Metal cofactor level chassis engineering enables aerobic expression of tungsten formate dehydrogenases in Escherichia coli

Metal cofactor level chassis engineering enables aerobic expression of tungsten formate dehydrogenases in Escherichia coli | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Metal cofactor level chassis engineering enables aerobic expression of tungsten formate dehydrogenases in Escherichia coli Yong Hwan Kim, Uyen Phan, Jun-Min Lee, Toan Vo, Min-Kyu Oh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8913862/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Tungsten-dependent formate dehydrogenases catalyze CO₂ reduction, yet their heterologous production remains limited by the absence of tungsten-specific cofactor maturation in standard laboratory hosts. Although molybdenum-dependent formate dehydrogenases have been implemented in Escherichia coli , analogous maturation of tungsten-dependent enzymes has proven unsuccessful because native metal homeostasis does not support selective tungstate uptake or W-bis-MGD assembly. Here we reconstitute tungsten-specific metal metabolism in E. coli by reconstructing tungsten cofactor biosynthesis, installing a high-affinity tungstate uptake system, and reinforcing Fe–S cluster biogenesis. This enables aerobic production of catalytically competent tungsten-dependent formate dehydrogenase from Methylorubrum extorquens AM1, achieving native-level specific activity and a tungsten occupancy of 0.92 mol per mol enzyme—nearly double that of the native host. The engineered strain delivers a volumetric activity exceeding 3,500 U L⁻¹. In a coupled whole-cell CO-to-formate biotransformation, the strain generated 44 mM formate within 4 hours, outperforming the native host system by more than threefold. The platform also supports activation of W-bis-MGD-dependent enzymes from Cupriavidus necator H16 and Lutibaculum baratangense AMV1. This work establishes a general design framework for W-bis-MGD maturation, expanding access to tungsten-dependent biocatalysts for CO₂-to-formate and related reductive biotransformations. Biological sciences/Biotechnology/Metabolic engineering Biological sciences/Microbiology/Applied microbiology Formate dehydrogenase Tungsten cofactor W-bis-MGD Tungstate transport Iron–sulfur cluster CO₂ reduction Synthetic chassis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Carbon dioxide (CO 2 ) conversion has become a cornerstone of climate-aligned manufacturing, enabling fixation and valorization of a major greenhouse gas into valuable chemicals 1 . Among CO₂-derived products, formate is particularly attractive because it can be produced from CO₂ with high energy efficiency using technically straightforward routes 2 and already supports a mature market with established applications across chemical and agricultural sectors 3 . Motivated by rising atmospheric CO 2 levels and increasingly stringent decarbonization targets, these advantages have intensified efforts to develop selective, low-energy CO 2 -to-formate platforms that integrate biological systems with renewable power, thereby accelerating the transition from carbon capture to circular utilization 4 , 5 , 6 . Among available biocatalysts, formate dehydrogenases (FDHs) incorporating high-valent transition metal cofactors, most prominently molybdenum (Mo) and tungsten (W), have emerged as leading candidates for CO₂-to-formate conversion due to their high catalytic efficiency, favorable thermodynamics, and broad applicability in C1-based biomanufacturing 4 , 7 , 8 . Although Mo- and W-dependent FDHs share a conserved bis-molybdopterin guanine dinucleotide (bis-MGD) framework, the identity of the central metal atom profoundly affects catalytic behaviors. The tungsten center in W-FDHs exhibits a substantially lower redox potential than its molybdenum counterpart, expanding the low-potential operational window and enhancing hydride-transfer kinetics. As a result, W-FDHs thermodynamically favor the reductive conversion of CO₂ to formate, making them particularly suitable for CO₂ fixation and bio-electrochemical CO₂ reduction 7 , 9 , 10 , 11 , 12 , 13 . Despite these advantages, the broader deployment of W-FDHs has been constrained by fundamental challenges in heterologous maturation. Although Escherichia coli can support recombinant expression of Mo-FDHs by leveraging its native molybdenum cofactor (Moco) biosynthesis pathway 14 , 15 , analogous functional expression of authentic tungsten-dependent enzymes in this host has proven much more difficult. While recent studies have demonstrated E. coli –based implementations of metalloenzyme modules for CO₂ reduction and formate-driven C1 metabolism 16 , 17 , a broadly applicable aerobic, chassis-level solution that enables efficient tungsten-selective maturation of W-bis-MGD enzymes in E. coli has remained limited. These limitations arise from intrinsic features of E. coli metal homeostasis. Moreover, tungsten-dependent enzymes impose additional constraints 18 , including competition between molybdenum and tungsten for cofactor metalation, stringent control of tungstate availability, and reliance on iron–sulfur (Fe–S) cluster biogenesis. These processes are not inherently aligned with native E. coli physiology. As a result, heterologous expression of tungsten-containing oxidoreductases in E. coli frequently yields incomplete cofactor incorporation, metal misallocation, or catalytically inactive apoenzymes 19 , 20 , 21 . Here, we engineered a W-bis-MGD-cofactor–competent E. coli chassis through coordinated reprogramming of intracellular tungsten metabolism and Fe-S cluster assembly. This strategy combined reconstruction of tungsten-cofactor biosynthesis using key genes from Methylorubrum extorquens AM1, chromosomal integration of a high-affinity tungstate transporter (TupBCA ) from Sulfitobacter dubius NA4, and genomic reinforcement of Fe–S cluster biogenesis via targeted engineering of the isc operon. The resulting chassis supports soluble, catalytically active expression of multiple W-dependent enzymes under aerobic conditions, including MeFDH1 from M. extorquens AM1, CnFDW from Cupriavidus necator H16, and a structurally predicted LbFDH from Lutibaculum baratangense AMV1. By overcoming tungsten-specific maturation barriers in a host previously considered incompatible with W-enzyme assembly, this work converts E. coli into a programmable platform for W-bis-MGD enzyme production. The approach establishes a transferable design framework for aerobic maturation of tungsten-dependent redox enzymes and expands access to this underexplored metallocofactor chemistry for sustainable CO₂ reduction and related reductive biotransformations. Results Reconstitution of W-bis-MGD biosynthesis restores MeFDH1 activation in E. coli The tungsten-dependent formate dehydrogenase from Methylorubrum extorquens AM1 (MeFDH1) was selected as a stringent tungsten-dependent model oxidoreductase, as its catalytic activity strictly requires W-bis-MGD metalation. However, recombinant expression of MeFDH1 in Escherichia coli does not yield catalytically competent enzyme 22 , indicating that this host lacks a tungsten-compatible maturation environment. We therefore sought to reconstruct the cognate tungsten-specific cofactor biosynthetic machinery from M. extorquens AM1 in E. coli to establish a tungsten-dependent maturation pathway (Fig. 1 ). Prior genetic characterization of MeFDH1 maturation in M. extorquens AM1 (summarized in Supplementary Table S1 ) identified MoeA1, MobA, MobB, and FdhD as essential components of the native W-bis-MGD biosynthetic pathway. Among the five annotated moeA homologs, only moeA1 in M. extorquens AM1 was required for MeFDH1 activity, establishing MoeA1 as the sole tungsten-specific insertion factor for this enzyme. Deletion analyses further showed that mobB and fdhD are required for MeFDH1 activity, whereas mobA is essential for bis-MGD biosynthesis in the native host. These findings defined the minimal tungsten-specific maturation module guiding heterologous reconstruction. To assess potential coordination among these factors in the context of reconstruction, protein–protein interactions were evaluated using a bacterial adenylate cyclase two-hybrid (BACTH) assay (Supplementary Fig. S1 ). MoeA1 exhibited reproducible interactions with both MobA and MobB, while FdhD interacted preferentially with the Mob protein rather than directly with MoeA1. Collectively, this interaction pattern is consistent with a cooperative engagement of these factors during W-bis-MGD maturation and provides contextual support for the reconstruction strategy, as schematically summarized in Fig. 2 a. Guided by these observations, moeA1 , mobA , mobB , and fdhD from M. extorquens AM1 were codon-optimized and cloned into a pCDFDuet-1 vector under separate T7 promoters (Table 1 ). In parallel, the MeFDH1 α and β subunits were codon-optimized and cloned into a pETDuet-1 expression vector. E. coli BL21(DE3) was then co-transformed with MeFDH1 and various combinations of the tungsten cofactor (Wco) biosynthetic genes to assess their individual and combinatorial contributions to enzyme activation. As expected, expression of MeFDH1 alone (BL) resulted in negligible activity (< 1 U mg⁻¹), indicating predominant accumulation of apoenzyme due to insufficient availability of the W-bis-MGD cofactor. Similarly, strains expressing individual Wco genes, moeA1 (W1), mobA (WA), or mobB (WB), failed to produce measurable activity (Fig. 2 b), demonstrating that no single component is sufficient to support cofactor assembly or enzyme activation. Although MoeA1 is strictly required for MeFDH1 function in the native host, its expression alone was insufficient to restore activity in the heterologous background. The recovery of measurable activity (~ 6 U mg⁻¹) in the moeA1–mobB strain (W2) indicates that cooperative expression of these two proteins is sufficient to enable functional tungsten cofactor maturation in E. coli . Introduction of mobA into the moeA1 – mobB background (W3) led to a substantial increase in activity, suggesting that bis-MGD formation markedly enhances catalytic competence once tungsten has been integrated into the cofactor. Conversely, removal of mobB from the W3 background abolished MeFDH1 activity (Supplementary Fig. S2 ) indicating that MobB is required for productive W-bis-MGD maturation. Finally, inclusion of fdhD (W4) yielded the highest activity (~ 27 U mg⁻¹) and was accompanied by an approximately threefold increase in tungsten occupancy relative to the MeFDH1-only background (Table 2 ), consistent with improved completion of cofactor maturation. Accordingly, the full four-gene module ( moeA1 , mobA , mobB , and fdhD ) was adopted as the standard Wco biosynthetic configuration for all subsequent experiments. The progressive enhancement in activity from W2 to W4 is consistent with the interaction relationships detected by BACTH analysis and supports a coordinated maturation process in which functional W-bis-MGD biosynthesis in E. coli requires the integrated action of multiple pathway components rather than expression of individual biosynthetic genes. Nevertheless, the maximal activity achieved in E. coli remained substantially lower than that observed in M. extorquens AM1 (~ 50 U mg⁻¹), suggesting the presence of additional constraints, including limitations in tungsten uptake and iron–sulfur (Fe–S) cluster availability. This establishes the minimal tungsten maturation module. Selective tungstate transport enhances tungsten incorporation and enzyme performance Consistent with the residual activity gap observed after W-bis-MGD pathway reconstruction, MeFDH1 produced with the W4 module exhibited limited tungsten occupancy (~ 27% of theoretical stoichiometry). This incomplete loading suggested that tungsten availability, rather than cofactor assembly capacity alone, may constrain effective W-bis-MGD biosynthesis in E. coli . Given that BL21(DE3) lacks the high-affinity ModABC molybdate/tungstate transport system present in K-12 derivatives 23 , 24 , we next examined whether enhanced tungstate uptake could alleviate this limitation. Three candidate transport systems were evaluated by plasmid expression in tungstate-supplemented cultures, including the E. coli K-12 modABC operon, the tungsten-specific tupBCA operon from Sulfitobacter dubius NA4 25, 26 , and the tupBCA operon from M. extorquens AM1 27 . Among these, the S. dubius NA4–derived TupBCA produced the highest MeFDH1 specific activity, outperforming both ModABC from E. coli K-12 and the M. extorquens TupBCA (Supplementary Fig. S3 ). Based on this performance and to minimize plasmid burden in downstream experiments, the S. dubius NA4 transporter was integrated into the chromosome of E. coli BL21(DE3) under control of a T7 promoter, generating strain BL1 (Table 1 ). The selected tupBCA operon from S. dubius NA4 encodes an ATP-binding cassette (ABC)–type tungstate transporter composed of the periplasmic binding protein TupA, membrane permease TupB, and ATPase TupC 26 . Co-expression of the genomically integrated transporter with the W4 biosynthetic module resulted in a substantial increase in MeFDH1 activity (~ 35 U mg⁻¹) and improved soluble protein yield (~ 2.0 mg g⁻¹ WCW) (Fig. 3 ). Tungsten incorporation increased by approximately twofold in MeFDH1 purified from transporter-containing strains, reaching ~ 50% of the theoretical stoichiometry (Table 2 ), representing a substantial improvement over the transporter-deficient controls. To further assess the metal selectivity of the reconstructed tungsten cofactor biosynthetic pathway, MeFDH1 activity and metal profiling were examined under three conditions: tungstate alone (1 mM), mixed tungstate (0.5 mM) and molybdate (0.5 mM), or molybdate alone (1 mM). Cultures supplied exclusively with tungstate produced the highest MeFDH1 activity (~ 35 U mg⁻¹), whereas mixed-metal conditions led to a marked reduction in activity (~ 10 U mg⁻¹). No activity was observed under molybdate-only supplementation. Metal quantification showed that tungsten incorporation was highest under tungstate-only conditions (~ 0.51 mol W per mol enzyme) and decreased under mixed-metal supplementation, while molybdenum incorporation remained negligible across all treatments (Supplementary Table S2 ). The minimal Mo occupancy suggests that molybdenum does not efficiently substitute for tungsten in the mature enzyme population. Instead, the reduced activity under mixed conditions likely reflects competitive interference during metal allocation or early stages of W-bis-MGD biosynthesis, which impairs efficient cofactor assembly without substantial Mo incorporation into the purified enzyme. These results reveal that W-bis-MGD assembly in BL21(DE3) is highly sensitive to intracellular metal balance, as even modest molybdate supplementation can disrupt selective tungsten incorporation. Enhancing high-efficiency tungsten transport therefore promotes effective cofactor assembly and improved enzyme performance. These findings identify tungsten transport as a primary bottleneck in this host and establish dedicated tungstate acquisition as a critical design element for reliable expression of tungsten-dependent enzymes in synthetic systems. Reinforcement of iron-sulfur cluster biogenesis increases metalation and yield Beyond the W-bis-MGD cofactor at the active site, MeFDH1 relies on multiple iron–sulfur (Fe–S) clusters in both the α and β subunits to mediate electron transfer to physiological and artificial electron acceptors such as NADH or ethyl viologen (EV) (Supplementary Fig. S4 ). In E. coli , Fe–S cluster biosynthesis is mediated primarily by the ISC and SUF systems, with ISC tightly regulated by the global repressor IscR 28 , 29 . While sufficient for native metabolism, IscR-mediated regulation may not meet the elevated Fe–S cluster demand imposed by overexpression of multi–Fe–S enzymes such as MeFDH1. Consistent with this limitation, MeFDH1 produced prior to isc engineering contained only ~ 20–25% of the theoretical iron content (~ 20 mol Fe per mol enzyme; Table 2 ), indicating extensive under-metalation. In addition to delivering Fe–S cluster to target proteins, the ISC machinery has been implicated in bis-MGD cofactor biosynthesis in bacteria. Specifically, IscA functions as an Fe–S carrier that transfers clusters to MoaA, which catalyzes the first step of MPT synthesis, while IscS serves as a sulfur donor that regulates the synthesis rate 30 , 31 . Enhanced ISC activity is therefore expected to support not only Fe–S cluster assembly but also W-bis-MGD biosynthesis 32 . To test this hypothesis, we first enhanced Fe–S biosynthesis by plasmid-based co-expression of the isc operon using pRKISC 33 . Introduction of pRKISC into BL1 moderately improved MeFDH1 specific activity (46 U mg⁻¹) and soluble yield (4.5 mg g⁻¹ WCW), approaching levels observed the native M. extorquens AM1 (Fig. 4 ). We next implemented genome-level upregulation of endogenous Fe–S biogenesis by deleting iscR and replacing the native isc promoter with a strong synthetic promoter (P trc ), generating strain BL2 (Table 1 ). In BL2, MeFDH1 exhibited near-native specific activity (~ 48 U mg⁻¹) and substantially increased soluble yield (~ 10 mg g⁻¹ biomass), corresponding to a total volumetric activity exceeding 3500 U L⁻¹, approximately 15-fold higher than native AM1 (~ 240 U L⁻¹) and surpassing reported productivity in Cupriavidus necator H16 19 . A potential concern associated with global reprogramming of metal homeostasis is unintended metabolic burden or impaired cellular fitness. Growth profiling (Supplementary Fig. S5 ) showed that plasmid-based overexpression of the isc operon moderately reduced maximal cell density, consistent with increased metabolic load. In contrast, genome-level reinforcement of Fe–S biosynthesis in BL2 restored and further improved growth relative to the plasmid-based strain, indicating that chromosomal integration enables more balanced expression of maturation machinery. Importantly, no severe growth defects were observed under production conditions, suggesting that coordinated reconstruction of tungsten transport, cofactor assembly, and Fe–S biogenesis is physiologically tolerated. The improved growth of BL2 likely contributes to its enhanced volumetric productivity by supporting greater biomass accumulation alongside increased intracellular abundance of fully active enzymes. Elemental analysis confirmed functional improvements by revealing substantially enhanced metal content in MeFDH1 purified from the BL2 strain (Table 2 ). Iron occupancy increased from ~ 5 mol Fe mol⁻¹ enzyme in BL1 to ~ 11 mol in BL2, closely matching the level observed for enzyme purified from native AM1. Although structural annotation predicts ~ 20 mol Fe mol⁻¹ enzyme, both native and engineered preparations exhibited lower measured values, suggesting incomplete biogenesis or oxidative loss during aerobic purification step. Notably, MeFDH1 purified from E. coli BL2 displayed 92% tungsten occupancy, substantially higher than the 50% observed in native M. extorquens AM1 host. These coordinated increases in iron and tungsten incorporation are consistent with improved Fe–S cluster biogenesis in the engineered strain and support a functional link between Fe–S assembly and efficient W-bis-MGD maturation. Together, these findings indicate that bis-MGD cofactor synthesis remains tightly coupled to cellular Fe–S assembly capacity, even when molybdenum is replaced by tungsten. Successful production of fully metalated tungsten enzymes therefore requires not only reconstruction of tungsten-selective transport and cofactor biosynthesis, but also sufficient Fe–S cluster assembly to sustain both catalytic function and cofactor maturation. These results identify Fe–S supply as a critical remaining constraint and directly link Fe–S capacity to efficient W-bis-MGD maturation. Preservation of MeFDH1 catalytic competence in the engineered E. coli chassis To determine whether the engineered chassis preserves intrinsic catalytic properties, kinetic parameters for CO₂ reduction were measured using purified MeFDH1 from engineered E. coli BL2 and the native M. extorquens AM1 host (Table 3 ). Under CO₂-dependent conditions, turnover numbers (kcat) were comparable between BL2- and AM1-derived enzymes (141 s⁻¹ vs 152 s⁻¹), indicating preserved catalytic turnover following heterologous maturation. Although the apparent K M for CO 2 was moderately increased in BL2 (8.4 mM vs 3.6 mM), overall catalytic competence remained within the same order of magnitude, consistent with a modest decrease in substrate affinity rather than impaired catalytic chemistry. Similar trends were observed for EV-dependent measurements, where kcat values were largely maintained between preparations (304 s⁻¹ vs 325 s⁻¹), with modestly elevated K M values resulted in reduced catalytic efficiency in BL2. These data indicate that tungsten-specific cofactor reconstruction and enhanced Fe–S maturation in E. coli support formation of a catalytically competent enzyme without compromising turnover chemistry of MeFDH1. Whole-cell CO-to-formate conversion demonstrates system-level impact We next examined whether enhanced tungsten maturation translates into improved carbon conversion at the whole-cell level. A coupled CO-to-formate system was constructed in which E. coli expressing ChCODH (carbon monoxide dehydrogenase from Carboxydothermus hydrogenoformans ) to oxidizes CO to CO 2 and generate reduced ethyl viologen (EV•⁺), which subsequently transfers electrons to MeFDH1-expressing cells to drive CO₂ reduction (Fig. 5 a). Under matched biomass conditions, MeFDH1 produced in engineered BL2 generated 44 mM formate within 4 h, compared to 13 mM using enzyme derived from the native AM1 host (Fig. 5 b), corresponding a ~ 3.5-fold increase in final titer and a markedly higher apparent production rate. Given the comparable intrinsic kinetic parameters of purified enzymes, the improved whole-cell productivity observed with BL2-derived MeFDH1 therefore is attributable to the higher intracellular abundance of tungsten-metalated MeFDH1 in the engineered chassis. Engineered chassis supports heterologous expression of diverse tungsten-containing enzymes Having established efficient tungsten-selective maturation and enhanced catalytic performance with MeFDH1, we next assessed whether the engineered chassis supports functional assembly beyond its original native context. To this end, we expressed CnFDW from C. necator H16, a characterized W-FDH previously produced in M. extorquens PA1 34 . In parallel, we identified a putative FDH from Lutibaculum baratangense AMV1 (LbFDH) through Foldseek-based structure homology analysis 35 , which exhibits high structural similarity to characterized W-FDHs despite limited sequence identity (Supplementary Fig. S6 ). As summarized in Table 4 , the engineered BL2 chassis enabled functional expression of heterologous W-bis-MGD enzymes beyond MeFDH1. Expression of CnFDW in wild-type E. coli BL21(DE3) yielded no detectable activity, consistent with the absence of tungsten cofactor biosynthetic capacity. In contrast, CnFDW produced in BL2 exhibited substantial tungsten incorporation (~ 0.9 mol W per mol enzyme), confirming effective W-bis-MGD biosynthesis and insertion in the engineered host. Although full theoretical occupancy was not achieved, the high tungsten loading was sufficient to support maximal catalytic activity (~ 30 U mg − 1 ), comparable to values reported for expression in M. extorquens PA1 34 . A similar pattern was observed for the structure-predicted LbFDH, which was inactive in BL21(DE3) but exhibited measurable activity accompanied by stoichiometric tungsten incorporation when expressed in BL2 (Table 4 ). The measured tungsten content (1.17 mol W per mol enzyme) slightly exceeded the theoretical maximum (1.0), likely reflecting minor uncertainties in protein quantification or normalization for this previously uncharacterized enzyme. Nevertheless, the high tungsten occupancy confirms successful W-bis-MGD assembly and insertion into a structurally predicted enzyme, demonstrating that the reconstructed metal-cofactor network supports proper metalation beyond well-studied FDHs. The comparatively lower activity of LbFDH likely reflects intrinsic structural features, differences in native electron-transfer partners, or suboptimal folding in the heterologous host, rather than incomplete cofactor assembly. These results indicate that the engineered E. coli BL2 chassis is capable of efficiently maturing multiple W-FDHs under aerobic conditions, including both previously characterized proteins and structure-guided candidates. The transferable activation and high tungsten occupancy observed across distinct enzyme backgrounds suggest that the principal barrier to heterologous expression lies in host metal homeostasis rather than intrinsic enzyme incompatibility. Discussion The successful heterologous expression of MeFDH1 in Escherichia coli reported in this study addresses persistent challenges in the production of tungsten-containing metalloenzymes and demonstrates that tungsten-dependent enzyme maturation is not intrinsically incompatible with this host. Instead, functional expression is limited by the absence of an appropriately configured metal–cofactor environment. Our findings show that protein overexpression alone is insufficient when cofactor biosynthesis, metal selectivity, and auxiliary maturation pathways are not aligned with catalytic requirements. By integrating W-bis-MGD biosynthesis, tungsten-specific transport, and reinforcing Fe–S cluster assembly within a coordinated framework, we enabled aerobic maturation of non-native tungsten-dependent enzymes in E. coli through genome-level rewiring of trace-metal allocation. Systematic reconstruction of the W-bis-MGD biosynthetic machinery revealed that functional activation required cooperative action of MoeA1, MobA, MobB, and FdhD, consistent with an integrated maturation network rather than a linear insertion sequence. The progressive restoration of activity and corresponding increases in tungsten occupancy identify cofactor availability and maturation efficiency as principal bottlenecks in heterologous expression of tungsten-dependent oxidoreductases. In this context, MoeA1 and MobB promote early cofactor formation, MobA enhances bis-MGD synthesis to elevate catalytic competence, and FdhD contributes to completion of the mature tungsten cofactor; these roles echo the cooperative biosynthetic roles described for molybdenum cofactor assembly in bacteria 36 , 37 . These observations underscore W-bis-MGD cofactor biogenesis as a coordinated system rather than a set of isolated enzymatic steps. Metal discrimination emerged as a central yet often underappreciated challenge in heterologous metalloenzyme engineering. Tungsten and molybdenum, as closely related group 6 oxyanions, compete for overlapping transport and cofactor assembly pathways 38 , 39 , imposing stringent demands on selective uptake and insertion systems. To address this constraint, chromosomal integration of the high-affinity tungstate transporter from Sulfitobacter dubius NA4 (TupBCA) substantially increased tungsten incorporation and catalytic activity. Notably, MeFDH1 purified from the engineered E. coli BL2 strain exhibited nearly double the tungsten occupancy of enzyme isolated from its native host, indicating that synthetic control of metal uptake can rebalance endogenous W/Mo homeostatic trade-offs. The pronounced activity loss under mixed W/Mo supplementation highlights the inherent sensitivity of W-bis-MGD maturation to W/Mo competition. Because molybdenum is not appreciably incorporated into the mature enzyme, interference likely occurs upstream of final cofactor insertion rather than through direct active-site substitution. Such cross-talk may involve competitive binding to the TupA periplasmic binding protein, limiting tungstate influx despite its higher intrinsic affinity for tungsten 40 , or nonproductive engagement of the heterologous MoeA1 insertion machinery during early W-bis-MGD assembly, thereby impairing maturation of nascent cofactor intermediates 19 , 20 , 21 . Together, these findings position selective metal allocation and early pathway specificity as engineerable interfaces between trace-element physiology and synthetic chassis engineering. In parallel, Fe–S cluster biogenesis represented another systems-level constraint on tungsten enzyme maturation. Because W-FDHs rely on multiple Fe–S clusters for structural integrity and electron transfer, their assembly is embedded within broader redox and metal-trafficking networks. Reinforcement of the isc operon relieved this bottleneck, restoring activity to native-comparable levels and enabling volumetric productivity surpassing alternative production systems 8 , 34 , 41 , while maintaining cellular fitness. Given that Fe–S cluster assembly also contributes to early stages of bis-MGD cofactor biosynthesis 30 , 31 , 32 , these improvements likely reflect coordinated enhancement of both electron-transfer capacity and cofactor maturation efficiency. These findings highlight that efficient production of tungsten-dependent enzymes is governed not by protein expression alone, but by integrated host metal selectivity and cofactor metabolism. Importantly, reconstruction of tungsten-specific metal metabolism in E. coli preserved the intrinsic catalytic properties of MeFDH1. Although modest differences in apparent substrate affinity were observed, turnover rates between enzymes derived from the engineered E. coli BL2 strain and the native M. extorquens AM1 host remained unchanged, suggesting intact cofactor insertion and structure. Instead, they likely arise from subtle differences in local microenvironment or mediator interactions. The enhanced whole-cell CO-to-formate conversion is therefore best explained by improved maturation efficiency and increased intracellular abundance of fully active enzymes rather than altered enzyme chemistry. The successful activation of both a canonical W-FDH (CnFDW) and a putative FDH (LbFDH) further demonstrates that the engineered chassis supports maturation across multiple W-bis-MGD–dependent enzymes rather than being tailored to a single target. Despite belonging to the same structural superfamily, these enzymes originate from evolutionarily separated bacterial lineages and were functionally restored without enzyme-specific optimization, supporting the conclusion that activation results from reconstruction of host metal metabolism rather than target-specific tuning. While validation across additional tungsten enzyme families will be important, these results establish a transferable capacity for W-bis-MGD–dependent maturation in an engineered host. Collectively, this work extends chassis engineering beyond genetic circuit design to the rational reconfiguration of intracellular metal homeostasis. By reconciling host metal metabolism with cofactor-dependent catalysis, we establish a generalizable strategy for activating W-bis-MGD enzymes that are inaccessible in standard laboratory strains. Although demonstrated here with FDHs, the underlying design principles—coordinated control of metal uptake, cofactor biosynthesis, and auxiliary cluster assembly—provide a foundation for expanding the functional expression of tungsten-dependent redox enzymes in synthetic hosts, This platform enables aerobic production of multiple W-FDHs and establishes a general design framework for W-bis-MGD maturation, offering new opportunities to enhance biocatalytic carbon conversion and redox biomanufacturing under oxygen-tolerant conditions. Methods Strains, growth medium, and culture conditions Escherichia coli DH5α was used for routine plasmid construction and propagation. E. coli BL21(DE3) served as the parental strain for heterologous protein expression and all subsequent chassis engineering. All strains and plasmids used in this study are listed in Table 1 . Seed cultures were initiated in Luria–Bertani (LB) medium (10 g L⁻¹ tryptone, 5 g L⁻¹ yeast extract, 10 g L⁻¹ NaCl) supplemented, where appropriate, with carbenicillin (100 µg mL⁻¹) or spectinomycin (100 µg mL⁻¹). For protein expression, cultures were transferred to Terrific Broth (TB; 12 g L⁻¹ tryptone, 24 g L⁻¹ yeast extract, 9.4 g L⁻¹ K₂HPO₄, 2.2 g L⁻¹ KH₂PO₄) with appropriate antibiotics. Sodium tungstate (Na₂WO₄) was added to a final concentration of 1 mM, and FeSO₄ (1 mM) was included where indicated. Plasmid construction All heterologous genes were codon-optimized for E. coli expression and synthesized commercially by Bionics (Seoul, Republic of Korea). The identity and putative function of each heterologous gene used in this study are summarized in Supplementary Table S3 . Gene fragments were assembled into the indicated plasmid backbones using Gibson Assembly Master Mix (New England Biolabs, MA, USA) following the manufacturer’s instructions. All constructs were propagated in E. coli DH5α and verified by colony PCR and Sanger sequencing prior to use. MeFDH1 expression vectors were constructed by assembling Methylorubrum extorquens AM1 genes fdh1A and fdh1B into the pETDuet-1 backbone under separate T7 promoters. Expression vectors for Cupriavidus necator H16 fdwAB and Lutibaculum baratangense AMV1 fdhAB were constructed similarly in the pETDuet-1 backbone. Genes involved in W-bis-MGD biosynthesis ( moeA1 , mobB , mobA , and fdhD ) were assembled individually or in defined combinations into pCDFDuet-1 under T7 promoters to enable modular co-expression. For comparative analysis of tungstate uptake systems, the modABC operon from E. coli K-12 was amplified from genomic DNA using Q5® High-Fidelity DNA Polymerase (New England Biolabs, MA, USA), while the tupBCA operons from M. extorquens AM1 and Sulfitobacter dubius NA4 were codon-optimized and synthesized. All transporter operons were cloned into the pZS vector for plasmid-based expression. Strain engineering All strain engineering was performed in E. coli BL21(DE3). For transporter engineering, the tupBCA operon from S. dubius NA4 was chromosomally integrated using λ-Red recombination. The operon was inserted into the proP locus using the pKD4/pRedET system, followed by excision of the selection marker with the pCP20 plasmid. The resulting strain carrying chromosomally encoded tupBCA was designated BL1. To enhance Fe–S cluster biosynthesis, iscR was deleted and the native isc promoter was replaced with the synthetic P trc promoter via the two-step inactivation method using the pKOV plasmid 42 . The final engineered strain incorporating chromosomal tupBCA and upregulated isc expression was designated BL2 and used as the expression chassis in subsequent protein expression and activity assays. All genomic modifications were validated by colony PCR and verified by Sanger sequencing. Protein expression and purification Recombinant E. coli strains were cultivated by inoculating 1:100 dilutions of overnight seed cultures into 200 mL of TB medium in 1 L baffled flasks. Cultures were incubated at 37°C with shaking to mid-exponential phase (OD₆₀₀ ≈ 0.5–0.6), and protein expression was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Following induction, cells were incubated at 25°C for 16–20 h. All cultivation and protein expression experiments were performed under aerobic conditions. Cell lysis and protein purification were also conducted aerobically. Cells were harvested by centrifugation at 7,000 × g for 15 min at 4°C and resuspended in BugBuster Protein Extraction Reagent (Millipore Sigma) and lysed according to the manufacturer’s protocol. Lysates were clarified by a second centrifugation step at 11,000 × g for 30 min at 4°C. Recombinant enzymes were purified from the soluble fraction using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Column was equilibrated and washed with 50 mM MOPS buffer (pH 7.0) containing 250 mM NaCl and 20 mM imidazole, and bound proteins were eluted with 300 mM imidazole in the same buffer. Protein concentrations were determined spectrophotometrically (NanoDrop, Thermo Scientific) using calculated extinction coefficient specific to each enzyme (ε = 8.94 L·mg⁻¹·cm⁻¹ for MeFDH1, ε = 8.07 L·mg⁻¹·cm⁻¹ for CnFDW, and ε = 9.59 L·mg⁻¹·cm⁻¹ for LbFDH). Enzyme activity assays Enzyme activity was measured spectrophotometrically by monitoring NAD⁺ reduction at 340 nm (ε = 6.22 mM⁻¹·cm⁻¹) using a UV–Vis spectrophotometer under aerobic conditions. Assays were conducted in quartz cuvettes with a total volume of 2.0 mL at 30°C. The standard reaction mixture contained 50 mM MOPS buffer (pH 7.0), 30 mM potassium formate, 0.5 mM NAD⁺, and 10–20 µg of purified enzyme. One unit (U) of activity was defined as the amount of enzyme catalyzing the formation of 1 µmol NADH per minute under these conditions. Metal content analysis The tungsten, molybdenum, and iron contents of purified enzymes were quantified using inductively coupled plasma–mass spectrometry (ICP-MS). Approximately 1 mL of each protein sample (1.0–2.0 mg mL⁻¹) was subjected to acid digestion in Teflon vessels containing 12 mL of trace metal–grade nitric acid (HNO₃) and 1 mL of hydrofluoric acid (HF). Digestion was carried out at 200°C for 2–3 h. After cooling to room temperature, the digested samples were diluted to a final volume of 25 mL with 1% HNO₃. Metal concentrations were measured using a PerkinElmer NexION 5000 ICP-MS calibrated against PerkinElmer multi-element standards 3 and 5. Measured values were normalized to protein concentration and expressed as molar equivalents of metal per αβ heterodimer. Kinetic analysis of MeFDH1 for CO₂ reduction CO₂-reduction kinetics were measured anaerobically using reduced ethyl viologen (EV•⁺) as the electron donor, monitored spectrophotometrically at 600 nm (ε = 10.22 mM⁻¹ cm⁻¹). All assays were conducted inside an anaerobic chamber to prevent the abiotic oxidation of EV•⁺ by oxygen, which would otherwise result in overestimated reaction rates. Reaction mixtures contained phosphate buffer (200 mM, pH 6.3), EV•⁺ (0.015–0.12 mM), and bicarbonate (3–100 mM). Sodium bicarbonate served as the CO₂ source and the relative concentrations of dissolved CO₂(aq), bicarbonate (HCO₃⁻), and carbonate (CO₃²⁻) were calculated from the total inorganic carbon concentration (c) according carbonate equilibrium relationships 43 , using dissociation constants pKa ₁ = 6.3 and pKa ₂ = 10.3: Initial rates (v₀) were determined from the linear portion of absorbance traces. Kinetic parameters (kcat, K M , and kcat/K M ) were obtained by nonlinear regression of v₀ versus substrate concentration to the Michaelis–Menten equation using OriginPro. Representative kinetic curves and model fits are shown in Supplementary Fig. S7 . Whole-cell CO-to-formate conversion Whole-cell formate production was evaluated using MeFDH1 expressed in either E. coli or M. extorquens AM1. The biocatalytic system consisted of two components: (i) an E. coli strain expressing carbon monoxide dehydrogenase from Carboxydothermus hydrogenoformans (ChCODH) and (ii) either E. coli expressing MeFDH1 or M. extorquens AM1 cells expressing MeFDH1. The two cell populations were combined at a fixed CODH-to-FDH activity ratio of 1:10 based on cell dry weight equivalents. Reactions were performed under anaerobic conditions in 10-mL serum vials continuously sparged with CO/CO₂ gas mixture (1:1, v/v) at 3 vvm. The reaction mixture was maintained at pH 6.5, and ethyl viologen (EV) was added to a final concentration of 10 mM as an electron mediator. Whole-cell biotransformations were conducted for 4 h at 30°C with continuous agitation to ensure homogeneous gas–liquid transfer. Formate concentrations were quantified by high-performance liquid chromatography (HPLC) using an Aminex HPX-87H column (Bio-Rad) operated at 35°C, with 5 mM H₂SO₄ at a flow rate of 0.6 mL min⁻¹ and detection using a refractive index (RI) detector. Calibration curves were generated using authentic sodium formate standards over a concentration range of 0–500 mM. Statistical analysis and reproducibility Statistical analyses were performed using OriginPro. All experiments were conducted with at least three independent replicates unless otherwise stated. Data are presented as mean ± standard deviation (s.d.), as indicated in the figure legends. Statistical significance was evaluated using an unpaired two-tailed Student’s t -test, and P values < 0.05 were considered statistically significant. Nonlinear regression for kinetic parameters (kcat and K M ) was performed by unweighted least-squares fitting to the Michaelis–Menten equation. No statistical methods were used to predetermine sample size. No data were excluded from the analysis. Experiments were not randomized, and investigators were not blinded to group allocation during experiments or outcome assessments. Declarations Data availability Data supporting the findings of this study are available within the Article and its Supplementary Information files and source data are provided with this paper. Acknowledgements This research was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (RS-2020-NR049543, RS-2024-00396026, RS-2024-00466474, RS-2024-00453085, RS-2025-16070008). We thank UNIST Central Research Facilities for assistance with ICP-MS measurements and access to instrumentation. Author contribution U. T. P. : Conceptualization, validation, formal analysis, investigation, writing – original draft, writing – review, and editing. J.-M. L. : Conceptualization, validation, formal analysis, investigation, writing – original draft, writing – review, and editing. T. M. V. : formal analysis, investigation. M.-K. O. : Supervision, project administration, formal analysis, writing – original draft, writing – review, and editing. Y. H. K. : Supervision, project administration, formal analysis, writing – original draft, writing – review, and editing. Competing interests The authors declare no competing interests. References Birdja YY et al (2019) Advances and challenges in understanding the electrocatalytic conversion of carbon dioxide to fuels. Nat Energy 4:732–745 Lai W, Qiao Y, Zhang J, Lin Z, Huang H (2022) Design strategies for markedly enhancing energy efficiency in the electrocatalytic CO2 reduction reaction. Energy Environ Sci 15:3603–3629 Sordakis K et al (2018) Homogeneous catalysis for sustainable hydrogen storage in formic acid and alcohols. Chem Rev 118:372–433 Bar-Even A (2016) Formate assimilation: the metabolic architecture of natural and synthetic pathways. Biochemistry 55:3851–3863 Zhang L, Liu J, Ong J, Li SF (2016) Specific and sustainable bioelectro-reduction of carbon dioxide to formate on a novel enzymatic cathode. Chemosphere 162:228–234 Alissandratos A, Kim HK, Easton CJ (2014) Formate production through carbon dioxide hydrogenation with recombinant whole cell biocatalysts. Bioresour Technol 164:7–11 Niks D, Hille R (2019) Molybdenum- and tungsten-containing formate dehydrogenases and formylmethanofuran dehydrogenases: structure, mechanism, and cofactor insertion. Protein Sci 28:111–122 Laukel M, Chistoserdova L, Lidstrom ME, Vorholt JA (2003) The tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1: purification and properties. Eur J Biochem 270:325–333 Seelmann CS, Willistein M, Heider J, Boll M (2020) Tungstoenzymes: occurrence, catalytic diversity and cofactor synthesis. Inorganics 8:44 Majumdar A (2014) Structural and functional models in molybdenum and tungsten bioinorganic chemistry: description of selected model complexes, present scenario and possible future scopes. Dalton Trans 43:8990–9003 Hille R, Hall J, Basu P (2014) The mononuclear molybdenum enzymes. Chem Rev 114:3963–4038 Bevers LE, Hagedoorn P-L, Hagen WR (2009) The bioinorganic chemistry of tungsten. Coord Chem Rev 253:269–290 Raaijmakers H et al (2002) Gene sequence and the 1.8 Å crystal structure of the tungsten-containing formate dehydrogenase from Desulfovibrio gigas . Structure 10:1261–1272 Schulz M et al (2025) Functional expression of a Mo-dependent formate dehydrogenase in Escherichia coli under aerobic conditions. PLoS ONE 20:e0334613 Cowan AE et al (2025) Fast growth and high-titer bioproduction from renewable formate via metal-dependent formate dehydrogenase in Escherichia coli . Nat Commun 16:5908 Shi HL et al (2024) Engineered Escherichia coli whole cell-mediated electro-biocatalysis for carbon dioxide to formic acid conversion. ACS Sustain Chem Eng 12:5544–5554 Liu W et al (2024) Bioelectrocatalytic carbon dioxide reduction by an engineered formate dehydrogenase from Thermoanaerobacter kivui . Nat Commun 15:9962 Szaleniec M, Heider J (2025) Obligately tungsten-dependent enzymes—catalytic mechanisms, models and applications. Biochemistry 64:2154–2172 Ryu H, Nguyen CNM, Lee SK, Park S (2024) Development of Cupriavidus necator H16 as a host for heterologous production of formate dehydrogenase I of Methylorubrum extorquens : possibilities and limitations. Bioresour Technol 394:130187 Bevers LE et al (2008) Function of MoaB proteins in the biosynthesis of the molybdenum and tungsten cofactors. Biochemistry 47:949–956 Leimkühler S (2023) Metal-containing formate dehydrogenases, a personal view. Molecules 28:5338 Park J et al (2024) Structure of recombinant formate dehydrogenase from Methylobacterium extorquens (MeFDH1). Sci Rep 14:3819 Pinske C, Bonn M, Kruger S, Lindenstrauss U, Sawers RG (2011) Metabolic deficiencies revealed in the biotechnologically important model bacterium Escherichia coli BL21(DE3). PLoS ONE 6:e22830 Han MJ (2016) Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular compartments. J Biosci Bioeng 122:1–9 Coimbra C, Branco R, Morais PV (2019) Efficient bioaccumulation of tungsten by Escherichia coli cells expressing the Sulfitobacter dubius TupBCA system. Syst Appl Microbiol 42:126001 Coimbra C, Farias P, Branco R, Morais PV (2017) Tungsten accumulation by highly tolerant marine hydrothermal Sulfitobacter dubius strains carrying a tupBCA cluster. Syst Appl Microbiol 40:388–395 Vuilleumier S et al (2009) Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of C1 compounds from natural and industrial sources. PLoS ONE 4:e5584 Outten FW, Djaman O, Storz G (2004) A suf operon requirement for Fe–S cluster assembly during iron starvation in Escherichia coli . Mol Microbiol 52:861–872 Mettert EL, Kiley PJ (2014) Coordinate regulation of the Suf and Isc Fe–S cluster biogenesis pathways by IscR is essential for viability of Escherichia coli . J Bacteriol 196:4315–4323 Zupok A, Iobbi-Nivol C, Mejean V, Leimkühler S (2019) The regulation of Moco biosynthesis and molybdoenzyme gene expression by molybdenum and iron in bacteria. Metallomics 11:1602–1624 Mendel RR, Hercher TW, Zupok A, Hasnat MA, Leimkühler S (2020) The requirement of inorganic Fe–S clusters for the biosynthesis of the organometallic molybdenum cofactor. Inorganics 8:43 Leimkühler S (2021) Transition metals in catalysis: the functional relationship of Fe–S clusters and molybdenum or tungsten cofactor-containing enzyme systems. Inorganics 9:6 Nakamura M, Saeki K, Takahashi Y (1999) Hyperproduction of recombinant ferredoxins in Escherichia coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster. J Biochem 126:10–18 Ryu H et al (2025) Production and characterization of CO2 reducing and tungsten-containing formate dehydrogenase of Cupriavidus necator H16. Biotechnol Bioprocess Eng 30:363–376 van Kempen M et al (2024) Fast and accurate protein structure search with Foldseek. Nat Biotechnol 42:243–246 Leimkühler S (2020) The biosynthesis of the molybdenum cofactors in Escherichia coli . Environ Microbiol 22:2007–2026 Magalon A, Mendel RR (2008) Biosynthesis and insertion of the molybdenum cofactor. EcoSal Plus 3 Bevers LE, Schwarz G, Hagen WR (2011) A molecular basis for tungstate selectivity in prokaryotic ABC transport systems. J Bacteriol 193:4999–5001 Hagen WR (2011) Cellular uptake of molybdenum and tungsten. Coord Chem Rev 255:1117–1128 Otrelo-Cardoso AR et al (2017) Highly selective tungstate transporter protein TupA from Desulfovibrio alaskensis G20. Sci Rep 7:5798 Jang J, Jeon BW, Kim YH (2018) Bioelectrochemical conversion of CO2 to value added product formate using engineered Methylobacterium extorquens . Sci Rep 8:7211 Link AJ, Phillips D, Church GM (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli : application to open reading frame characterization. J Bacteriol 179:6228–6237 Jeon BW et al (2024) Enzymatic conversion of CO2 to formate: the potential of tungsten-containing formate dehydrogenase in flow reactor system. J CO2 Util 82:102754 Tables Table 1. Strains and plasmids used in this study. Strains Description Source /Reference BL21(DE3) E. coli BL21(DE3) expression host RBC Real Biotech BL BL21(DE3) expressing Methylorubrum extorquens FDH1 This study BL(W1) BL21(DE3) expressing MeFDH1 with MoeA1 This study BL(WA) BL21(DE3) expressing MeFDH1 with MobA This study BL(WB) BL21(DE3) expressing MeFDH1 with MobB This study BL(W2) BL21(DE3) expressing MeFDH1 with MoeA1 and MobB This study BL(W3) BL21(DE3) expressing MeFDH1 with MoeA1, MobA and MobB This study BL(W4) BL21(DE3) expressing MeFDH1 with MoeA1, MobA, MobB and FdhD (W4 machinery) This study BL1 BL21(DE3) with chromosomal tungstate transporter ( ΔproP ::PT7– tupBCA from Sulfitobacter dubius NA4) This study BL1(W4) BL1 expressing MeFDH1 with W4 machinery This study BL1(W4+ISC) BL1(W4) harboring pRKISC ( isc operon) This study BL2 BL1 with chromosomally upregulated isc operon ( ΔiscR::Ptrc–iscSUA–hscBA–fdx ) This study BL2(W4) BL2 expressing MeFDH1 with W4 machinery This study BL(Cn) BL expressing Cupriavidus necator FDW This study BL2(W4+Cn) BL2 expressing CnFDW together with W4 machinery This study BL(Lb) BL expressing Lutibaculum baratangense FDH This study BL2(W4+Lb) BL2 expressing LbFDH together with W4 machinery This study Plasmids Description Source /Reference pETDuet-1 Dual T7 expression backbone, Ampᴿ Expressys pF1 pETDuet-1 carrying M. extorquens FDH1 α/β expression cassette ( fdh1A -6×His– fdh1B ) This study pF2 pETDuet-1 carrying C. necator FDW α/β expression cassette ( fdwA -6×His– fdwB ) This study pF3 pETDuet-1 carrying L. baratangense FDH α/β expression cassette ( fdhA -6×His– fd h B ) This study pCDFDuet-1 Cloning/expression backbone, Smᴿ Expressys pW1 pCDFDuet-1 carrying moeA1 from M. extorquens AM1 This study pWA pCDFDuet-1 carrying mobA from M. extorquens AM1 This study pWB pCDFDuet-1 carrying mobB from M. extorquens AM1 This study pW1A pCDFDuet-1 carrying moeA1–mob A from M. extorquens AM1 This study pW1B pCDFDuet-1 carrying moeA1–mobB from M. extorquens AM1 This study pWAB pCDFDuet-1 carrying mobA–mobB from M. extorquens AM1 This study pW3 pCDFDuet-1 carrying moeA1–mobA–mobB from M. extorquens AM1 This study pW4 pCDFDuet-1 carrying moeA1–mobA–mobB–fdhD from M. extorquens AM1 This study pZ1 pZS21 carrying modABC from E. coli K-12 This study pZ2 pZS21 carrying tupBCA from S. dubius NA4 This study pZ3 pZS21 carrying tupBCA from M. extorquens AM1 This study pRKISC iscSUA–hscBA–fdx operon, Tetᴿ Nakamura et al . 33 Table 2. Metal content of MeFDH1 across engineered E. coli strains and the native M . extorquens AM1 host. Metal incorporation into purified MeFDH1 was quantified by inductively coupled plasma–mass spectrometry (ICP–MS). Iron (Fe), tungsten (W), and molybdenum (Mo) contents are shown for enzymes produced in strains expressing progressively expanded tungsten-cofactor biosynthetic and accessory modules: BL (MeFDH1 only), BL(W4) (MoeA1, MobA, MobB and FdhD), BL1(W4) (W4 machinery with chromosomal tupBCA ), BL1(W4+ISC) (additional plasmid-borne isc operon), BL2(W4) (chromosomally upregulated isc operon), and the native AM1 host. Values represent mean ± s.d. (n = 3). Strain Metal content (mol metal mol -1 enzyme) Fe W Mo BL 1.75 ± 0.03 0.01 ± 0.01 0.02 ± 0.01 BL(W4) 5.50 ± 0.09 0.27 ± 0.05 0.05 ± 0.04 BL1(W4) 5.14 ± 0.10 0.55 ± 0.05 0.09 ± 0.05 BL1(W4+ISC) 8.30 ± 0.10 0.75 ± 0.03 0.05 ± 0.05 BL2(W4) 10.8 ± 0.09 0.92 ± 0.01 0.02 ± 0.01 AM1 11.6 ± 0.21 0.50 ± 0.05 0.02 ± 0.02 Table 3. Kinetic parameters of MeFDH1 in expressed engineered E. coli compared with the enzyme from M. extorquens AM1 Turnover numbers (k cat ), Michaelis constants (K M ), and catalytic efficiencies (k cat /K M ) were determined for CO₂ reduction using either dissolved CO₂ or reduced ethyl viologen (EV⁺·) as the electron mediator. MeFDH1 was purified from E. coli BL2 (BL2) or isolated from the native AM1 host. Values represent mean ± s.d. (n = 3). k cat (s⁻¹) K M (mM) k cat /K M (s⁻¹ mM⁻¹) CO₂ BL2 141 ± 10 8.4 ± 1.4 17 ± 3 AM1 152 ± 5 3.6 ± 0.1 42 ± 1 EV•⁺ BL2 304 ± 39 0.06 ± 0.02 5067 ± 1810 AM1 325 ± 6 0.02 ± 0.01 16250 ± 8131 Table 4. Functional expression of heterologous tungsten-dependent dehydrogenases in engineered E. coli chassis. CnFDW from C. necator H16 and LbFDH from L. baratangense AMV1 were expressed in BL21(DE3) or the engineered BL2 chassis. BL2 carries a chromosomally integrated tungstate transporter ( tupBCA ), chromosomal upregulation of the isc operon, and plasmid-encoded W-cofactor biosynthetic genes ( moeA1 , mobA , mobB , and fdhD ). Specific activities were determined by enzymatic assay, and metal contents (Fe, W, Mo) were quantified by ICP–MS. ND, not detected. Values represent mean ± s.d. (n = 3). Strain Specific activity (U mg⁻¹ enzyme) Metal (mol mol⁻¹ enzyme) Fe W Mo BL(Cn) ND 2.72 ± 0.01 0.03 ± 0.01 0.04 ± 0.01 BL2(W4+Cn) 38 ± 4 18.6 ± 0.02 0.89 ± 0.02 0.03 ± 0.02 BL(Lb) ND 5.68 ± 0.08 0.01 ± 0.01 0.24 ± 0.01 BL2(W4+Lb) 8 ± 3 12.8 ± 0.04 1.17 ± 0.02* 0.01 ± 0.01 * W occupancy slightly above 1.0 reflects minor analytical uncertainty in protein quantification. Additional Declarations There is NO Competing Interest. Supplementary Files 260219SImeFDH1E.colifinalversion.docx Supplementary information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8913862","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":594356355,"identity":"29b3d179-2ee2-431f-94dd-e26acd67f57e","order_by":0,"name":"Yong Hwan Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIie3RMQrCMBiG4V8KcRG6xskTCJFC6VDxIC6/FJxajyCCYMeuOUZBcG4I6JIDZFPo6lC3utnWzSEWXBzyLqGQh3xQAJvtX7sVFNzmLN6f5Nt9BwAbMt61BPsTANY90YdM00xUqIKlpxMhnjVM3B1ZVybiK+lQ1DQ56Q3KEcKMF0RyI9ERUKxaEjPZDBvkMNwbh/nX0qlbcuQxEzXC4jvRDumG5TRmRTNslQORZqIiP0BFE67uTI7WNOKSRGZyEaWuztskS2PvUYfhPEsPnpF8RrsfZbPZbLYfewEliU0HPIyxUQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4729-693X","institution":"UNIST","correspondingAuthor":true,"prefix":"","firstName":"Yong","middleName":"Hwan","lastName":"Kim","suffix":""},{"id":594356356,"identity":"8e3e74c9-a296-47ad-adb6-a9ffd1dacf99","order_by":1,"name":"Uyen Phan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Uyen","middleName":"","lastName":"Phan","suffix":""},{"id":594356357,"identity":"f284dc23-e9c8-43b0-9dbe-d0d39e793acd","order_by":2,"name":"Jun-Min Lee","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Jun-Min","middleName":"","lastName":"Lee","suffix":""},{"id":594356358,"identity":"0025dfb0-357f-4640-92ef-0e94eb2af8a4","order_by":3,"name":"Toan Vo","email":"","orcid":"","institution":"UNIST","correspondingAuthor":false,"prefix":"","firstName":"Toan","middleName":"","lastName":"Vo","suffix":""},{"id":594356359,"identity":"d64b7288-ff92-42ad-91bf-d045bcd37bb6","order_by":4,"name":"Min-Kyu Oh","email":"","orcid":"https://orcid.org/0000-0001-6406-3930","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Min-Kyu","middleName":"","lastName":"Oh","suffix":""}],"badges":[],"createdAt":"2026-02-19 04:41:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8913862/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8913862/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103195539,"identity":"16e01fe2-3349-4bcb-86fb-4d4da682dbb3","added_by":"auto","created_at":"2026-02-23 03:45:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":174732,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative biogenesis of molybdenum- and tungsten-containing bis-MGD cofactors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSchematic comparison of Mo-bis-MGD biosynthesis in native\u003c/em\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e \u003cem\u003eand W-bis-MGD biosynthesis in M. extorquens\u003c/em\u003e AM1\u003cem\u003e. The native E. coli\u003c/em\u003e \u003cem\u003epathways support molybdenum insertion but is incompatible with tungsten-dependent enzymes. \u003c/em\u003eIn contrast, the \u003cem\u003eM. extorquens\u003c/em\u003e AM1 pathway, involving MoeA1, MobA, MobB and FdhD, enables selective tungstate incorporation, guanylylation and sulfur transfer, resulting in the formation of catalytically competent W-bis-MGD.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8913862/v1/8aca7ccba1c64d56c5568a0a.png"},{"id":103195544,"identity":"1cd2a173-7b43-47fd-b2ee-cd52784d0aed","added_by":"auto","created_at":"2026-02-23 03:45:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":133210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStepwise reconstitution of the tungsten-cofactor (Wco) biosynthetic pathway and its impact on MeFDH1 activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eBL21(DE3).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Schematic representation of the reconstructed W-bis-MGD maturation pathway.AlphaFold3-predicted structures of MoeA1, MobA, MobB, and FdhD from \u003cem\u003eM. extorquens\u003c/em\u003eAM1 were arranged to reflect interaction relationships observed in the BACTH assay (Supplementary Fig. S1). Strong interactions between MoeA1–MobA and MoeA1–MobB (solid lines) indicate a tightly associated core complex for W-bis-MGD biogenesis, whereas the weaker MoeA1–FdhD interaction (dashed line) suggests transient recruitment, consistent with sulfur transfer to the nascent cofactor.\u003cbr\u003e\n \u003cstrong\u003eb,\u003c/strong\u003e Specific activity of recombinant MeFDH1 expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) with stepwise combinations of Wco biosynthetic genes: BL (MeFDH1 only), W1 (+MoeA1), WA (+MobA), WB (+MobB), W2 (+MoeA1+MobB), W3 (+MoeA1+MobA+MobB), and W4 (+MoeA1+MobA+MobB+FdhD). Activity of MeFDH1 in the native \u003cem\u003eM. extorquens\u003c/em\u003e AM1 host is shown for comparison. Bars represent mean ± s.d. (n = 3).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8913862/v1/f4570b571e01d02abca6a5b5.png"},{"id":103504752,"identity":"3166d5e0-80a7-451e-ba31-f3d5eb21b70d","added_by":"auto","created_at":"2026-02-26 13:21:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":122650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosomal expression of the TupBCA transporter enhances tungstate uptake and MeFDH1 maturation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003cbr\u003e\n\u003c/strong\u003e a, Schematic of heterologous chromosomal integration of the \u003cem\u003eSulfitobacter dubius\u003c/em\u003e NA4 \u003cem\u003etupBCA\u003c/em\u003e operon, encoding a high-affinity tungstate transport system. This modification facilitates tungsten uptake across the cell membrane, supporting intracellular cofactor biosynthesis and activation of W-dependent enzymes such as MeFDH1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb,\u003c/strong\u003e Specific activity (U mg⁻¹) of MeFDH1 expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) carrying the W4 biosynthetic module alone (W4) or combined with chromosomal \u003cem\u003etupBCA\u003c/em\u003e [BL1(W4)], compared with the native \u003cem\u003eM.\u003c/em\u003e \u003cem\u003eextorquens\u003c/em\u003e AM1 host.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec,\u003c/strong\u003e Corresponding soluble MeFDH1 yield (mg g⁻¹ WCW). Data represent mean ± s.d. (n = 3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8913862/v1/4166a170793da9a844fc2d12.png"},{"id":103195542,"identity":"85e741f1-7264-4ed5-af4c-59a515315a8d","added_by":"auto","created_at":"2026-02-23 03:45:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReinforcement of Fe–S cluster biogenesis further enhances MeFDH1 performance in engineered \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003eSchematic of enhancement of the \u003cem\u003eisc\u003c/em\u003e pathway through deletion of the transcriptional repressor \u003cem\u003eiscR\u003c/em\u003e and \u003cem\u003ePtrc\u003c/em\u003e-driven expression of the \u003cem\u003eiscSUA–hscBA–fdx\u003c/em\u003eoperon, increasing cellular Fe–S cluster assembly capacity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb–d,\u003c/strong\u003e Specific activity, soluble protein yield, and volumetric activity of MeFDH1 expressed in \u003cem\u003eE. coli\u003c/em\u003e strains with stepwise optimization of Fe–S cluster biogenesis: BL1(W4) (chromosomal \u003cem\u003etupBCA\u003c/em\u003e), BL1(W4+ISC) (plasmid-borne \u003cem\u003eisc\u003c/em\u003e operon), and BL2(W4) (chromosomally upregulated \u003cem\u003eisc\u003c/em\u003e operon), compared with the native \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eextorquens\u003c/em\u003e AM1 host. Bars represent mean ± s.d. (n = 3).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8913862/v1/eaf36d91223ffc32e104bd91.png"},{"id":103195540,"identity":"134d531c-58a1-4073-b7fe-e2a3361c7f44","added_by":"auto","created_at":"2026-02-23 03:45:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":155645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWhole-cell CO-to-formate conversion using MeFDH1 expressed in engineered \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e BL2 and the native \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eextorquens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e AM1 host.\u003cbr\u003e\n\u003c/strong\u003ea, Schematic of the coupled whole-cell system. \u003cem\u003eE. coli\u003c/em\u003e expressing ChCODH oxidizes CO to CO₂, generating reduced ethyl viologen (EV•⁺), which mediates electron transfer to \u003cem\u003eE. coli\u003c/em\u003e expressing MeFDH1 and drives the reductive conversion of CO₂ to formate. Oxidized EV²⁺ is recycled back to the ChCODH-expressing cells, completing the electron-transfer cycle.\u003cbr\u003e\nb, Time course of formate production in the coupled system using ChCODH-expressing cells combined with MeFDH1 produced in either \u003cem\u003eE. coli\u003c/em\u003e BL2 or \u003cem\u003eM. extorquens\u003c/em\u003e AM1. Cell densities were normalized based on dry weight equivalents. Data represent mean ± s.d.(n = 3).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8913862/v1/c8b20a83ce966dcc807b2c6d.png"},{"id":104407246,"identity":"b6a3cd95-b7e3-4cc6-a200-2d5b690f743d","added_by":"auto","created_at":"2026-03-11 12:36:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2122312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8913862/v1/49a95b9e-7af3-4847-97ff-a116f75aa0b1.pdf"},{"id":104397484,"identity":"fa4a0063-337f-4d9f-bc1e-b8a073d3dc89","added_by":"auto","created_at":"2026-03-11 11:49:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3699945,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"260219SImeFDH1E.colifinalversion.docx","url":"https://assets-eu.researchsquare.com/files/rs-8913862/v1/4ff5bb97477c45024e0bfc72.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Metal cofactor level chassis engineering enables aerobic expression of tungsten formate dehydrogenases in Escherichia coli","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCarbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) conversion has become a cornerstone of climate-aligned manufacturing, enabling fixation and valorization of a major greenhouse gas into valuable chemicals\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among CO₂-derived products, formate is particularly attractive because it can be produced from CO₂ with high energy efficiency using technically straightforward routes\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and already supports a mature market with established applications across chemical and agricultural sectors\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Motivated by rising atmospheric CO\u003csub\u003e2\u003c/sub\u003e levels and increasingly stringent decarbonization targets, these advantages have intensified efforts to develop selective, low-energy CO\u003csub\u003e2\u003c/sub\u003e-to-formate platforms that integrate biological systems with renewable power, thereby accelerating the transition from carbon capture to circular utilization\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong available biocatalysts, formate dehydrogenases (FDHs) incorporating high-valent transition metal cofactors, most prominently molybdenum (Mo) and tungsten (W), have emerged as leading candidates for CO₂-to-formate conversion due to their high catalytic efficiency, favorable thermodynamics, and broad applicability in C1-based biomanufacturing\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although Mo- and W-dependent FDHs share a conserved bis-molybdopterin guanine dinucleotide (bis-MGD) framework, the identity of the central metal atom profoundly affects catalytic behaviors. The tungsten center in W-FDHs exhibits a substantially lower redox potential than its molybdenum counterpart, expanding the low-potential operational window and enhancing hydride-transfer kinetics. As a result, W-FDHs thermodynamically favor the reductive conversion of CO₂ to formate, making them particularly suitable for CO₂ fixation and bio-electrochemical CO₂ reduction\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite these advantages, the broader deployment of W-FDHs has been constrained by fundamental challenges in heterologous maturation. Although \u003cem\u003eEscherichia coli\u003c/em\u003e can support recombinant expression of Mo-FDHs by leveraging its native molybdenum cofactor (Moco) biosynthesis pathway\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, analogous functional expression of authentic tungsten-dependent enzymes in this host has proven much more difficult. While recent studies have demonstrated \u003cem\u003eE. coli\u003c/em\u003e\u0026ndash;based implementations of metalloenzyme modules for CO₂ reduction and formate-driven C1 metabolism\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, a broadly applicable aerobic, chassis-level solution that enables efficient tungsten-selective maturation of W-bis-MGD enzymes in \u003cem\u003eE. coli\u003c/em\u003e has remained limited.\u003c/p\u003e \u003cp\u003eThese limitations arise from intrinsic features of \u003cem\u003eE. coli\u003c/em\u003e metal homeostasis. Moreover, tungsten-dependent enzymes impose additional constraints\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, including competition between molybdenum and tungsten for cofactor metalation, stringent control of tungstate availability, and reliance on iron\u0026ndash;sulfur (Fe\u0026ndash;S) cluster biogenesis. These processes are not inherently aligned with native \u003cem\u003eE. coli\u003c/em\u003e physiology. As a result, heterologous expression of tungsten-containing oxidoreductases in \u003cem\u003eE. coli\u003c/em\u003e frequently yields incomplete cofactor incorporation, metal misallocation, or catalytically inactive apoenzymes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we engineered a W-bis-MGD-cofactor\u0026ndash;competent \u003cem\u003eE. coli\u003c/em\u003e chassis through coordinated reprogramming of intracellular tungsten metabolism and Fe-S cluster assembly. This strategy combined reconstruction of tungsten-cofactor biosynthesis using key genes from \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e AM1, chromosomal integration of a high-affinity tungstate transporter (TupBCA\u003cem\u003e)\u003c/em\u003e from \u003cem\u003eSulfitobacter dubius\u003c/em\u003e NA4, and genomic reinforcement of Fe\u0026ndash;S cluster biogenesis via targeted engineering of the \u003cem\u003eisc\u003c/em\u003e operon. The resulting chassis supports soluble, catalytically active expression of multiple W-dependent enzymes under aerobic conditions, including MeFDH1 from \u003cem\u003eM. extorquens\u003c/em\u003e AM1, CnFDW from \u003cem\u003eCupriavidus necator\u003c/em\u003e H16, and a structurally predicted LbFDH from \u003cem\u003eLutibaculum baratangense\u003c/em\u003e AMV1.\u003c/p\u003e \u003cp\u003eBy overcoming tungsten-specific maturation barriers in a host previously considered incompatible with W-enzyme assembly, this work converts E. coli into a programmable platform for W-bis-MGD enzyme production. The approach establishes a transferable design framework for aerobic maturation of tungsten-dependent redox enzymes and expands access to this underexplored metallocofactor chemistry for sustainable CO₂ reduction and related reductive biotransformations.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eReconstitution of W-bis-MGD biosynthesis restores MeFDH1 activation in\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe tungsten-dependent formate dehydrogenase from \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e AM1 (MeFDH1) was selected as a stringent tungsten-dependent model oxidoreductase, as its catalytic activity strictly requires W-bis-MGD metalation. However, recombinant expression of MeFDH1 in \u003cem\u003eEscherichia coli\u003c/em\u003e does not yield catalytically competent enzyme\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, indicating that this host lacks a tungsten-compatible maturation environment. We therefore sought to reconstruct the cognate tungsten-specific cofactor biosynthetic machinery from \u003cem\u003eM. extorquens\u003c/em\u003e AM1 in \u003cem\u003eE. coli\u003c/em\u003e to establish a tungsten-dependent maturation pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrior genetic characterization of MeFDH1 maturation in \u003cem\u003eM. extorquens\u003c/em\u003e AM1 (summarized in Supplementary \u003cb\u003eTable S1\u003c/b\u003e) identified MoeA1, MobA, MobB, and FdhD as essential components of the native W-bis-MGD biosynthetic pathway. Among the five annotated \u003cem\u003emoeA\u003c/em\u003e homologs, only \u003cem\u003emoeA1\u003c/em\u003e in \u003cem\u003eM. extorquens\u003c/em\u003e AM1 was required for MeFDH1 activity, establishing MoeA1 as the sole tungsten-specific insertion factor for this enzyme. Deletion analyses further showed that \u003cem\u003emobB\u003c/em\u003e and \u003cem\u003efdhD\u003c/em\u003e are required for MeFDH1 activity, whereas \u003cem\u003emobA\u003c/em\u003e is essential for bis-MGD biosynthesis in the native host. These findings defined the minimal tungsten-specific maturation module guiding heterologous reconstruction.\u003c/p\u003e \u003cp\u003eTo assess potential coordination among these factors in the context of reconstruction, protein\u0026ndash;protein interactions were evaluated using a bacterial adenylate cyclase two-hybrid (BACTH) assay (Supplementary \u003cb\u003eFig. S1\u003c/b\u003e). MoeA1 exhibited reproducible interactions with both MobA and MobB, while FdhD interacted preferentially with the Mob protein rather than directly with MoeA1. Collectively, this interaction pattern is consistent with a cooperative engagement of these factors during W-bis-MGD maturation and provides contextual support for the reconstruction strategy, as schematically summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea.\u003c/p\u003e \u003cp\u003eGuided by these observations, \u003cem\u003emoeA1\u003c/em\u003e, \u003cem\u003emobA\u003c/em\u003e, \u003cem\u003emobB\u003c/em\u003e, and \u003cem\u003efdhD\u003c/em\u003e from \u003cem\u003eM. extorquens\u003c/em\u003e AM1 were codon-optimized and cloned into a pCDFDuet-1 vector under separate T7 promoters (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In parallel, the MeFDH1 α and β subunits were codon-optimized and cloned into a pETDuet-1 expression vector. \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) was then co-transformed with MeFDH1 and various combinations of the tungsten cofactor (Wco) biosynthetic genes to assess their individual and combinatorial contributions to enzyme activation.\u003c/p\u003e \u003cp\u003eAs expected, expression of MeFDH1 alone (BL) resulted in negligible activity (\u0026lt;\u0026thinsp;1 U mg⁻\u0026sup1;), indicating predominant accumulation of apoenzyme due to insufficient availability of the W-bis-MGD cofactor. Similarly, strains expressing individual Wco genes, \u003cem\u003emoeA1\u003c/em\u003e (W1), \u003cem\u003emobA\u003c/em\u003e (WA), or \u003cem\u003emobB\u003c/em\u003e (WB), failed to produce measurable activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), demonstrating that no single component is sufficient to support cofactor assembly or enzyme activation. Although MoeA1 is strictly required for MeFDH1 function in the native host, its expression alone was insufficient to restore activity in the heterologous background.\u003c/p\u003e \u003cp\u003eThe recovery of measurable activity (~\u0026thinsp;6 U mg⁻\u0026sup1;) in the \u003cem\u003emoeA1\u0026ndash;mobB\u003c/em\u003e strain (W2) indicates that cooperative expression of these two proteins is sufficient to enable functional tungsten cofactor maturation in \u003cem\u003eE. coli\u003c/em\u003e. Introduction of \u003cem\u003emobA\u003c/em\u003e into the \u003cem\u003emoeA1\u003c/em\u003e\u0026ndash;\u003cem\u003emobB\u003c/em\u003e background (W3) led to a substantial increase in activity, suggesting that bis-MGD formation markedly enhances catalytic competence once tungsten has been integrated into the cofactor. Conversely, removal of \u003cem\u003emobB\u003c/em\u003e from the W3 background abolished MeFDH1 activity (Supplementary \u003cb\u003eFig. S2\u003c/b\u003e) indicating that MobB is required for productive W-bis-MGD maturation. Finally, inclusion of \u003cem\u003efdhD\u003c/em\u003e (W4) yielded the highest activity (~\u0026thinsp;27 U mg⁻\u0026sup1;) and was accompanied by an approximately threefold increase in tungsten occupancy relative to the MeFDH1-only background (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), consistent with improved completion of cofactor maturation. Accordingly, the full four-gene module (\u003cem\u003emoeA1\u003c/em\u003e, \u003cem\u003emobA\u003c/em\u003e, \u003cem\u003emobB\u003c/em\u003e, and \u003cem\u003efdhD\u003c/em\u003e) was adopted as the standard Wco biosynthetic configuration for all subsequent experiments.\u003c/p\u003e \u003cp\u003eThe progressive enhancement in activity from W2 to W4 is consistent with the interaction relationships detected by BACTH analysis and supports a coordinated maturation process in which functional W-bis-MGD biosynthesis in \u003cem\u003eE. coli\u003c/em\u003e requires the integrated action of multiple pathway components rather than expression of individual biosynthetic genes. Nevertheless, the maximal activity achieved in \u003cem\u003eE. coli\u003c/em\u003e remained substantially lower than that observed in \u003cem\u003eM. extorquens\u003c/em\u003e AM1 (~\u0026thinsp;50 U mg⁻\u0026sup1;), suggesting the presence of additional constraints, including limitations in tungsten uptake and iron\u0026ndash;sulfur (Fe\u0026ndash;S) cluster availability. This establishes the minimal tungsten maturation module.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSelective tungstate transport enhances tungsten incorporation and enzyme performance\u003c/h2\u003e \u003cp\u003eConsistent with the residual activity gap observed after W-bis-MGD pathway reconstruction, MeFDH1 produced with the W4 module exhibited limited tungsten occupancy (~\u0026thinsp;27% of theoretical stoichiometry). This incomplete loading suggested that tungsten availability, rather than cofactor assembly capacity alone, may constrain effective W-bis-MGD biosynthesis in \u003cem\u003eE. coli\u003c/em\u003e. Given that BL21(DE3) lacks the high-affinity ModABC molybdate/tungstate transport system present in K-12 derivatives\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, we next examined whether enhanced tungstate uptake could alleviate this limitation.\u003c/p\u003e \u003cp\u003eThree candidate transport systems were evaluated by plasmid expression in tungstate-supplemented cultures, including the \u003cem\u003eE. coli\u003c/em\u003e K-12 \u003cem\u003emodABC\u003c/em\u003e operon, the tungsten-specific \u003cem\u003etupBCA\u003c/em\u003e operon from \u003cem\u003eSulfitobacter dubius\u003c/em\u003e NA4\u003csup\u003e25, 26\u003c/sup\u003e, and the \u003cem\u003etupBCA\u003c/em\u003e operon from \u003cem\u003eM. extorquens\u003c/em\u003e AM1\u003csup\u003e27\u003c/sup\u003e. Among these, the \u003cem\u003eS. dubius\u003c/em\u003e NA4\u0026ndash;derived TupBCA produced the highest MeFDH1 specific activity, outperforming both ModABC from \u003cem\u003eE. coli\u003c/em\u003e K-12 and the \u003cem\u003eM. extorquens\u003c/em\u003e TupBCA (Supplementary \u003cb\u003eFig. S3\u003c/b\u003e). Based on this performance and to minimize plasmid burden in downstream experiments, the \u003cem\u003eS. dubius\u003c/em\u003e NA4 transporter was integrated into the chromosome of \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) under control of a T7 promoter, generating strain BL1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe selected \u003cem\u003etupBCA\u003c/em\u003e operon from \u003cem\u003eS. dubius\u003c/em\u003e NA4 encodes an ATP-binding cassette (ABC)\u0026ndash;type tungstate transporter composed of the periplasmic binding protein TupA, membrane permease TupB, and ATPase TupC\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Co-expression of the genomically integrated transporter with the W4 biosynthetic module resulted in a substantial increase in MeFDH1 activity (~\u0026thinsp;35 U mg⁻\u0026sup1;) and improved soluble protein yield (~\u0026thinsp;2.0 mg g⁻\u0026sup1; WCW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Tungsten incorporation increased by approximately twofold in MeFDH1 purified from transporter-containing strains, reaching\u0026thinsp;~\u0026thinsp;50% of the theoretical stoichiometry (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), representing a substantial improvement over the transporter-deficient controls.\u003c/p\u003e \u003cp\u003eTo further assess the metal selectivity of the reconstructed tungsten cofactor biosynthetic pathway, MeFDH1 activity and metal profiling were examined under three conditions: tungstate alone (1 mM), mixed tungstate (0.5 mM) and molybdate (0.5 mM), or molybdate alone (1 mM). Cultures supplied exclusively with tungstate produced the highest MeFDH1 activity (~\u0026thinsp;35 U mg⁻\u0026sup1;), whereas mixed-metal conditions led to a marked reduction in activity (~\u0026thinsp;10 U mg⁻\u0026sup1;). No activity was observed under molybdate-only supplementation.\u003c/p\u003e \u003cp\u003eMetal quantification showed that tungsten incorporation was highest under tungstate-only conditions (~\u0026thinsp;0.51 mol W per mol enzyme) and decreased under mixed-metal supplementation, while molybdenum incorporation remained negligible across all treatments (Supplementary \u003cb\u003eTable S2\u003c/b\u003e). The minimal Mo occupancy suggests that molybdenum does not efficiently substitute for tungsten in the mature enzyme population. Instead, the reduced activity under mixed conditions likely reflects competitive interference during metal allocation or early stages of W-bis-MGD biosynthesis, which impairs efficient cofactor assembly without substantial Mo incorporation into the purified enzyme.\u003c/p\u003e \u003cp\u003eThese results reveal that W-bis-MGD assembly in BL21(DE3) is highly sensitive to intracellular metal balance, as even modest molybdate supplementation can disrupt selective tungsten incorporation. Enhancing high-efficiency tungsten transport therefore promotes effective cofactor assembly and improved enzyme performance. These findings identify tungsten transport as a primary bottleneck in this host and establish dedicated tungstate acquisition as a critical design element for reliable expression of tungsten-dependent enzymes in synthetic systems.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReinforcement of iron-sulfur cluster biogenesis increases metalation and yield\u003c/h3\u003e\n\u003cp\u003eBeyond the W-bis-MGD cofactor at the active site, MeFDH1 relies on multiple iron\u0026ndash;sulfur (Fe\u0026ndash;S) clusters in both the α and β subunits to mediate electron transfer to physiological and artificial electron acceptors such as NADH or ethyl viologen (EV) (Supplementary \u003cb\u003eFig. S4\u003c/b\u003e). In \u003cem\u003eE. coli\u003c/em\u003e, Fe\u0026ndash;S cluster biosynthesis is mediated primarily by the ISC and SUF systems, with ISC tightly regulated by the global repressor IscR\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. While sufficient for native metabolism, IscR-mediated regulation may not meet the elevated Fe\u0026ndash;S cluster demand imposed by overexpression of multi\u0026ndash;Fe\u0026ndash;S enzymes such as MeFDH1. Consistent with this limitation, MeFDH1 produced prior to \u003cem\u003eisc\u003c/em\u003e engineering contained only\u0026thinsp;~\u0026thinsp;20\u0026ndash;25% of the theoretical iron content (~\u0026thinsp;20 mol Fe per mol enzyme; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating extensive under-metalation.\u003c/p\u003e \u003cp\u003eIn addition to delivering Fe\u0026ndash;S cluster to target proteins, the ISC machinery has been implicated in bis-MGD cofactor biosynthesis in bacteria. Specifically, IscA functions as an Fe\u0026ndash;S carrier that transfers clusters to MoaA, which catalyzes the first step of MPT synthesis, while IscS serves as a sulfur donor that regulates the synthesis rate\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Enhanced ISC activity is therefore expected to support not only Fe\u0026ndash;S cluster assembly but also W-bis-MGD biosynthesis\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo test this hypothesis, we first enhanced Fe\u0026ndash;S biosynthesis by plasmid-based co-expression of the \u003cem\u003eisc\u003c/em\u003e operon using pRKISC\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Introduction of pRKISC into BL1 moderately improved MeFDH1 specific activity (46 U mg⁻\u0026sup1;) and soluble yield (4.5 mg g⁻\u0026sup1; WCW), approaching levels observed the native \u003cem\u003eM. extorquens\u003c/em\u003e AM1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We next implemented genome-level upregulation of endogenous Fe\u0026ndash;S biogenesis by deleting \u003cem\u003eiscR\u003c/em\u003e and replacing the native \u003cem\u003eisc\u003c/em\u003e promoter with a strong synthetic promoter (P\u003csub\u003e\u003cem\u003etrc\u003c/em\u003e\u003c/sub\u003e), generating strain BL2 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In BL2, MeFDH1 exhibited near-native specific activity (~\u0026thinsp;48 U mg⁻\u0026sup1;) and substantially increased soluble yield (~\u0026thinsp;10 mg g⁻\u0026sup1; biomass), corresponding to a total volumetric activity exceeding 3500 U L⁻\u0026sup1;, approximately 15-fold higher than native AM1 (~\u0026thinsp;240 U L⁻\u0026sup1;) and surpassing reported productivity in \u003cem\u003eCupriavidus necator\u003c/em\u003e H16\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA potential concern associated with global reprogramming of metal homeostasis is unintended metabolic burden or impaired cellular fitness. Growth profiling (Supplementary \u003cb\u003eFig. S5\u003c/b\u003e) showed that plasmid-based overexpression of the \u003cem\u003eisc\u003c/em\u003e operon moderately reduced maximal cell density, consistent with increased metabolic load. In contrast, genome-level reinforcement of Fe\u0026ndash;S biosynthesis in BL2 restored and further improved growth relative to the plasmid-based strain, indicating that chromosomal integration enables more balanced expression of maturation machinery. Importantly, no severe growth defects were observed under production conditions, suggesting that coordinated reconstruction of tungsten transport, cofactor assembly, and Fe\u0026ndash;S biogenesis is physiologically tolerated. The improved growth of BL2 likely contributes to its enhanced volumetric productivity by supporting greater biomass accumulation alongside increased intracellular abundance of fully active enzymes.\u003c/p\u003e \u003cp\u003eElemental analysis confirmed functional improvements by revealing substantially enhanced metal content in MeFDH1 purified from the BL2 strain (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Iron occupancy increased from ~\u0026thinsp;5 mol Fe mol⁻\u0026sup1; enzyme in BL1 to ~\u0026thinsp;11 mol in BL2, closely matching the level observed for enzyme purified from native AM1. Although structural annotation predicts\u0026thinsp;~\u0026thinsp;20 mol Fe mol⁻\u0026sup1; enzyme, both native and engineered preparations exhibited lower measured values, suggesting incomplete biogenesis or oxidative loss during aerobic purification step. Notably, MeFDH1 purified from \u003cem\u003eE. coli\u003c/em\u003e BL2 displayed 92% tungsten occupancy, substantially higher than the 50% observed in native \u003cem\u003eM. extorquens\u003c/em\u003e AM1 host. These coordinated increases in iron and tungsten incorporation are consistent with improved Fe\u0026ndash;S cluster biogenesis in the engineered strain and support a functional link between Fe\u0026ndash;S assembly and efficient W-bis-MGD maturation.\u003c/p\u003e \u003cp\u003eTogether, these findings indicate that bis-MGD cofactor synthesis remains tightly coupled to cellular Fe\u0026ndash;S assembly capacity, even when molybdenum is replaced by tungsten. Successful production of fully metalated tungsten enzymes therefore requires not only reconstruction of tungsten-selective transport and cofactor biosynthesis, but also sufficient Fe\u0026ndash;S cluster assembly to sustain both catalytic function and cofactor maturation. These results identify Fe\u0026ndash;S supply as a critical remaining constraint and directly link Fe\u0026ndash;S capacity to efficient W-bis-MGD maturation.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreservation of MeFDH1 catalytic competence in the engineered\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003echassis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether the engineered chassis preserves intrinsic catalytic properties, kinetic parameters for CO₂ reduction were measured using purified MeFDH1 from engineered \u003cem\u003eE. coli\u003c/em\u003e BL2 and the native \u003cem\u003eM. extorquens\u003c/em\u003e AM1 host (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Under CO₂-dependent conditions, turnover numbers (kcat) were comparable between BL2- and AM1-derived enzymes (141 s⁻\u0026sup1; vs 152 s⁻\u0026sup1;), indicating preserved catalytic turnover following heterologous maturation. Although the apparent K\u003csub\u003eM\u003c/sub\u003e for CO\u003csub\u003e2\u003c/sub\u003e was moderately increased in BL2 (8.4 mM vs 3.6 mM), overall catalytic competence remained within the same order of magnitude, consistent with a modest decrease in substrate affinity rather than impaired catalytic chemistry. Similar trends were observed for EV-dependent measurements, where kcat values were largely maintained between preparations (304 s⁻\u0026sup1; vs 325 s⁻\u0026sup1;), with modestly elevated K\u003csub\u003eM\u003c/sub\u003e values resulted in reduced catalytic efficiency in BL2. These data indicate that tungsten-specific cofactor reconstruction and enhanced Fe\u0026ndash;S maturation in \u003cem\u003eE. coli\u003c/em\u003e support formation of a catalytically competent enzyme without compromising turnover chemistry of MeFDH1.\u003c/p\u003e\n\u003ch3\u003eWhole-cell CO-to-formate conversion demonstrates system-level impact\u003c/h3\u003e\n\u003cp\u003eWe next examined whether enhanced tungsten maturation translates into improved carbon conversion at the whole-cell level. A coupled CO-to-formate system was constructed in which \u003cem\u003eE. coli\u003c/em\u003e expressing ChCODH (carbon monoxide dehydrogenase from \u003cem\u003eCarboxydothermus hydrogenoformans\u003c/em\u003e) to oxidizes CO to CO\u003csub\u003e2\u003c/sub\u003e and generate reduced ethyl viologen (EV\u0026bull;⁺), which subsequently transfers electrons to MeFDH1-expressing cells to drive CO₂ reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Under matched biomass conditions, MeFDH1 produced in engineered BL2 generated 44 mM formate within 4 h, compared to 13 mM using enzyme derived from the native AM1 host (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), corresponding a\u0026thinsp;~\u0026thinsp;3.5-fold increase in final titer and a markedly higher apparent production rate. Given the comparable intrinsic kinetic parameters of purified enzymes, the improved whole-cell productivity observed with BL2-derived MeFDH1 therefore is attributable to the higher intracellular abundance of tungsten-metalated MeFDH1 in the engineered chassis.\u003c/p\u003e\n\u003ch3\u003eEngineered chassis supports heterologous expression of diverse tungsten-containing enzymes\u003c/h3\u003e\n\u003cp\u003eHaving established efficient tungsten-selective maturation and enhanced catalytic performance with MeFDH1, we next assessed whether the engineered chassis supports functional assembly beyond its original native context. To this end, we expressed CnFDW from \u003cem\u003eC. necator\u003c/em\u003e H16, a characterized W-FDH previously produced in \u003cem\u003eM. extorquens\u003c/em\u003e PA1\u003csup\u003e34\u003c/sup\u003e. In parallel, we identified a putative FDH from \u003cem\u003eLutibaculum baratangense\u003c/em\u003e AMV1 (LbFDH) through Foldseek-based structure homology analysis\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, which exhibits high structural similarity to characterized W-FDHs despite limited sequence identity (Supplementary \u003cb\u003eFig. S6\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAs summarized in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the engineered BL2 chassis enabled functional expression of heterologous W-bis-MGD enzymes beyond MeFDH1. Expression of CnFDW in wild-type \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) yielded no detectable activity, consistent with the absence of tungsten cofactor biosynthetic capacity. In contrast, CnFDW produced in BL2 exhibited substantial tungsten incorporation (~\u0026thinsp;0.9 mol W per mol enzyme), confirming effective W-bis-MGD biosynthesis and insertion in the engineered host. Although full theoretical occupancy was not achieved, the high tungsten loading was sufficient to support maximal catalytic activity (~\u0026thinsp;30 U mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), comparable to values reported for expression in \u003cem\u003eM. extorquens\u003c/em\u003e PA1\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA similar pattern was observed for the structure-predicted LbFDH, which was inactive in BL21(DE3) but exhibited measurable activity accompanied by stoichiometric tungsten incorporation when expressed in BL2 (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The measured tungsten content (1.17 mol W per mol enzyme) slightly exceeded the theoretical maximum (1.0), likely reflecting minor uncertainties in protein quantification or normalization for this previously uncharacterized enzyme. Nevertheless, the high tungsten occupancy confirms successful W-bis-MGD assembly and insertion into a structurally predicted enzyme, demonstrating that the reconstructed metal-cofactor network supports proper metalation beyond well-studied FDHs. The comparatively lower activity of LbFDH likely reflects intrinsic structural features, differences in native electron-transfer partners, or suboptimal folding in the heterologous host, rather than incomplete cofactor assembly.\u003c/p\u003e \u003cp\u003eThese results indicate that the engineered \u003cem\u003eE. coli\u003c/em\u003e BL2 chassis is capable of efficiently maturing multiple W-FDHs under aerobic conditions, including both previously characterized proteins and structure-guided candidates. The transferable activation and high tungsten occupancy observed across distinct enzyme backgrounds suggest that the principal barrier to heterologous expression lies in host metal homeostasis rather than intrinsic enzyme incompatibility.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe successful heterologous expression of MeFDH1 in \u003cem\u003eEscherichia coli\u003c/em\u003e reported in this study addresses persistent challenges in the production of tungsten-containing metalloenzymes and demonstrates that tungsten-dependent enzyme maturation is not intrinsically incompatible with this host. Instead, functional expression is limited by the absence of an appropriately configured metal\u0026ndash;cofactor environment. Our findings show that protein overexpression alone is insufficient when cofactor biosynthesis, metal selectivity, and auxiliary maturation pathways are not aligned with catalytic requirements. By integrating W-bis-MGD biosynthesis, tungsten-specific transport, and reinforcing Fe\u0026ndash;S cluster assembly within a coordinated framework, we enabled aerobic maturation of non-native tungsten-dependent enzymes in \u003cem\u003eE. coli\u003c/em\u003e through genome-level rewiring of trace-metal allocation.\u003c/p\u003e \u003cp\u003eSystematic reconstruction of the W-bis-MGD biosynthetic machinery revealed that functional activation required cooperative action of MoeA1, MobA, MobB, and FdhD, consistent with an integrated maturation network rather than a linear insertion sequence. The progressive restoration of activity and corresponding increases in tungsten occupancy identify cofactor availability and maturation efficiency as principal bottlenecks in heterologous expression of tungsten-dependent oxidoreductases. In this context, MoeA1 and MobB promote early cofactor formation, MobA enhances bis-MGD synthesis to elevate catalytic competence, and FdhD contributes to completion of the mature tungsten cofactor; these roles echo the cooperative biosynthetic roles described for molybdenum cofactor assembly in bacteria\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. These observations underscore W-bis-MGD cofactor biogenesis as a coordinated system rather than a set of isolated enzymatic steps.\u003c/p\u003e \u003cp\u003eMetal discrimination emerged as a central yet often underappreciated challenge in heterologous metalloenzyme engineering. Tungsten and molybdenum, as closely related group 6 oxyanions, compete for overlapping transport and cofactor assembly pathways\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, imposing stringent demands on selective uptake and insertion systems. To address this constraint, chromosomal integration of the high-affinity tungstate transporter from \u003cem\u003eSulfitobacter dubius\u003c/em\u003e NA4 (TupBCA) substantially increased tungsten incorporation and catalytic activity. Notably, MeFDH1 purified from the engineered \u003cem\u003eE. coli\u003c/em\u003e BL2 strain exhibited nearly double the tungsten occupancy of enzyme isolated from its native host, indicating that synthetic control of metal uptake can rebalance endogenous W/Mo homeostatic trade-offs.\u003c/p\u003e \u003cp\u003eThe pronounced activity loss under mixed W/Mo supplementation highlights the inherent sensitivity of W-bis-MGD maturation to W/Mo competition. Because molybdenum is not appreciably incorporated into the mature enzyme, interference likely occurs upstream of final cofactor insertion rather than through direct active-site substitution. Such cross-talk may involve competitive binding to the TupA periplasmic binding protein, limiting tungstate influx despite its higher intrinsic affinity for tungsten\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, or nonproductive engagement of the heterologous MoeA1 insertion machinery during early W-bis-MGD assembly, thereby impairing maturation of nascent cofactor intermediates\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Together, these findings position selective metal allocation and early pathway specificity as engineerable interfaces between trace-element physiology and synthetic chassis engineering.\u003c/p\u003e \u003cp\u003eIn parallel, Fe\u0026ndash;S cluster biogenesis represented another systems-level constraint on tungsten enzyme maturation. Because W-FDHs rely on multiple Fe\u0026ndash;S clusters for structural integrity and electron transfer, their assembly is embedded within broader redox and metal-trafficking networks. Reinforcement of the \u003cem\u003eisc\u003c/em\u003e operon relieved this bottleneck, restoring activity to native-comparable levels and enabling volumetric productivity surpassing alternative production systems\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, while maintaining cellular fitness. Given that Fe\u0026ndash;S cluster assembly also contributes to early stages of bis-MGD cofactor biosynthesis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, these improvements likely reflect coordinated enhancement of both electron-transfer capacity and cofactor maturation efficiency. These findings highlight that efficient production of tungsten-dependent enzymes is governed not by protein expression alone, but by integrated host metal selectivity and cofactor metabolism.\u003c/p\u003e \u003cp\u003eImportantly, reconstruction of tungsten-specific metal metabolism in \u003cem\u003eE. coli\u003c/em\u003e preserved the intrinsic catalytic properties of MeFDH1. Although modest differences in apparent substrate affinity were observed, turnover rates between enzymes derived from the engineered \u003cem\u003eE. coli\u003c/em\u003e BL2 strain and the native \u003cem\u003eM. extorquens\u003c/em\u003e AM1 host remained unchanged, suggesting intact cofactor insertion and structure. Instead, they likely arise from subtle differences in local microenvironment or mediator interactions. The enhanced whole-cell CO-to-formate conversion is therefore best explained by improved maturation efficiency and increased intracellular abundance of fully active enzymes rather than altered enzyme chemistry.\u003c/p\u003e \u003cp\u003eThe successful activation of both a canonical W-FDH (CnFDW) and a putative FDH (LbFDH) further demonstrates that the engineered chassis supports maturation across multiple W-bis-MGD\u0026ndash;dependent enzymes rather than being tailored to a single target. Despite belonging to the same structural superfamily, these enzymes originate from evolutionarily separated bacterial lineages and were functionally restored without enzyme-specific optimization, supporting the conclusion that activation results from reconstruction of host metal metabolism rather than target-specific tuning. While validation across additional tungsten enzyme families will be important, these results establish a transferable capacity for W-bis-MGD\u0026ndash;dependent maturation in an engineered host.\u003c/p\u003e \u003cp\u003eCollectively, this work extends chassis engineering beyond genetic circuit design to the rational reconfiguration of intracellular metal homeostasis. By reconciling host metal metabolism with cofactor-dependent catalysis, we establish a generalizable strategy for activating W-bis-MGD enzymes that are inaccessible in standard laboratory strains. Although demonstrated here with FDHs, the underlying design principles\u0026mdash;coordinated control of metal uptake, cofactor biosynthesis, and auxiliary cluster assembly\u0026mdash;provide a foundation for expanding the functional expression of tungsten-dependent redox enzymes in synthetic hosts, This platform enables aerobic production of multiple W-FDHs and establishes a general design framework for W-bis-MGD maturation, offering new opportunities to enhance biocatalytic carbon conversion and redox biomanufacturing under oxygen-tolerant conditions.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eStrains, growth medium, and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α was used for routine plasmid construction and propagation. \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) served as the parental strain for heterologous protein expression and all subsequent chassis engineering. All strains and plasmids used in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Seed cultures were initiated in Luria\u0026ndash;Bertani (LB) medium (10 g L⁻\u0026sup1; tryptone, 5 g L⁻\u0026sup1; yeast extract, 10 g L⁻\u0026sup1; NaCl) supplemented, where appropriate, with carbenicillin (100 \u0026micro;g mL⁻\u0026sup1;) or spectinomycin (100 \u0026micro;g mL⁻\u0026sup1;). For protein expression, cultures were transferred to Terrific Broth (TB; 12 g L⁻\u0026sup1; tryptone, 24 g L⁻\u0026sup1; yeast extract, 9.4 g L⁻\u0026sup1; K₂HPO₄, 2.2 g L⁻\u0026sup1; KH₂PO₄) with appropriate antibiotics. Sodium tungstate (Na₂WO₄) was added to a final concentration of 1 mM, and FeSO₄ (1 mM) was included where indicated.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmid construction\u003c/h3\u003e\n\u003cp\u003eAll heterologous genes were codon-optimized for \u003cem\u003eE. coli\u003c/em\u003e expression and synthesized commercially by Bionics (Seoul, Republic of Korea). The identity and putative function of each heterologous gene used in this study are summarized in Supplementary \u003cb\u003eTable S3\u003c/b\u003e. Gene fragments were assembled into the indicated plasmid backbones using Gibson Assembly Master Mix (New England Biolabs, MA, USA) following the manufacturer\u0026rsquo;s instructions. All constructs were propagated in \u003cem\u003eE. coli\u003c/em\u003e DH5α and verified by colony PCR and Sanger sequencing prior to use.\u003c/p\u003e \u003cp\u003eMeFDH1 expression vectors were constructed by assembling \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e AM1 genes \u003cem\u003efdh1A\u003c/em\u003e and \u003cem\u003efdh1B\u003c/em\u003e into the pETDuet-1 backbone under separate T7 promoters. Expression vectors for \u003cem\u003eCupriavidus necator\u003c/em\u003e H16 \u003cem\u003efdwAB\u003c/em\u003e and \u003cem\u003eLutibaculum baratangense\u003c/em\u003e AMV1 \u003cem\u003efdhAB\u003c/em\u003e were constructed similarly in the pETDuet-1 backbone. Genes involved in W-bis-MGD biosynthesis (\u003cem\u003emoeA1\u003c/em\u003e, \u003cem\u003emobB\u003c/em\u003e, \u003cem\u003emobA\u003c/em\u003e, and \u003cem\u003efdhD\u003c/em\u003e) were assembled individually or in defined combinations into pCDFDuet-1 under T7 promoters to enable modular co-expression.\u003c/p\u003e \u003cp\u003eFor comparative analysis of tungstate uptake systems, the \u003cem\u003emodABC\u003c/em\u003e operon from \u003cem\u003eE. coli\u003c/em\u003e K-12 was amplified from genomic DNA using Q5\u0026reg; High-Fidelity DNA Polymerase (New England Biolabs, MA, USA), while the \u003cem\u003etupBCA\u003c/em\u003e operons from \u003cem\u003eM. extorquens\u003c/em\u003e AM1 and \u003cem\u003eSulfitobacter dubius\u003c/em\u003e NA4 were codon-optimized and synthesized. All transporter operons were cloned into the pZS vector for plasmid-based expression.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStrain engineering\u003c/h2\u003e \u003cp\u003eAll strain engineering was performed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). For transporter engineering, the \u003cem\u003etupBCA\u003c/em\u003e operon from \u003cem\u003eS. dubius\u003c/em\u003e NA4 was chromosomally integrated using λ-Red recombination. The operon was inserted into the \u003cem\u003eproP\u003c/em\u003e locus using the pKD4/pRedET system, followed by excision of the selection marker with the pCP20 plasmid. The resulting strain carrying chromosomally encoded \u003cem\u003etupBCA\u003c/em\u003e was designated BL1.\u003c/p\u003e \u003cp\u003eTo enhance Fe\u0026ndash;S cluster biosynthesis, \u003cem\u003eiscR\u003c/em\u003e was deleted and the native \u003cem\u003eisc\u003c/em\u003e promoter was replaced with the synthetic P\u003cem\u003etrc\u003c/em\u003e promoter via the two-step inactivation method using the pKOV plasmid \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The final engineered strain incorporating chromosomal \u003cem\u003etupBCA\u003c/em\u003e and upregulated \u003cem\u003eisc\u003c/em\u003e expression was designated BL2 and used as the expression chassis in subsequent protein expression and activity assays. All genomic modifications were validated by colony PCR and verified by Sanger sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression and purification\u003c/h2\u003e \u003cp\u003eRecombinant \u003cem\u003eE. coli\u003c/em\u003e strains were cultivated by inoculating 1:100 dilutions of overnight seed cultures into 200 mL of TB medium in 1 L baffled flasks. Cultures were incubated at 37\u0026deg;C with shaking to mid-exponential phase (OD₆₀₀ \u0026asymp; 0.5\u0026ndash;0.6), and protein expression was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Following induction, cells were incubated at 25\u0026deg;C for 16\u0026ndash;20 h. All cultivation and protein expression experiments were performed under aerobic conditions.\u003c/p\u003e \u003cp\u003eCell lysis and protein purification were also conducted aerobically. Cells were harvested by centrifugation at 7,000 \u0026times; g for 15 min at 4\u0026deg;C and resuspended in BugBuster Protein Extraction Reagent (Millipore Sigma) and lysed according to the manufacturer\u0026rsquo;s protocol. Lysates were clarified by a second centrifugation step at 11,000 \u0026times; g for 30 min at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eRecombinant enzymes were purified from the soluble fraction using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Column was equilibrated and washed with 50 mM MOPS buffer (pH 7.0) containing 250 mM NaCl and 20 mM imidazole, and bound proteins were eluted with 300 mM imidazole in the same buffer. Protein concentrations were determined spectrophotometrically (NanoDrop, Thermo Scientific) using calculated extinction coefficient specific to each enzyme (ε\u0026thinsp;=\u0026thinsp;8.94 L\u0026middot;mg⁻\u0026sup1;\u0026middot;cm⁻\u0026sup1; for MeFDH1, ε\u0026thinsp;=\u0026thinsp;8.07 L\u0026middot;mg⁻\u0026sup1;\u0026middot;cm⁻\u0026sup1; for CnFDW, and ε\u0026thinsp;=\u0026thinsp;9.59 L\u0026middot;mg⁻\u0026sup1;\u0026middot;cm⁻\u0026sup1; for LbFDH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme activity assays\u003c/h2\u003e \u003cp\u003eEnzyme activity was measured spectrophotometrically by monitoring NAD⁺ reduction at 340 nm (ε\u0026thinsp;=\u0026thinsp;6.22 mM⁻\u0026sup1;\u0026middot;cm⁻\u0026sup1;) using a UV\u0026ndash;Vis spectrophotometer under aerobic conditions. Assays were conducted in quartz cuvettes with a total volume of 2.0 mL at 30\u0026deg;C. The standard reaction mixture contained 50 mM MOPS buffer (pH 7.0), 30 mM potassium formate, 0.5 mM NAD⁺, and 10\u0026ndash;20 \u0026micro;g of purified enzyme. One unit (U) of activity was defined as the amount of enzyme catalyzing the formation of 1 \u0026micro;mol NADH per minute under these conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMetal content analysis\u003c/h2\u003e \u003cp\u003eThe tungsten, molybdenum, and iron contents of purified enzymes were quantified using inductively coupled plasma\u0026ndash;mass spectrometry (ICP-MS). Approximately 1 mL of each protein sample (1.0\u0026ndash;2.0 mg mL⁻\u0026sup1;) was subjected to acid digestion in Teflon vessels containing 12 mL of trace metal\u0026ndash;grade nitric acid (HNO₃) and 1 mL of hydrofluoric acid (HF). Digestion was carried out at 200\u0026deg;C for 2\u0026ndash;3 h. After cooling to room temperature, the digested samples were diluted to a final volume of 25 mL with 1% HNO₃. Metal concentrations were measured using a PerkinElmer NexION 5000 ICP-MS calibrated against PerkinElmer multi-element standards 3 and 5. Measured values were normalized to protein concentration and expressed as molar equivalents of metal per αβ heterodimer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eKinetic analysis of MeFDH1 for CO₂ reduction\u003c/h2\u003e \u003cp\u003eCO₂-reduction kinetics were measured anaerobically using reduced ethyl viologen (EV\u0026bull;⁺) as the electron donor, monitored spectrophotometrically at 600 nm (ε\u0026thinsp;=\u0026thinsp;10.22 mM⁻\u0026sup1; cm⁻\u0026sup1;). All assays were conducted inside an anaerobic chamber to prevent the abiotic oxidation of EV\u0026bull;⁺ by oxygen, which would otherwise result in overestimated reaction rates. Reaction mixtures contained phosphate buffer (200 mM, pH 6.3), EV\u0026bull;⁺ (0.015\u0026ndash;0.12 mM), and bicarbonate (3\u0026ndash;100 mM).\u003c/p\u003e \u003cp\u003eSodium bicarbonate served as the CO₂ source and the relative concentrations of dissolved CO₂(aq), bicarbonate (HCO₃⁻), and carbonate (CO₃\u0026sup2;⁻) were calculated from the total inorganic carbon concentration (c) according carbonate equilibrium relationships\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, using dissociation constants pKa\u003csub\u003e₁\u003c/sub\u003e = 6.3 and pKa\u003csub\u003e₂\u003c/sub\u003e = 10.3:\u003c/p\u003e\u003cp\u003e\u003cimg 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Yf/+/d29aUkaIzf6E9KNm4OyWv342+F0c7fTnG7u1sNHcBeLRRaPx5mmaSwSiVSNFwkGg13rs80qN2f5VCLBYFC8J+sxME1TpDejkX3UK1OesMq+kt6iGj8hXTQ0NARN0/DSSy8BlSe6Xb9+Xcy4aFRG+U5PT+PatWu2kbGd9NFHH+HatWsYGxvDlStXoCgKxsbGRDu8z+cTbf7PP/98U+NtttpHfmV08OBBsa+kx+QzASHd7BZo7ZrZzXU3UytvhHVkb6fXTch2c7Fm+q4RQgjZ9aiphxBC+gwFfkII6TMU+AkhpM9Q4CeEkD5DgZ8QQvoMBX5CCOkzFPgJIaTPUOAnhJA+Q4GfEEL6DAV+QgjpMxT4CSGkz1DgJ4SQPkOBnxBC+gwFfkII6TMU+AkhpM9Q4CeEkD5DgZ8QQvoMBX5CCOkzFPgJIaTPUOAnhJA+Q4GfEEL6DAV+QgjpMxT4CSGkz1DgJ4SQPkOBn+x5hmHA5/PJyYT0LQr8hBDSZyjwkx3N5XKJxTAMObst0WhUrDsajcrZhOxZFPjJjpfJZMAYg9frBQAEAoGqk8GlS5eq0nw+H1wuF0ZGRpBKpaqC/OTkJBhjiEQilq39z8DAgCiPSnMR/39gYEAuTsiuQoGf7Dpff/01ACASiYiTwdWrVwEA4XBYpC0uLoIxhkwmA13XwRgDYwyTk5OWtTn7/vvvgcpJBwC8Xi80TYPf70c2m5VKE7K7UOAnPVcqlRCNRkUte6sa9aNHjwAAuq6LtEKhAAB48803RVo7Hj58CFVV4fV6USqVEAqFcOrUKSQSCQwODsrFCdlVKPCTnvP5fPjxxx+RzWZhmiZUVZWL2KysrEBVVQwNDYm01dVVKIoiavvtWl5exujoKAzDwNjYGE6fPt3QlQIhuwEFftJTiUQCpmlicXERg4ODGBwcxNramlzMZnl5GaZpijZ3l8uFDz74AKOjo3JRoNJMs7i4KCfXlU6nMT09jZGREaCyDkL2Cgr8pKdmZ2cRCATgdrvlrJrS6TRisZhos2eMQdM0HD9+XC7aknw+j3K5jFwuh1gshlQq1fEeRYT0EgV+0nMvvvii+LtUKtnyZDwonzhxQqQVCgVks1kcPXrUVrZVKysrUBQFQ0NDmJiYgK7r+OSTT2xleLu/y+WCx+OBx+Ox5ScSCQwPD9MJg+xIXQ/81r7SLperagTl8PCwyLP+eKzd51x1+lkbhmHr3jcwMACfzydu9sny+TwCgYC4kejxeGquuxHW9+iqdP0jzfnll1+ASgD3+Xx1g2UqlQIA2w3W+fl5AMC+fftEWjtu376Nw4cPi/8vX76MbDaLmZkZkTY2NgYAKBaL+PTTT6EoishLJBIdey9kb8jn87bvz1YMw2iqfNNYl0UiEQaAZTIZOYsxxlixWGQAmKqqrFgs2vLi8TgDwPx+vy2dCwaDDACLRCKsWCyyYrHI/H4/A1C1LmZZXzAYZKZpMsYYi8ViYh1Wuq4zADUXGd9P0pxcLsdUVWUAmK7rLJfL2fKt351MJiM+/2AwWJWm67rttY2KRCLi+PPjCIDF4/GqtEwmw3K5HFMURXzHIpEIC4fDtnWyyneo1vee9I94PF4VX2KxmPjeK4oiYphVMpkU3/NO63qkajTwO/1w6r2WB+xYLGZLz2QyTFVVWxpPtwYMK03THF/TDAr83VHr+HeSNfA3IhaL2U4yuq6Lk4QVBX6SyWSqKiQ86PPvHT8BOFVwa1Uq2tX1pp6trK+vAzX6Xy8tLQEOPSoKhQIuXrwIXdcxMTFhy/N6vdjY2LClAcD4+DgURcGVK1fkLLjdbpimKScTUhO/F3Hp0iVsbGxg3759WFhYsOWXSiU8ffrU8irSb8bHx3Hu3Dlb2oMHD7CxsYHJyUlMTk7iP//5D1RVxdzcXFUT9ZkzZzA9PV23+bMVPQ/8d+/edex/XSqVkEqlbIN0uPn5eZTLZVy+fFnOcsS7DF64cKGp3iNkZxgZGYGri3P1nD9/Xs6q68SJEzBNEx6PB++//z40TcP4+Dj+/Oc/A5X1Pv/888hms3jrrbfo3k+f4nHn5MmTtnQ+ypxzu934+OOPAQB//PFHVd7o6Ci++uorW3rb5EuATqvXXMMaaEt3usxRVbWpphne7s/b9WX8PbSDmnoIIVaaplU189TCm3yc8NhSK361ouc1/lQqhUgkYuuTbZ0464033rCVz+fzME2z5mAdJ+l0Gqqq1hxqn0qloGmanNwVcm+lrZZO13IJIdsjm83iueeek5MdLS0t4caNG3IyAGD//v0AgF9//VXOallPA38+nwcAvPrqq3KW6OJ35MgRWzq/J2Adrl9PoVBAuVyuGdh5u2wzJ5J2eL3eqpNcvUVuArOSTxK00NLNhTSOV9gOHTokZ1UxDAODg4NVTUIc7xp87949OatlPQ38KysrAICXX35ZzkI2m4WmaVVt8g8ePAAAvPLKK7b0Wh4/fgzUOQB37twBALz//vtA5UQRCoXg8XjgqvTz72p/2jbIJwlaaOnmQjqvVCrh7t27mJqakrO6qqeBf3l5GYqiVDXBFAoFmKaJ4eFhW3qnFQoFTE9PQ9d1cQXx+PFjrK2tIZ1OgzEGv9+Ps2fPVt1tbxU19RBCUAn68/PzPZn8r6eBP51O20ZIcrwt69ixY3KWaBbiU/NypVIJHo9HNB9xBw4cAADcv3/flg4AoVAIiqLYzrZerxdra2viZMTvMTz77LOiTDs62dRDCNmZ+O+Wd0mX8aAvd0d3wrsE87b+TuhZ4F9YWEC5XHacm4Xf5Hjy5ImchSNHjkBVVXz55ZeiFp7P5+Hz+bC5uVnV9u92uxEOh219YfnUAKurq1heXq664uAWFhZw48YNJJPJqiYnQgipR9d1x/hWKBQwNjaGJ0+eIBqNiuXSpUsIhUJycdG271RJbpnczafTnLpzWofZQ5ougZfni1M30FwuJ7po8kXX9apRvFaRSIQpisJQGSJtnbbBiXW9Tu9Bttu7cza6nzuN08hIQnYCPkWMlWmaIg45Lclk0laeVbqjd/o73vVI5RT4d4tisSjmA6p3kmA9DPzySbRVtQK/07qTySTTNI3BMs9IK6zrdtp2I2oFfmsFotX3R0i7VFV1DObNUBSl5d9HLT1r6tmpCoWCaEJyu91iigfeO2in4vcEOPlh4ZBmQm2UvF5ULj1HR0fBGMMXX3yB69ev2/KtfD5f3dlPW32Q+lZ2woPU673fmZkZsU3++fCHw7tcrrZ7km3XPpL6bt26hc8++0xObtjMzAyCwWDn7/XJZ4JO2201/kgkwjRNs828iF1Q45clk8mqz52X5TUQ+WrBusic0lhlv50mKON0Xa9Z45bfH7NM2ie/BtIo7lojvuXXRRwmYHP6bDRNY36/f8vj3Ax5GzJ5ZGcymWSKotT9PBu1XftIthaPxx0nh9xKLpdznLmgE7atxs/nW5Hn499pdF3H8PAwXnrpJbhcLvz4449IJpM1bwDzWlSz871028OHD6vmQOI9ofhAEWsPI13XRe1bruHXEgqFsH//fgQCATmrZY0+SH1xcRGMMWQyGei6Lt53I13jdsqD1E3TxPj4OFCpkd+4cQPLy8sd+Tx3yj6S/13FTkxMIJFIyFk15fN5rK+vV83r0yldD/z8kpsvzT77dLsNDQ1hamoKm5ubYIxhbW2t5og6/K8abFt2CqcA8u9//xt+v9+W1iqfz4eJiQkEAgHHngit6pcHqfMniR04cADRaBQ//PADbt68WdUrrVU7YR/J/xkaGqr6PdbTbPmmyZcAZHep1dRTq+dArZ5PtW7uctZtWG+c8kUuW2uxNm3AoSlE7q3FF6e5ylmdm7ucU1OP9bOp99pm1Ws6g9QMxZ8nwRf5c2hXt/aR7A3VEYPsKk6BP5fLMQC2p1nxtnP5CVeNkrfRqGbb+BVFqTo5aZpWldYoOfBbPxsefOX3YO3N5TQTbDweZ5qmVb1O5rRuzu/3M7/fz4rFIlNV1TE4J5NJ29PJrPthmiYLBoOOJ8R297FTvbbIztX1ph6y/ZyaS37++WfxAPGdaqc8SH07nqebTqdx/PhxuN1uzM7OIpVK2R7kYhgGPvzwQ8zOzqJYLGJjY0OMWuejPo8dO4bff//dstb/aXcfm+m1RXYnCvx70O3bt20PrgeA2dlZqKpqS9tpdsKD1PP5PFZXV3HlyhW43W48efLENnNrIBCA1+ttayS3YRgol8tiokGv1wtd13Hu3Dkx0vOrr77ChQsXxLZM0xQz1brdbkxOTtb8TNrdx8nJSXFT8cmTJ9s+gRjpPgr8e0w0GkUqlUIqlRK9CHhaNput26e+lwzDED2j+M1iaxqvobbD6bO5e/cuAODs2bMwDAMrKys4fPiwCOxLS0t47bXXbOtp18jICFB5LB+XSqVgmqbYz3Q6jddffx2ofA6qqjZ0sunkPnaj1xbZIeS2H7K7OLXxd0M3tuHU9txpchv/VmKxGNM0jTHGWDgcZqqqskwmYxt9WSwWmaZpbY/IrEdRFJZMJlkul2O6rjNd11kul7P1wef3GprVyD7y7THGWuqDTna2zv+aybaiwF9fs4Gfz6WiqqqYE4r/zRro0dQp4XCYoTJojd+stQ7mkd/DbtxH0jsutpM6n5OmGYYhmg7wv1+oLb9dfMg/urzuTCbTsX76qDR58GaiSCRCfdgJsaDATwghfYZu7hJCSJ+hwE8IIX2GAj8hhPQZCvyEENJnKPATQkifocBPCCF9hgI/IYT0GQr8hBDSZyjwE0JIn6HATwghfYYCPyGE9BkK/IQQ0mco8BNCSJ+hwE8IIX2GAj8hhPQZCvyEENJnKPATQkifocBPCCF9hgI/IYT0GQr8hBDSZyjwE0JIn6HATwghfYYCPyGE9BkK/IQQ0mco8JMdJxAIwOVyweVywTAMABD/u1wu5PN5+SVNGRgYEOsCAMMwxP8DAwNycUL2HAr8ZMf5+uuvAQCRSARerxcAEA6HoWkaMpkMhoaGpFc05/vvvwcAZDIZAIDX64WmafD7/chms1JpQvYeCvykJwqFAkKhkKhpBwIBkffo0SMAwHvvvQcAmJmZgWmaWFxcFCeCdjx8+BCqqsLr9aJUKiEUCuHUqVNIJBIYHByUixOy51DgJ9uuUChA0zQAQLFYRDweh6qqIn9lZUX8z08IiUQCbrdblGnH8vIyRkdHYRgGxsbGcPr0aUxOTsrFCNmzXIwxJifuJfl8HjMzM0gkEiiXy9B1HVNTU1Sz66FQKIRCoYDFxUU5C6gE+7m5OQCAoij47bffOhb0UWnjL5fLAABd12u+D0L2qj1f4z9//jyGhoawubmJXC6H1dVV/P3vf5eLkW00PT2N8fFxOVlIp9OIxWLI5XIol8v49ttv5SIty+fzKJfLyOVyiMViSKVS4gYyIf1izwf+xcVFTExMAACGhoZw+PBhbG5uysXINtu3b5/4u1Qqib95YD569CiGhoYQiURw/fp1FAoFUQYAFhYW4PF44HK54PP5EI1GRR6/f2C9b8CtrKxAURQMDQ1hYmICuq7jk08+sZXh7f4ulwsejwcej0fkRaNRsV2Px4OFhQXbawnZDboe+KPRqK0rns/nk4sAlS511m58AwMD8Pl8VT94Lp/PIxAIiK55Ho/H9uN3UiqVsLq6io8++kjO2pJ1H1yVboCkdf/973+BynE/cuSICP6pVAqonKQB4MyZM0ClVw9nGAY+/PBDzM7OolgsYmNjA6+++ipQOcbz8/M4duwYfv/9d/Ea7vbt2zh8+LD4//Lly8hms5iZmRFpY2NjQOX+w6effgpFUUTe0tISbty4AcYYNE3Dd999J/IIqYU3OTfKMIymyjeNdVkkEmEAWCaTkbOEYDDIALBIJMKKxSIrFovM7/czAKxYLMrFWTweZwBYMBhkpmkyxhiLxWJiHbUEg0EWDAZtaQBqLrqu28oyy/6Q1sXjcaYoijiG/BhnMhnx2fPjZE3jx9bv99uOs9P3JJPJVB0/fuwAsHg8XpWWyWRYLpdjiqKI9UUiERYOh23r4fx+P8vlcnIyITbxeLwqLmUyGaZpGgPAFEWpymeMsWQyWRWvOqXrEWyrwM8DdiwWs6VnMhmmqqotjadbA4OVpmmOr2E1gn4rKPD3nqIo4vtU73siB/5GxGIx2+t0XRcnCa5YLDJd1ynoky05fQ9zuRwLBoMsmUyyZDLJdF23VUas6lU82tH1pp56CoUCLl68CF3XRTs85/V6sbGxYUsDgPHxcSiKgitXrshZcLvdME1TTkYoFAIATE1NyVlkl3r69Cny+Tw+//xzeDwe5PN5W7Pgo0ePbPcOmsFfd+nSJWxsbGDfvn2iLb9QKGBsbAw3b97E0NCQ+G4R4mR8fBznzp2zpa2vr2NqagonT57EyZMncfPmTQDAgwcPbOVQaeqcnp7ufAcE+UzQafVq/PXynPAmHqfLIlapncm7FI/HmaZp4n/ejNQqqvH3XjgcZgBYOBxmuVxO/M3xphu+1Pq+ODFNkymKwlRVZblcjvn9fvE3s3zH+CLX5gjheLzaimmaDEDNK0i/399WzHKy9btqU73grqqq42V6Lbzdn7fry5wCP29L7tSPlQI/IaQRmqbVjTXFYlFUTJ2aeTgec2rFvVb0rKknn8/DNE2Mjo7KWTWl02moqlpz8FUqlRIjQrnNzU1UTnBi2Y4BO9aJvxpZOn4pRwjpqWw2i+eee05OFsbGxvDBBx8gm81idna2Zg/G/fv3AwB+/fVXOatlPQv86+vrgKXb3lYKhQLK5XJVYOd4G2wzJ5Ju8nq9VSeceku9OWjkkwQttHRzIe3jFblDhw7JWcLi4iJM00QwGEQqlYLf75eLAJYxL/fu3ZOzWtazwM9vZLzyyitylqPHjx8DdT7IO3fuAADef/99wDKIxzrYpqv9YrtIPknQQks3F7J9BgcHMTU1hWAwiGw22/aU443qWeDvpEKhgOnpaei6Lq4gHj9+jLW1NaTTaTDG4Pf7cfbs2ZqXU51GTT2EkEadPn0aAPDMM8/IWV3Rs8DPR1ryKXi5UqkkuudZHThwAABw//59Wzoq3TUVRbF11/R6vVhbWxP3A9544w0AwLPPPivKdFMnm3oIIbsL/z0vLS3JWY6ePn1a8/7l06dPAUtbfyf0LPAfOXIEqqriyy+/FLXwfD4Pn8+Hzc3NqrZ/t9uNcDhs69NaKBTg8/mwurqK5eVlxw8Nlfb/GzduIJlMdnSWR0IIqUXXdcexJIFAAKFQSFRuDcPAuXPncOvWLbkoYGnbt0410q6eBX63241bt25BVVWoqgqXy4WDBw/C7Xbjiy++kIsDAK5evYpIJIK//vWvcLlc0DQNg4ODyGazVScKzuVy4a233gIA/OlPf5Kz+47P59uVzUqGYdSc54mQnWh8fNzxiW6qqmJ6ehoHDx6Ex+PBDz/8gHQ6XTOG/fLLL9B1vWbFtiVy/85Oq9ePf7sUi0UxH1C7fWF71Y/fOmdNO9vXdd3xWDitO5lMbjmfSCOs63badiOchr4zaUBVq+smpFtUVWXJZFJObop1ipJO6VmNv9sKhYJoQnK73WKKB947aLeSe17IDw4HgOHh4aq0rcjrReUSc3R0FIwxfPHFF7h+/bot30qeGlmWyWRs9zKcHqh+6dKlqrStLC4ugjEGXddt6fl8XqyLXylYZ4p1mrK5VfXer9N+8v9d9OD4Pe/WrVv47LPP5OSGzczMIBgMdv4eoHwm6LRe1fgjkQjTNM02yyL2QI1flkwmqz5fXpbXNOSrBesic0pjlf2uN7pQ1/WaVwTy+2OVqzA4TKcAafoFeYoEvsivc7qSCYfDtpk2rduUZ/Nsh9P+cU77GQ6HmaZpNV/TDKfjr2ka8/v9bX/XSWfE4/GWJojM5XJdmaCNbWeNf2RkxFb76jZd1zE8PIyXXnoJLpcLP/74I5LJZMvtZLwWdf78eTmrpx4+fAhFUWw1At5T6uTJk4DUw0jXdVH7lmv4tYRCIezfv7+jtWT+Hq01dX6F9uabb4o0XqPPZDLQdV2870aekWuaJgKBANxut5hcLZlMYnJycttu8tOD40kgEMDExAQSiYScVVM+n8f6+jquXr0qZ3VE1wP/5OSk+LGybZouAZURwVNTU2LKhrW1NREIW2Hdh0YD5nZYXl6uCsj//ve/a44CbJbP58PExIToidAp/IHq1htaq6urVSexdqTTaRw7dgyJRAKhUEjMiLid6MHxBJV4JP9O62m2fNPkSwCyM9Vq6nGahA4OzzfgnJpErKzb4M1a1kUuW2ux3oiFQ1MIn3BPXmrNQpipcXOXk/eLz9rJF7lpqB31ms7kbVn309rs1CnW41/v8yHEqjqSkB3JKfDz4GadzpW3Kdea4nUr8jYa1Wwbv6IoVScnTdOq0holB/5YLMYURWGsEnydgq61t5fTTLF85kT5vcuc9o/j+8mPldNnlEwmmaqqInhby5imyYLBoOMJ0Xr8+QON5PdRbx8jkYjYbid6n5Ddo+tNPaR7nJpLfv75Z/Ew8Z2KP1D9xIkTIq1QKCCbzeLo0aO2sq3iTSAA8PXXXwMA/vGPf9jK1Hu2biKRsD0QvhWNPDjeaOP5wY08OL7ePtLzg/sXBf5d7Pbt2/B4PLa02dlZ0aa8U/EHqltvPs7PzwOWmQjbUSqVkE6nxYR+brcbFy5cwLVr10T3yXw+j9XVVVy5cgVutxtPnjyxzewaCATg9Xrbaotv5MHxX331FS5cuCC2ZZomjhw5AlTe9+TkZM3PZKsHx2+1j4uLi7Z7HhcvXhR/k72NAv8uFY1GkUqlkEqlRG8BnpbNZuv2qe8lwzBEzyh+s9iaxmuo7RgbG0O5XMb58+dF7ZrPmXLw4EGgUls+fPiwCOxLS0t47bXXLGtpj9N+rq+vo1wuY25uThyfdDqN119/XbxGVdWGTjZOx//u3bsAgLNnz8IwjIb2sVQqwefz4eLFizv6KpF0mNz2Q3Ympzb+bujGNpzanjtNbuPfSiwWE4/kDIfDTFVVlslkbO3cxWKRaZrW1bZvRVFYMplkuVyO6brO9MpD3K198OXHhzZqq300TZPpui7ufbTS15zsTp3/lZOuoMBfX7OBf6tn627Vo6lT6PnBpBdcbCd1Sic1GYaBkZER8X+nDxsf8o8urzuTyXSsnz4q4wx4W3qn103IXkWBnxBC+gzd3CWEkD5DgZ8QQvoMBX5CCOkzFPgJIaTPUOAnhJA+Q4GfEEL6DAV+QgjpMxT4CSGkz1DgJ4SQPvP/Aadh3XwqGkj9AAAAAElFTkSuQmCC\" width=\"382\" height=\"178\"\u003e\u003c/p\u003e\u003cp\u003eInitial rates (v₀) were determined from the linear portion of absorbance traces. Kinetic parameters (kcat, K\u003csub\u003eM\u003c/sub\u003e, and kcat/K\u003csub\u003eM\u003c/sub\u003e) were obtained by nonlinear regression of v₀ versus substrate concentration to the Michaelis\u0026ndash;Menten equation using OriginPro. Representative kinetic curves and model fits are shown in Supplementary \u003cb\u003eFig. S7\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eWhole-cell CO-to-formate conversion\u003c/h2\u003e \u003cp\u003eWhole-cell formate production was evaluated using MeFDH1 expressed in either \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eM. extorquens\u003c/em\u003e AM1. The biocatalytic system consisted of two components: (i) an \u003cem\u003eE. coli\u003c/em\u003e strain expressing carbon monoxide dehydrogenase from \u003cem\u003eCarboxydothermus hydrogenoformans\u003c/em\u003e (ChCODH) and (ii) either \u003cem\u003eE. coli\u003c/em\u003e expressing MeFDH1 or \u003cem\u003eM. extorquens\u003c/em\u003e AM1 cells expressing MeFDH1. The two cell populations were combined at a fixed CODH-to-FDH activity ratio of 1:10 based on cell dry weight equivalents.\u003c/p\u003e \u003cp\u003eReactions were performed under anaerobic conditions in 10-mL serum vials continuously sparged with CO/CO₂ gas mixture (1:1, v/v) at 3 vvm. The reaction mixture was maintained at pH 6.5, and ethyl viologen (EV) was added to a final concentration of 10 mM as an electron mediator. Whole-cell biotransformations were conducted for 4 h at 30\u0026deg;C with continuous agitation to ensure homogeneous gas\u0026ndash;liquid transfer.\u003c/p\u003e \u003cp\u003eFormate concentrations were quantified by high-performance liquid chromatography (HPLC) using an Aminex HPX-87H column (Bio-Rad) operated at 35\u0026deg;C, with 5 mM H₂SO₄ at a flow rate of 0.6 mL min⁻\u0026sup1; and detection using a refractive index (RI) detector. Calibration curves were generated using authentic sodium formate standards over a concentration range of 0\u0026ndash;500 mM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis and reproducibility\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using OriginPro. All experiments were conducted with at least three independent replicates unless otherwise stated. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (s.d.), as indicated in the figure legends. Statistical significance was evaluated using an unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, and \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Nonlinear regression for kinetic parameters (kcat and K\u003csub\u003eM\u003c/sub\u003e) was performed by unweighted least-squares fitting to the Michaelis\u0026ndash;Menten equation. No statistical methods were used to predetermine sample size. No data were excluded from the analysis. Experiments were not randomized, and investigators were not blinded to group allocation during experiments or outcome assessments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available within the Article and its Supplementary Information files and source data are provided with this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT \u0026amp; Future Planning (RS-2020-NR049543, RS-2024-00396026, RS-2024-00466474, RS-2024-00453085, RS-2025-16070008). We thank UNIST Central Research Facilities for assistance with ICP-MS measurements and access to instrumentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eU. T.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eP.\u003c/strong\u003e: Conceptualization, validation, formal analysis, investigation, writing \u0026ndash; original draft, writing \u0026ndash; review, and editing. \u003cstrong\u003eJ.-M. L.\u003c/strong\u003e: Conceptualization, validation, formal analysis, investigation, writing \u0026ndash; original draft, writing \u0026ndash; review, and editing. \u003cstrong\u003eT. M. V.\u003c/strong\u003e: formal analysis, investigation. \u003cstrong\u003eM.-K. O.\u003c/strong\u003e: Supervision, project administration, formal analysis, writing \u0026ndash; original draft, writing \u0026ndash; review, and editing. \u003cstrong\u003eY. H. K.\u003c/strong\u003e: Supervision, project administration, formal analysis, writing \u0026ndash; original draft, writing \u0026ndash; review, and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBirdja YY et al (2019) Advances and challenges in understanding the electrocatalytic conversion of carbon dioxide to fuels. Nat Energy 4:732\u0026ndash;745\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLai W, Qiao Y, Zhang J, Lin Z, Huang H (2022) Design strategies for markedly enhancing energy efficiency in the electrocatalytic CO2 reduction reaction. Energy Environ Sci 15:3603\u0026ndash;3629\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSordakis K et al (2018) Homogeneous catalysis for sustainable hydrogen storage in formic acid and alcohols. Chem Rev 118:372\u0026ndash;433\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBar-Even A (2016) Formate assimilation: the metabolic architecture of natural and synthetic pathways. Biochemistry 55:3851\u0026ndash;3863\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Liu J, Ong J, Li SF (2016) Specific and sustainable bioelectro-reduction of carbon dioxide to formate on a novel enzymatic cathode. Chemosphere 162:228\u0026ndash;234\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlissandratos A, Kim HK, Easton CJ (2014) Formate production through carbon dioxide hydrogenation with recombinant whole cell biocatalysts. Bioresour Technol 164:7\u0026ndash;11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiks D, Hille R (2019) Molybdenum- and tungsten-containing formate dehydrogenases and formylmethanofuran dehydrogenases: structure, mechanism, and cofactor insertion. Protein Sci 28:111\u0026ndash;122\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaukel M, Chistoserdova L, Lidstrom ME, Vorholt JA (2003) The tungsten-containing formate dehydrogenase from \u003cem\u003eMethylobacterium extorquens\u003c/em\u003e AM1: purification and properties. Eur J Biochem 270:325\u0026ndash;333\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeelmann CS, Willistein M, Heider J, Boll M (2020) Tungstoenzymes: occurrence, catalytic diversity and cofactor synthesis. Inorganics 8:44\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMajumdar A (2014) Structural and functional models in molybdenum and tungsten bioinorganic chemistry: description of selected model complexes, present scenario and possible future scopes. Dalton Trans 43:8990\u0026ndash;9003\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHille R, Hall J, Basu P (2014) The mononuclear molybdenum enzymes. Chem Rev 114:3963\u0026ndash;4038\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBevers LE, Hagedoorn P-L, Hagen WR (2009) The bioinorganic chemistry of tungsten. Coord Chem Rev 253:269\u0026ndash;290\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaaijmakers H et al (2002) Gene sequence and the 1.8 \u0026Aring; crystal structure of the tungsten-containing formate dehydrogenase from \u003cem\u003eDesulfovibrio gigas\u003c/em\u003e. Structure 10:1261\u0026ndash;1272\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchulz M et al (2025) Functional expression of a Mo-dependent formate dehydrogenase in \u003cem\u003eEscherichia coli\u003c/em\u003e under aerobic conditions. PLoS ONE 20:e0334613\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCowan AE et al (2025) Fast growth and high-titer bioproduction from renewable formate via metal-dependent formate dehydrogenase in \u003cem\u003eEscherichia coli\u003c/em\u003e. Nat Commun 16:5908\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi HL et al (2024) Engineered \u003cem\u003eEscherichia coli\u003c/em\u003e whole cell-mediated electro-biocatalysis for carbon dioxide to formic acid conversion. ACS Sustain Chem Eng 12:5544\u0026ndash;5554\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu W et al (2024) Bioelectrocatalytic carbon dioxide reduction by an engineered formate dehydrogenase from \u003cem\u003eThermoanaerobacter kivui\u003c/em\u003e. Nat Commun 15:9962\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzaleniec M, Heider J (2025) Obligately tungsten-dependent enzymes\u0026mdash;catalytic mechanisms, models and applications. Biochemistry 64:2154\u0026ndash;2172\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyu H, Nguyen CNM, Lee SK, Park S (2024) Development of \u003cem\u003eCupriavidus necator\u003c/em\u003e H16 as a host for heterologous production of formate dehydrogenase I of \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e: possibilities and limitations. Bioresour Technol 394:130187\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBevers LE et al (2008) Function of MoaB proteins in the biosynthesis of the molybdenum and tungsten cofactors. Biochemistry 47:949\u0026ndash;956\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeimk\u0026uuml;hler S (2023) Metal-containing formate dehydrogenases, a personal view. Molecules 28:5338\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark J et al (2024) Structure of recombinant formate dehydrogenase from \u003cem\u003eMethylobacterium extorquens\u003c/em\u003e (MeFDH1). Sci Rep 14:3819\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinske C, Bonn M, Kruger S, Lindenstrauss U, Sawers RG (2011) Metabolic deficiencies revealed in the biotechnologically important model bacterium \u003cem\u003eEscherichia coli\u003c/em\u003e BL21(DE3). PLoS ONE 6:e22830\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan MJ (2016) Exploring the proteomic characteristics of the \u003cem\u003eEscherichia coli\u003c/em\u003e B and K-12 strains in different cellular compartments. J Biosci Bioeng 122:1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoimbra C, Branco R, Morais PV (2019) Efficient bioaccumulation of tungsten by \u003cem\u003eEscherichia coli\u003c/em\u003e cells expressing the \u003cem\u003eSulfitobacter dubius\u003c/em\u003e TupBCA system. Syst Appl Microbiol 42:126001\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoimbra C, Farias P, Branco R, Morais PV (2017) Tungsten accumulation by highly tolerant marine hydrothermal \u003cem\u003eSulfitobacter dubius\u003c/em\u003e strains carrying a \u003cem\u003etupBCA\u003c/em\u003e cluster. Syst Appl Microbiol 40:388\u0026ndash;395\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVuilleumier S et al (2009) Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of C1 compounds from natural and industrial sources. PLoS ONE 4:e5584\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOutten FW, Djaman O, Storz G (2004) A \u003cem\u003esuf\u003c/em\u003e operon requirement for Fe\u0026ndash;S cluster assembly during iron starvation in \u003cem\u003eEscherichia coli\u003c/em\u003e. Mol Microbiol 52:861\u0026ndash;872\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMettert EL, Kiley PJ (2014) Coordinate regulation of the Suf and Isc Fe\u0026ndash;S cluster biogenesis pathways by IscR is essential for viability of \u003cem\u003eEscherichia coli\u003c/em\u003e. J Bacteriol 196:4315\u0026ndash;4323\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZupok A, Iobbi-Nivol C, Mejean V, Leimk\u0026uuml;hler S (2019) The regulation of Moco biosynthesis and molybdoenzyme gene expression by molybdenum and iron in bacteria. Metallomics 11:1602\u0026ndash;1624\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMendel RR, Hercher TW, Zupok A, Hasnat MA, Leimk\u0026uuml;hler S (2020) The requirement of inorganic Fe\u0026ndash;S clusters for the biosynthesis of the organometallic molybdenum cofactor. Inorganics 8:43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeimk\u0026uuml;hler S (2021) Transition metals in catalysis: the functional relationship of Fe\u0026ndash;S clusters and molybdenum or tungsten cofactor-containing enzyme systems. Inorganics 9:6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura M, Saeki K, Takahashi Y (1999) Hyperproduction of recombinant ferredoxins in \u003cem\u003eEscherichia coli\u003c/em\u003e by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster. J Biochem 126:10\u0026ndash;18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyu H et al (2025) Production and characterization of CO2 reducing and tungsten-containing formate dehydrogenase of \u003cem\u003eCupriavidus necator\u003c/em\u003e H16. Biotechnol Bioprocess Eng 30:363\u0026ndash;376\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Kempen M et al (2024) Fast and accurate protein structure search with Foldseek. Nat Biotechnol 42:243\u0026ndash;246\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeimk\u0026uuml;hler S (2020) The biosynthesis of the molybdenum cofactors in \u003cem\u003eEscherichia coli\u003c/em\u003e. Environ Microbiol 22:2007\u0026ndash;2026\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagalon A, Mendel RR (2008) Biosynthesis and insertion of the molybdenum cofactor. EcoSal Plus 3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBevers LE, Schwarz G, Hagen WR (2011) A molecular basis for tungstate selectivity in prokaryotic ABC transport systems. J Bacteriol 193:4999\u0026ndash;5001\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHagen WR (2011) Cellular uptake of molybdenum and tungsten. Coord Chem Rev 255:1117\u0026ndash;1128\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOtrelo-Cardoso AR et al (2017) Highly selective tungstate transporter protein TupA from \u003cem\u003eDesulfovibrio alaskensis\u003c/em\u003e G20. Sci Rep 7:5798\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJang J, Jeon BW, Kim YH (2018) Bioelectrochemical conversion of CO2 to value added product formate using engineered \u003cem\u003eMethylobacterium extorquens\u003c/em\u003e. Sci Rep 8:7211\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLink AJ, Phillips D, Church GM (1997) Methods for generating precise deletions and insertions in the genome of wild-type \u003cem\u003eEscherichia coli\u003c/em\u003e: application to open reading frame characterization. J Bacteriol 179:6228\u0026ndash;6237\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeon BW et al (2024) Enzymatic conversion of CO2 to formate: the potential of tungsten-containing formate dehydrogenase in flow reactor system. J CO2 Util 82:102754\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Strains and plasmids used in this study.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eStrains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003cstrong\u003e/Reference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL21(DE3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eBL21(DE3) expression host\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRBC Real Biotech\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL21(DE3) expressing \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e FDH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL(W1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL21(DE3) expressing MeFDH1 with MoeA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL(WA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL21(DE3) expressing MeFDH1 with MobA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL(WB)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL21(DE3) expressing MeFDH1 with MobB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL(W2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL21(DE3) expressing MeFDH1 with MoeA1 and MobB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL(W3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL21(DE3) expressing MeFDH1 with MoeA1, MobA and MobB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL(W4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL21(DE3) expressing MeFDH1 with MoeA1, MobA, MobB and FdhD\u0026nbsp;(W4 machinery)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL21(DE3) with chromosomal tungstate transporter (\u003cem\u003e\u0026Delta;proP\u003c/em\u003e::PT7\u0026ndash;\u003cem\u003etupBCA\u003c/em\u003e from \u003cem\u003eSulfitobacter\u003c/em\u003e\u003cem\u003e\u0026nbsp;dubius\u003c/em\u003e NA4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL1(W4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL1 expressing MeFDH1 with W4 machinery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL1(W4+ISC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL1(W4) harboring pRKISC (\u003cem\u003eisc\u003c/em\u003e operon)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL1 with chromosomally upregulated isc operon (\u003cem\u003e\u0026Delta;iscR::Ptrc\u0026ndash;iscSUA\u0026ndash;hscBA\u0026ndash;fdx\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL2(W4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL2 expressing MeFDH1 with W4 machinery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL(Cn)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL expressing \u003cem\u003eCupriavidus\u003c/em\u003e\u003cem\u003e\u0026nbsp;necator\u003c/em\u003e FDW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL2(W4+Cn)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL2 expressing CnFDW together with W4 machinery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL(Lb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL expressing \u003cem\u003eLutibaculum\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003ebaratangense\u003c/em\u003e FDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBL2(W4+Lb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBL2 expressing LbFDH together with W4 machinery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePlasmids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003cstrong\u003e/Reference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epETDuet-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eDual T7 expression backbone, Ampᴿ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eExpressys\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epETDuet-1 carrying \u003cem\u003eM. extorquens\u003c/em\u003e FDH1 \u0026alpha;/\u0026beta; expression cassette (\u003cem\u003efdh1A\u003c/em\u003e-6\u0026times;His\u0026ndash;\u003cem\u003efdh1B\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epETDuet-1 carrying \u003cem\u003eC. necator\u003c/em\u003e FDW \u0026alpha;/\u0026beta; expression cassette (\u003cem\u003efdwA\u003c/em\u003e-6\u0026times;His\u0026ndash;\u003cem\u003efdwB\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epETDuet-1 carrying \u003cem\u003eL.\u0026nbsp;\u003c/em\u003e\u003cem\u003ebaratangense\u003c/em\u003e FDH \u0026alpha;/\u0026beta; expression cassette (\u003cem\u003efdhA\u003c/em\u003e-6\u0026times;His\u0026ndash;\u003cem\u003efd\u003c/em\u003e\u003cem\u003eh\u003c/em\u003e\u003cem\u003eB\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epCDFDuet-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eCloning/expression backbone, Smᴿ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eExpressys\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epW1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epCDFDuet-1 carrying \u003cem\u003emoeA1\u003c/em\u003e from \u003cem\u003eM. extorquens\u003c/em\u003e AM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epWA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epCDFDuet-1 carrying \u003cem\u003emobA\u003c/em\u003e from \u003cem\u003eM. extorquens\u003c/em\u003e AM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epWB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epCDFDuet-1 carrying \u003cem\u003emobB\u003c/em\u003e from \u003cem\u003eM. extorquens\u003c/em\u003e AM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epW1A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epCDFDuet-1 carrying \u003cem\u003emoeA1\u0026ndash;mob\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e from \u003cem\u003eM. extorquens\u003c/em\u003e AM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epW1B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epCDFDuet-1 carrying \u003cem\u003emoeA1\u0026ndash;mobB\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003efrom \u003cem\u003eM. extorquens\u003c/em\u003e AM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epWAB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epCDFDuet-1 carrying \u003cem\u003emobA\u0026ndash;mobB\u003c/em\u003e from \u003cem\u003eM. extorquens\u003c/em\u003e AM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epW3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epCDFDuet-1 carrying \u003cem\u003emoeA1\u0026ndash;mobA\u0026ndash;mobB\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003efrom \u003cem\u003eM. extorquens\u003c/em\u003e AM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epW4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epCDFDuet-1 carrying \u003cem\u003emoeA1\u0026ndash;mobA\u0026ndash;mobB\u0026ndash;fdhD\u003c/em\u003e from \u003cem\u003eM. extorquens\u003c/em\u003e AM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epZ1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epZS21 carrying \u003cem\u003emodABC\u003c/em\u003e from \u003cem\u003eE. coli\u003c/em\u003e K-12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epZ2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epZS21 carrying \u003cem\u003etupBCA\u0026nbsp;\u003c/em\u003efrom \u003cem\u003eS. dubius\u003c/em\u003e NA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epZ3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003epZS21 carrying \u003cem\u003etupBCA\u0026nbsp;\u003c/em\u003efrom \u003cem\u003eM. extorquens\u0026nbsp;\u003c/em\u003eAM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16px;\"\u003e\n \u003cp\u003epRKISC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cem\u003eiscSUA\u0026ndash;hscBA\u0026ndash;fdx\u003c/em\u003e operon, Tetᴿ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eNakamura \u003cem\u003eet al\u003c/em\u003e. \u003csup\u003e33\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Metal content of MeFDH1 across engineered \u003cem\u003eE. coli\u003c/em\u003e strains and the native \u003cem\u003eM\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003eextorquens\u0026nbsp;\u003c/em\u003eAM1 host.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetal incorporation into purified MeFDH1 was quantified by inductively coupled plasma\u0026ndash;mass spectrometry (ICP\u0026ndash;MS). Iron (Fe), tungsten (W), and molybdenum (Mo) contents are shown for enzymes produced in strains expressing progressively expanded tungsten-cofactor biosynthetic and accessory modules: BL (MeFDH1 only), BL(W4) (MoeA1, MobA, MobB and FdhD), BL1(W4) (W4 machinery with chromosomal \u003cem\u003etupBCA\u003c/em\u003e), BL1(W4+ISC) (additional plasmid-borne \u003cem\u003eisc\u003c/em\u003e operon), BL2(W4) (chromosomally upregulated \u003cem\u003eisc\u003c/em\u003e operon), and the native AM1 host. Values represent mean \u0026plusmn; s.d. (n = 3).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"616\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 154px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 462px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetal content (mol metal mol\u003csup\u003e-1\u003c/sup\u003e enzyme)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFe\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003eBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e1.75 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.01\u0026nbsp;\u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.02\u0026nbsp;\u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003eBL(W4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e5.50 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.27 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.05 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003eBL1(W4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e5.14 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.55 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.09 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003eBL1(W4+ISC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e8.30 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.75\u0026nbsp;\u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.05 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003eBL2(W4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e10.8 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.92 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.02 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003eAM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e11.6 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.50 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e0.02 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Kinetic parameters of MeFDH1 in expressed engineered \u003cem\u003eE. coli\u003c/em\u003e compared with the enzyme from \u003cem\u003eM. extorquens\u003c/em\u003e AM1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTurnover numbers (k\u003csub\u003ecat\u003c/sub\u003e), Michaelis constants (K\u003csub\u003eM\u003c/sub\u003e), and catalytic efficiencies (k\u003csub\u003ecat\u003c/sub\u003e/K\u003csub\u003eM\u003c/sub\u003e) were determined for CO₂ reduction using either dissolved CO₂ or reduced ethyl viologen (EV⁺\u0026middot;) as the electron mediator. MeFDH1 was purified from \u003cem\u003eE. coli\u003c/em\u003e BL2 (BL2) or isolated from the native AM1 host. Values represent mean \u0026plusmn; s.d. (n = 3).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ek\u003csub\u003ecat\u003c/sub\u003e (s⁻\u0026sup1;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003csub\u003eM\u0026nbsp;\u003c/sub\u003e(mM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ek\u003csub\u003ecat\u003c/sub\u003e/K\u003csub\u003eM\u003c/sub\u003e (s⁻\u0026sup1; mM⁻\u0026sup1;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCO₂\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eBL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e141 \u0026plusmn; 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e8.4 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e17\u0026nbsp;\u0026plusmn;\u0026nbsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eAM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e152 \u0026plusmn; 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e3.6 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e42 \u0026plusmn; 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEV\u0026bull;⁺\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eBL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e304\u0026nbsp;\u0026plusmn;\u0026nbsp;39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e0.06 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e5067 \u0026plusmn; 1810\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eAM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e325 \u0026plusmn; 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21px;\"\u003e\n \u003cp\u003e0.02 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003e16250 \u0026plusmn; 8131\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. Functional expression of heterologous tungsten-dependent dehydrogenases in engineered \u003cem\u003eE. coli\u003c/em\u003e chassis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCnFDW from \u003cem\u003eC. necator\u003c/em\u003e H16 and LbFDH from \u003cem\u003eL. baratangense\u003c/em\u003e AMV1 were expressed in BL21(DE3) or the engineered BL2 chassis. BL2 carries a chromosomally integrated tungstate transporter (\u003cem\u003etupBCA\u003c/em\u003e), chromosomal upregulation of the isc operon, and plasmid-encoded W-cofactor biosynthetic genes (\u003cem\u003emoeA1\u003c/em\u003e, \u003cem\u003emobA\u003c/em\u003e, \u003cem\u003emobB\u003c/em\u003e, and \u003cem\u003efdhD\u003c/em\u003e). Specific activities were determined by enzymatic assay, and metal contents (Fe, W, Mo) were quantified by ICP\u0026ndash;MS. ND, not detected. Values represent mean \u0026plusmn; s.d. (n = 3).\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecific activity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(U mg⁻\u0026sup1; enzyme)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 51px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMetal (mol mol⁻\u0026sup1; enzyme)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFe\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eBL(Cn)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e2.72 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e0.04 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eBL2(W4+Cn)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e38 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e18.6 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e0.89 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eBL(Lb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e5.68 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e0.24 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eBL2(W4+Lb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e8 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18px;\"\u003e\n \u003cp\u003e12.8 \u003cstrong\u003e\u0026plusmn;\u0026nbsp;\u003c/strong\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17px;\"\u003e\n \u003cp\u003e1.17 \u0026plusmn; 0.02*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e* W occupancy slightly above 1.0 reflects minor analytical uncertainty in protein quantification.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Formate dehydrogenase, Tungsten cofactor, W-bis-MGD, Tungstate transport, Iron–sulfur cluster, CO₂ reduction, Synthetic chassis","lastPublishedDoi":"10.21203/rs.3.rs-8913862/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8913862/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTungsten-dependent formate dehydrogenases catalyze CO₂ reduction, yet their heterologous production remains limited by the absence of tungsten-specific cofactor maturation in standard laboratory hosts. Although molybdenum-dependent formate dehydrogenases have been implemented in \u003cem\u003eEscherichia coli\u003c/em\u003e, analogous maturation of tungsten-dependent enzymes has proven unsuccessful because native metal homeostasis does not support selective tungstate uptake or W-bis-MGD assembly. Here we reconstitute tungsten-specific metal metabolism in \u003cem\u003eE. coli\u003c/em\u003e by reconstructing tungsten cofactor biosynthesis, installing a high-affinity tungstate uptake system, and reinforcing Fe\u0026ndash;S cluster biogenesis. This enables aerobic production of catalytically competent tungsten-dependent formate dehydrogenase from \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e AM1, achieving native-level specific activity and a tungsten occupancy of 0.92 mol per mol enzyme\u0026mdash;nearly double that of the native host. The engineered strain delivers a volumetric activity exceeding 3,500 U L⁻\u0026sup1;. In a coupled whole-cell CO-to-formate biotransformation, the strain generated 44 mM formate within 4 hours, outperforming the native host system by more than threefold. The platform also supports activation of W-bis-MGD-dependent enzymes from \u003cem\u003eCupriavidus necator\u003c/em\u003e H16 and \u003cem\u003eLutibaculum baratangense\u003c/em\u003e AMV1. This work establishes a general design framework for W-bis-MGD maturation, expanding access to tungsten-dependent biocatalysts for CO₂-to-formate and related reductive biotransformations.\u003c/p\u003e","manuscriptTitle":"Metal cofactor level chassis engineering enables aerobic expression of tungsten formate dehydrogenases in Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 03:45:06","doi":"10.21203/rs.3.rs-8913862/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0eff88e6-0962-417f-9e6f-5dedc270ac3b","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63251012,"name":"Biological sciences/Biotechnology/Metabolic engineering"},{"id":63251013,"name":"Biological sciences/Microbiology/Applied microbiology"}],"tags":[],"updatedAt":"2026-03-18T09:35:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-23 03:45:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8913862","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8913862","identity":"rs-8913862","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}