethanol production in the engineered homoethanologen strain. We identify a set of approximately interchangeable AdhE mutations (G544D, T597K, T597I, and T605I), whose function is to disrupt the activity of the alcohol dehydrogenase (ADH) domain of AdhE. This reduces NADH-linked ADH activity, which dramatically increases ethanol tolerance and changes the overall stoichiometry of acetaldehyde to ethanol conversion. Furthermore, our improved understanding of the function of these AdhE mutations calls into question a proposed feature of AdhE enzymes known as substrate channeling—direct transfer of acetaldehyde between the two domains of the AdhE enzyme. This improved the understanding of the role of AdhE mutations in T. saccharolyticum and provides deeper insights into the function of the unique ethanol production pathway in this organism. IMPORTANCE Many anaerobic bacteria maintain redox equilibrium by producing reduced organic compounds such as ethanol. The final two steps of ethanol production are mediated by a bifunctional enzyme, AdhE, and this enzyme is a frequent target of mutations in strains engineered for increased ethanol production. Paradoxically, these mutations increase ethanol production by eliminating the activity of one domain of the AdhE enzyme (the ADH domain). This provides additional support for a redox-imbalance theory of alcohol tolerance, which challenges the prevailing hypothesis that alcohol tolerance is associated with cell membrane effects.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structure-driven development of a biomimetic rare earth artificial metalloprotein

The 2011 discovery of the first rare earth–dependent enzyme in methylotrophic Methylobacterium extorquens AM1 prompted intensive research toward understanding the unique chemistry at play in these systems. This enzyme, an alcohol dehydrogenase (ADH), features a La 3+ ion closely associated with redox-active coenzyme pyrroloquinoline quinone (PQQ) and is structurally homologous to the Ca 2+ -dependent ADH from the same organism. AM1 also produces a periplasmic PQQ-binding protein, PqqT, which we have now structurally characterized to 1.46-Å resolution by X-ray diffraction. This crystal structure reveals a Lys residue hydrogen-bonded to PQQ at the site analogously occupied by a Lewis acidic cation in ADH. Accordingly, we prepared K 142 A- and K 142 D-PqqT variants to assess the relevance of this site toward metal binding. Isothermal titration calorimetry experiments and titrations monitored by UV–Vis absorption and emission spectroscopies support that K 142 D-PqqT binds tightly (Kd = 0.6 ± 0.2 μM) to La 3+ in the presence of bound PQQ and produces spectral signatures consistent with those of ADH enzymes. These spectral signatures are not observed for WT- or K 142 A-variants or upon addition of Ca 2+ to PQQ ⸦ K 142 D-PqqT. Addition of benzyl alcohol to La 3+ -bound PQQ ⸦ K 142 D-PqqT (but not Ca 2+ -bound PQQ ⸦ K 142 D-PqqT, or La 3+ -bound PQQ ⸦ WT-PqqT) produces spectroscopic changes associated with PQQ reduction, and chemical trapping experiments reveal the production of benzaldehyde, supporting ADH activity. By creating a metal binding site that mimics native ADH enzymes, we present a rare earth-dependent artificial metalloenzyme primed for future mechanistic, biocatalytic, and biosensing applications.

Science & Technology - Other Topics↗
DOE OSTI2025

Expanding on the ability of trivalent actinides to support microbial alcohol metabolism in evolved methylotrophic bacterium

The 4 f elements, known as the lanthanides, have only recently been recognized as biologically essential metals. These elements can be found in the active site of certain alcohol dehydrogenase (ADH) enzymes, which are used by methylotrophic bacteria to metabolize simple alcohols such as methanol and ethanol. The 5 f elements, known as the actinides, often show similar chemical behaviour to the lanthanides and recent reports demonstrated that americium (Am) and curium (Cm) can replace lanthanides in certain ADHs to yield a catalytically competent enzyme and support bacterial life. In this work, we expand on these results to show the trivalent ions actinium (Ac3+), americium (Am3+), curium (Cm3+), berkelium (Bk3+), and californium (Cf3+) all support growth of an evolved strain of the model methylotroph Methylobacterium extorquens AM1. Further in vitro experiments using a reconstituted ethanol dehydrogenase enzyme confirm the ability of this enzyme to utilize trivalent actinide ions.

Woods, Joshua J↗
DOE OSTI2025

A polyketide-based biosynthetic platform for diols, amino alcohols and hydroxy acids

Medium- and branched-chain diols and amino alcohols are important industrial solvents, polymer building blocks, cosmetics and pharmaceutical ingredients, yet biosynthetically challenging to produce. Here we present an approach that uses a modular polyketide synthase (PKS) platform for the efficient production of these compounds. This platform takes advantage of a versatile loading module from the rimocidin PKS and nicotinamide adenine dinucleotide phosphate-dependent terminal thioreductases. Reduction of the terminal aldehyde with alcohol dehydrogenases enables the production of diols, oxidation enables the production of hydroxy acids and specific transaminases allow the production of various amino alcohols. Furthermore, replacement of the malonyl-coenzyme A-specific acyltransferase in the extension module with methyl- or ethylmalonyl-coenzyme A-specific acyltransferase enables the production of branched-chain diols, amino alcohols and carboxylic acids in high titres. Use of our PKS platform in Streptomyces albus demonstrated the high tunability and efficiency of the platform.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Prediction of non-intuitive metabolic targets with bayesian metabolic control analysis to improve 3-hydroxypropionic acid production in Aspergillus niger

Development of efficient bioconversion processes is limited by the ability to predictably improve metabolic flux. Here we deployed Bayesian Metabolic Control Analysis as a platform to integrate multi-omics data with metabolic modeling and evaluated its ability to predict genetic interventions that improve metabolic flux. Global Metabolomics and proteomics data was collected from 17 Aspergillus niger strains engineered to produce the platform biochemical 3-hydroxypropionic acid from which seven actional genetic interventions were predicted from significant flux control coefficients. Of the suggested genetic interventions, two were present within the intuitively designed strains used for training (malonic semialdehyde dehydrogenase and pyruvate carboxylase) while five predicted targets were present within non-intuitive areas of the metabolic network including 5-formyltetrahydrofolate deformylase and four mitochondrial enzymes, alcohol dehydrogenase, succinyl-CoA ligase, aspartate aminotransferase, and malate dehydrogenase. Six of the targets were validated in the highest performing 3-HP strain used for multi-omics data generation which contained a prior disruption of the highest scoring target malonic semialdehyde dehydrogenase. Predicted directional perturbation of five of the six tested targets significantly improved titer and rate of 3-HP production and two significantly improved yield. The greatest improvements were observed following disruption of the non-intuitive target succinyl-CoA ligase which increased titer by 39% and yield by 29% (to 20.4 g/L 3-HP and 0.31 g 3-HP/g glucose) over the strains used for training. This study demonstrates the utility of Bayesian Metabolic Control Analysis and highlights the ability to predict meaningful genetic targets in unexpected areas of metabolism to improve engineered strains for bioconversion.

3-hydroxypropionic acid↗
DOE OSTI2024

Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism

Many bacteria secrete metallophores, low-molecular-weight organic compounds that bind ions with high selectivity and affinity, in order to access essential metals from the environment. Previous work has elucidated the structures and biosynthetic machinery of metallophores specific for iron, zinc, nickel, molybdenum, and copper. No physiologically relevant lanthanide-binding metallophore has been discovered despite the knowledge that lanthanide metals (Ln) have been revealed to be essential cofactors for certain alcohol dehydrogenases across a diverse range of phyla. Here, we report the biosynthetic machinery, the structure, and the physiological relevance of a lanthanophore, methylolanthanin. The structure of methylolanthanin exhibits a unique 4-hydroxybenzoate moiety which has not previously been described in other metallophores. We find that production of methylolanthanin is required for normal levels of Ln accumulation in the methylotrophic bacterium Methylobacterium extorquens AM1, while overexpression of the molecule greatly increases bioaccumulation and adsorption. Our results provide a clearer understanding of how Ln-utilizing bacteria sense, scavenge, and store Ln; essential processes in the environment where Ln are poorly bioavailable. More broadly, the identification of this lanthanophore opens doors for study of how biosynthetic gene clusters are repurposed for additional functions and the complex relationship between metal homeostasis and fitness.

Science & Technology - Other Topics↗
DOE OSTI2025

Variation in Flooding Tolerance in Populus deltoides ‘D-124’ and P. trichocarpa x P. deltoides Hybrid ‘52–225’

Flooding poses a substantial challenge to plant survival and productivity, particularly in riparian genus like Populus. This study examines the physiological, morphological, metabolic, and molecular responses of Populus deltoides ‘D-124’ and P. trichocarpa x P. deltoides hybrid clone ‘52–225’ under control and inundated conditions to identify differences in flooding tolerance. Under flooding conditions, physiological and cellular stress was more pronounced in P. deltoides ‘D-124’ than in the hybrid clone ‘52–225,’ as evidenced by lower transpiration (E), photosynthesis (A), and chlorophyll content. In contrast, ‘52–225’ showed reduced ROS accumulation suggesting better cellular function under stress. Morphologically, ‘52–225’ produced more shoot-born roots, which likely enhance oxygen transport and metabolic activity during flooding. Metabolite profiling revealed both overlapping and distinct patterns of sugar and amino acid accumulation between genotypes. Gene expression analysis revealed that flooding-responsive genes, including ALCOHOL DEHYDROGENASE 1 and HYPOXIA RESPONSIVE ERF 2, were activated in both genotypes, with a more pronounced response noted in ‘52–225.’ These findings extend our understanding of flooding tolerance mechanisms in Populus by connecting physiological traits, stress responses, and genetic regulation. This research contributes to the development of more flooding-resilient poplar varieties, with potential applications in breeding and restoration programs for flooding-prone environments.

Flooding stress↗
DOE OSTI2025

Biosensor-driven strain engineering reveals key cellular processes for maximizing isoprenol production in Pseudomonas putida

Synthetic biology generates vast combinatorial designs, yet high-throughput analytical methods to screen them are poorly matched to interrogate this search space. We address this challenge by developing a biosensor-driven, growth-coupled selection strategy in Pseudomonas putida for isoprenol, a potential aviation fuel precursor. We found and characterized a noncanonical signaling pathway, revealing a functional and physical complex between a hybrid histidine kinase and an alcohol dehydrogenase, whose activity is tuned by heterodimerization. Leveraging this biosensor in a pooled CRISPRi library selection, we identified key host limitations. Iterative combinatorial strain engineering derived from these hits yielded a 36-fold titer increase to ~900 milligrams per liter. Integrated omics analysis revealed that metabolic rewiring toward amino acid catabolism was crucial for this improvement. This observation was found to be beneficial by technoeconomic analysis. Our modular workflow provides a powerful strategy for optimizing complex heterologous pathways and uncovering emergent host biology.

CRISPRi↗
DOE OSTI2024

Structural Characterization and Dynamics of AdhE Ultrastructures from Clostridium thermocellum Show a Containment Strategy for Toxic Intermediates

Clostridium thermocellum, a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production. C. thermocellum is among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe; however, ethanol production using C. thermocellum is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in C. thermocellum involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the C. thermocellum AdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 A structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role of C. thermocellum AdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.

09 BIOMASS FUELS↗
DOE OSTI2026

Adaptive_Evolution_2026

Thermoanaerobacterium saccharolyticum, an anaerobic and thermophilic bacterium capable of metabolizing sugar monomers and soluble oligomers into ethanol, has been proposed for use in consolidated bioprocessing in coculture with compatible cellulolytic bacteria such asClostridium thermocellum. Although the mixed acid fermentation of both of these strains has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far byC. thermocellum, about 3% (w/v) ethanol, is half that produced byT. saccharolyticum. There is thus motivation to understand the mechanistic basis of the robust ethanol pathway inT. saccharolyticumso that key features can be recapitulated inC. thermocellumand other organisms.Previously, we characterized theindividualrole of the main genes responsible for electron transfer in the ethanol production ofT. saccharolyticum. However, the consequences of thecombinedloss of function of all these genes have not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. In this work, we combined knockouts of ferredoxin nicotinamide oxidoreductase (fnor) genes (nfnAandnfnB) and hydrogenase genes (hydAandhfsD) and studied their effects on fermentation and grow. We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth, and most notably, we observed a single nucleotide variation in the alcohol dehydrogenaseadhAgene. Through molecular dynamics simulations and enzymatic assays, we determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of the functioning of electron transfer pathways in this organism.

alcohol dehydrogenase↗
DOE OSTI2026

Genetic and metabolic drivers of membrane remodeling in Clostridium thermocellum under alcohol stress

Clostridium thermocellum is a leading candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels due to its native cellulolytic capabilities. Beyond ethanol, C. thermocellum is being developed as a platform for producing higher-chain alcohols such as isobutanol and n-butanol. However, its physiological adaptations to alcohol stress remain poorly understood. Here, we investigate how C. thermocellum remodels its membrane lipid composition in response to exogenous ethanol, n-butanol, isobutanol, and butyrate. Exposure to linear alcohols such as n-butanol or to organic acids like butyrate increased the proportion of straight-chain fatty acids in the membrane at the expense of branched-chain species, whereas exposure to the branched alcohol isobutanol produced the opposite effect. Isotope tracer experiments demonstrated that C. thermocellum directly incorporates the carbon backbones of exogenous alcohols and acids into fatty acids, providing a mechanistic basis for these contrasting shifts. We show that the bifunctional aldehyde/alcohol dehydrogenase AdhE is essential for the assimilation of exogenous alcohols into fatty acids, acting through its oxidative activity by first oxidizing alcohols to aldehydes and then converting them to acyl-CoA intermediates. Deletion of the pyruvate:ferredoxin oxidoreductase isozyme pfor4 abolished branched-chain fatty acid synthesis, but supplementation with isobutanol restored production, indicating that Pfor4 substitutes for the canonical branched-chain α-keto acid dehydrogenase complex. These findings reveal two distinct routes for branched-chain fatty acid production in C. thermocellum: a Pfor4-dependent pathway from α-keto acid intermediates derived from amino acid synthesis, and an AdhE-dependent salvage pathway that assimilates exogenous branched-chain alcohols.

Acetivibrio thermocellus↗
DOE OSTI2024

Catabolism of β-5 linked aromatics by Novosphingobium aromaticivorans

ABSTRACT Aromatic compounds are an important source of commodity chemicals traditionally produced from fossil fuels. Aromatics derived from plant lignin can potentially be converted into commodity chemicals through depolymerization followed by microbial funneling of monomers and low molecular weight oligomers. This study investigates the catabolism of the β-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans . We used genome-wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidate genes elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans . Specifically, a newly identified γ-formaldehyde lyase, PcfL, opens the phenylcoumaran ring to form a stilbene and formaldehyde. A lignostilbene dioxygenase, LsdD, then cleaves the stilbene to generate the aromatic monomers vanillin and 5-formylferulate (5-FF). We also showed that the aldehyde dehydrogenase FerD oxidizes 5-FF before it is decarboxylated by LigW, yielding ferulic acid. We found that some enzymes involved in the β-5 catabolism pathway can act on multiple substrates and that some steps in the pathway can be mediated by multiple enzymes, providing new insights into the robust flexibility of aromatic catabolism in N. aromaticivorans . A comparative genomic analysis predicted that the newly discovered β-5 aromatic catabolic pathway is common within the order Sphingomonadales. IMPORTANCE In the transition to a circular bioeconomy, the plant polymer lignin holds promise as a renewable source of industrially important aromatic chemicals. However, since lignin contains aromatic subunits joined by various chemical linkages, producing single chemical products from this polymer can be challenging. One strategy to overcome this challenge is using microbes to funnel a mixture of lignin-derived aromatics into target chemical products. This approach requires strategies to cleave the major inter-unit linkages of lignin to release monomers for funneling into valuable products. In this study, we report newly discovered aspects of a pathway by which the Novosphingobium aromaticivorans DSM12444 catabolizes aromatics joined by the second most common inter-unit linkage in lignin, the β-5 linkage. This work advances our knowledge of aromatic catabolic pathways, laying the groundwork for future metabolic engineering of this and other microbes for optimized conversion of lignin into products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Crystallographic Fragment Screening of a Bifunctional Proline Catabolic Enzyme Reveals New Inhibitor Templates for Proline Dehydrogenase and L-Glutamate-γ-semialdehyde Dehydrogenase

The proline catabolic pathway consisting of proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde (GSAL) dehydrogenase (GSALDH) catalyzes the four-electron oxidation of L-proline to L-glutamate. Chemical probes to these enzymes are of interest for their role in cancer and inherited metabolic disease. Here, we report the results of a crystallographic fragment-screening campaign targeting both enzymes. A unique aspect of our approach is the screening of both enzymes simultaneously using crystals of the bifunctional PRODH-GSALDH enzyme, proline utilization A (PutA). A 288-fragment library from Zenobia was screened in crystallo in cocktails of six fragments. Validation X-ray crystallography with individual fragments identified seven crystal hits distributed in the PRODH active site, GSALDH aldehyde substrate-binding site, and GSALDH NAD+ adenine-binding site. The fragment bound in the PRODH active site, 4-methoxybenzyl alcohol, is structurally distinct from all known PRODH inhibitors as it lacks an anionic anchor and stabilizes open conformations of the active site, motivating the study of eighteen analogs. In total, thirteen crystal structures with resolutions ranging from 1.32 Å to 1.80 Å were determined, resolving the poses and interactions of seven fragments from the Zenobia library and five analogs of 4-methoxybenzyl alcohol. These results expand the chemical space of probes targeting proline catabolic enzymes and provide new structural information for further inhibitor development.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Carbon Negative Synthesis of Amino Acids Using a Cell-Free-Based Biocatalyst

Biological systems can directly upgrade carbon dioxide (CO 2 ) into chemicals. The CO 2 fixation rate of autotrophic organisms, however, is too slow for industrial utility, and the breadth of engineered metabolic pathways for the synthesis of value-added chemicals is too limited. Biotechnology workhorse organisms with extensively engineered metabolic pathways have recently been engineered for CO 2 fixation. Yet, their low carbon fixation rate, compounded by the fact that living organisms split their carbon between cell growth and chemical synthesis, has led to only cell growth with no chemical synthesis achieved to date. Here, we engineer a lysate-based cell-free expression (CFE)-based multienzyme biocatalyst for the carbon negative synthesis of the industrially relevant amino acids glycine and serine from CO 2 equivalents–formate and bicarbonate–and ammonia. The formate-to-serine biocatalyst leverages tetrahydrofolate (THF)-dependent formate fixation, reductive glycine synthesis, serine synthesis, and phosphite dehydrogenase-dependent NAD(P)H regeneration to convert 30% of formate into serine and glycine, surpassing the previous 22% conversion using a purified enzyme system. We find that (1) the CFE-based biocatalyst is active even after 200-fold dilution, enabling higher substrate loading and product synthesis without incurring additional cell lysate cost, (2) NAD(P)H regeneration is pivotal to driving forward reactions close to thermodynamic equilibrium, (3) balancing the ratio of the formate-to-serine pathway genes added to the CFE is key to improving amino acid synthesis, and (4) efficient THF recycling enables lowering the loading of this cofactor, reducing the cost of the CFE-based biocatalyst. To our knowledge, this is the first synthesis of amino acids that can capture CO 2 equivalents for the carbon negative synthesis of amino acids using a CFE-based biocatalyst. Looking ahead, the CFE-based biocatalyst process could be extended beyond serine to pyruvate, a key intermediate, to access a variety of chemicals from aromatics and terpenes to alcohols and polymers.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗