Heterologous production of tungsten-dependent formate dehydrogenase I from Methylorubrum extorquens in Escherichia coli reveals α-subunit maturation as the major bottleneck

Heterologous production of tungsten-dependent formate dehydrogenase I from Methylorubrum extorquens in Escherichia coli reveals α-subunit maturation as the major bottleneck | 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 Research Article Heterologous production of tungsten-dependent formate dehydrogenase I from Methylorubrum extorquens in Escherichia coli reveals α-subunit maturation as the major bottleneck Ngoc Minh Chau Nguyen, Huichang Ryu, Joon Young Park, YongHwan Kim, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9133619/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Methylorubrum extorquens formate dehydrogenase I (Me-FDH1) is a tungsten-dependent heterodimeric enzyme with high activity for CO2/formate interconversion, making it an attractive biocatalyst for carbon capture, formate production, and bioelectrocatalysis. Its broader application, however, is limited by the lack of a heterologous production host for this complex metalloenzyme. Here, we systematically evaluated Escherichia coli as a host for Me-FDH1 production and compared its performance with that of the native host and a previous heterologous host. Across multiple E. coli strains, active Me-FDH1 was obtained only when tungstate uptake was supported by a functional ModABC system or by heterologous expression of the TupBCA transporter, demonstrating that E. coli can synthesize and incorporate the W-bis-MGD cofactor. Nevertheless, expression remained low (<1% of total cellular protein), and the purified enzyme displayed only 4–14 U mg-1 specific activity, far below the 80–100 U.mg-1 observed for the enzyme produced in M. extorquens. Operon redesign, altered gene order, stronger ribosome-binding sites, SUMO fusion, chaperone co-expression, and codon harmonization did not improve α-subunit production. In both E. coli and M. extorquens, the α-subunit was poorly produced in the absence of the β-subunit, indicating that the β-subunit contributes to α-subunit stabilization and/or maturation. Cell-free translation produced both subunits efficiently, showing that the principal barrier in E. coli is not transcription or translation, but post-translational instability and likely proteolytic loss of the α-subunit. These findings define the key bottleneck for Me-FDH1 production in E. coli and provide a roadmap for engineering hosts for tungsten-containing enzymes. Methylorubrum extorquens tungsten-dependent formate dehydrogenase W-bis-MGD tungstate transport α-subunit maturation post-translational instability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Points E. coli produced active Me-FDH1 when tungstate uptake was enabled. ModABC or TupBCA supported W-bis-MGD-dependent Me-FDH1 activity. The α-subunit was the major bottleneck in Me-FDH1 production. The β-subunit promoted α-subunit accumulation and/or maturation. Cell-free translation revealed post-translational loss of the α-subunit. Introduction The enzymatic reduction of carbon dioxide to formate is an attractive route for carbon capture and utilization because formate can serve both as a C1 chemical feedstock and as a liquid hydrogen carrier (Jang et al., 2018). Formate dehydrogenases (FDHs) are therefore important biocatalysts for sustainable CO 2 conversion. Among them, formate dehydrogenase I from Methylorubrum extorquens (Me-FDH1) is particularly attractive because it efficiently catalyzes the interconversion of CO 2 and formate and also supports direct electron-transfer bioelectrocatalysis, making it relevant to enzymatic and electrochemical carbon-recycling systems (Laukel et al., 2003; Jang et al., 2018; Yoshikawa et al., 2022). Me-FDH1 is unusual among FDHs in that it contains a tungsten cofactor rather than the more common molybdenum cofactor (Park et al., 2024). The enzyme is a heterodimer composed of an approximately 107 kDa α-subunit and a 62 kDa β-subunit. The α-subunit carries a tungsto-bis(molybdopterin guanine dinucleotide) cofactor (W-bis-MGD), whereas multiple Fe-S clusters are distributed across both subunits (Yoshikawa et al., 2022). This complex architecture likely underlies the high catalytic performance of Me-FDH1, but it also makes heterologous production demanding because active holoenzyme formation requires coordinated expression of two large subunits, correct folding, W-bis-MGD biosynthesis and insertion, and proper Fe-S cluster assembly. Me-FDH1 can be produced efficiently in its native host, M. extorquens , where recombinant expression under a strong promoter yields high activity and high intracellular accumulation (Jang et al., 2018; Ryu et al., 2024). However, homologous production in M. extorquens is not ideal for large-scale application because the organism grows relatively slowly, is less convenient for high-cell-density cultivation, and relies on less convenient carbon sources such as methanol or succinate (Ryu et al., 2024, 2025). More recently, Cupriavidus necator H16 was shown to be an effective heterologous host, yielding recombinant Me-FDH1 with near-native activity and high volumetric productivity (Ryu et al., 2024, 2025). Nevertheless, recombinant Me-FDH1 still underwent degradation during later cultivation, and deeper host engineering is less straightforward than in Escherichia coli (Park et al., 2024; Ryu et al., 2024, 2025). These considerations make E. coli an attractive candidate host. E. coli grows rapidly, reaches high cell density, and offers unmatched genetic accessibility. The key question, however, is whether E. coli can be adapted to produce a catalytically competent tungsten-containing metalloenzyme as complex as Me-FDH1. In this study, we systematically evaluated E. coli as a host for Me-FDH1 production and used this analysis to identify the dominant bottleneck. We tested multiple E. coli strains with different tungstate/molybdate transport capacities, examined operon architecture using a series of recombinant plasmids, and evaluated SUMO fusion, chaperone co-expression, and codon harmonization as strategies to improve α-subunit production. We further analyzed transcript levels by RT-PCR and used a cell-free translation system to distinguish between failure of α-subunit synthesis and loss of the α-subunit after synthesis. By comparing the results obtained in E. coli with those from M. extorquens and a previously developed heterologous host, we aimed to define the host functions that must be engineered to support robust production of Me-FDH1 and related tungsten-dependent enzymes. Materials and Methods Materials Various E. coli strains, including B, BL21 (DE3), BL21 Star™ (DE3) pLysS, BW25113, C ATCC8739, JM109 (DE3), K-12 MG1655, K-12 Shuffle ® T7, MC1061, W, W3110, XL-1 Blue and Rosetta 2 (DE3) and a derivative of M. extorquens PA1 lacking all four native FDH genes (MeP4; Δ fdh 1–4) were used in this study (Table 1). E. coli DH5α (Toyobo, Japan) was used for gene cloning. DNA sequencing and primer synthesis were performed by Macrogen (Korea). In-Fusion ® Snap Assembly Master Mix (TaKaRa, Japan), restriction endonucleases and T4 DNA ligase (New England Biolabs, USA), and Pfu-X DNA polymerase with standard PCR reagents (SolGent, Korea) were used for plasmid construction. Plasmids and genomic DNA were prepared with commercial kits according to the manufacturers’ instructions. Isopropyl-β-D-thiogalactopyranoside (IPTG; Bio Basic, Canada) was used for protein induction, and all other reagents were of analytical grade unless otherwise noted. Plasmid construction and strains preparation Genes encoding the Me-FDH1 α- and β-subunits ( fdh1 a and fdh1 b) were amplified from M. extorquens PA1 genomic DNA and cloned under the IPTG-inducible P L/O4 promoter in the pBBR1-derived broad-host-range vector pCM110, generating pCM2 (Figure 1; Table 1). The vector carries lac I and is compatible with E. coli , M. extorquens , and C. necator . Additional plasmids were constructed to evaluate the effects of gene organization, solubility tags, chaperone co-expression, and codon harmonization on heterologous expression. All plasmid backbones are summarized in Table 1, and all primers are listed in Table S1 (see Additional File). The reference construct pCM2 contained the native fdh1 b– fdh1 a arrangement, including the 52 nucleotides (52-nt) intergenic region, and a C-terminal hexa-histidine tag (His 6 -tag) on the α-subunit. RNA secondary structure of the 52-nt intergenic region was predicted using RNAfold ViennaRNA package (Lorenz et al., 2011) and the model was deposited in ModelArchive ma-ny5ny (see Additional File: Figure S1). In pCM3, the gene order was reversed and the β-subunit carried an N-terminal His 6 -tag. In pCM4, the native gene order was retained, but a strong synthetic ribosome-binding site (RBS) was introduced upstream of fdh1a . In pCM5, the α- and β-subunits were expressed from separate promoter-RBS units. Single-subunit plasmids were also constructed: pCM6 encoded His 6 -tagged fdh1b , whereas pCM7 encoded His 6 -tagged fdh1a with a strong RBS (Figure 1; Table 1). Plasmids pCM4, pCM6, and pCM7 were derived from pCM2 by restriction digestion, PCR amplification of the desired fragments, and In-Fusion- or ligase-based assembly. pCM3 and pCM5 were subsequently constructed from the pCM6 backbone by insertion of PCR-amplified fdh1a -containing fragments and, for pCM5, an additional promoter-terminator cassette. When appropriate restriction sites were unavailable, fragments were combined by overlap PCR. All constructs were verified by restriction analysis and DNA sequencing. For specific tungstate transport, the high-affinity tup BCA operon was amplified from M. extorquens PA1 and cloned into the tac-promoter vector pAC1, generating pAC2 (Figure 1; Table 1). The operon corresponds to Mext _2850– Mext _2852. This construct was used to complement E. coli strains lacking an effective endogenous tungstate uptake route. To examine whether general folding support improves Me-FDH1 production, the chaperone plasmids pG-KJE8 and pG-Tf2 (TaKaRa) were used. pG-KJE8 expresses DnaK - Dna J- Grp E together with Gro EL- Gro ES, whereas pG-Tf2 expresses Gro EL- Gro ES with trigger factor (Nishihara et al., 1998, 2000). Culture conditions and production of recombinant Me-FDH1 For E. coli , a modified M9 medium supplemented with 1 g/L yeast extract and 10 g/L glucose was used as described previously (Ryu et al., 2024, 2025). To support tungsten cofactor formation, sodium tungstate (Na 2 WO 4 , 30 μM) was added to the medium after autoclaving by filter sterilization (Ryu et al., 2025). Cultures were grown at 30 o C and 200 rpm to mid-log phase (OD 600 ~0.4–0.8), induced with 0.5 mM IPTG, and incubated at 30 o C for 16–18 h. When chaperone plasmids were used, arabinose (0.5 mg/mL) or tetracycline (10 ng/mL) was added together with IPTG according to the plasmid system. Antibiotics were used at the following concentrations: kanamycin 50 µg/mL, ampicillin 100 µg/mL, and tetracycline 10 µg/mL. Cultivation and induction of M. extorquens MeP4 carrying pCM plasmids were performed as described previously (Jang et al., 2018; Ryu et al., 2024, 2025) Protein extraction, purification and expression analysis Cells were harvested after induction, washed, and resuspended in lysis buffer (20 mM MOPS, 200 mM NaCl, 20 mM imidazole). Cell disruption was performed by sonication on ice, and soluble and insoluble fractions were separated by centrifugation (13000 g , 10–20 min, 4 o C) as described previously (Ryu et al., 2024, 2025). His 6 -tag proteins were purified from crude extracts under nondenaturing conditions using Nickel-Nitrilotriacetic Acid (Ni-NTA) Bind Resin column (Qiagen). Eluted proteins were concentrated and buffer-exchanged with Amicon ® Ultra-15 centrifugal filters (30 kDa cutoff; Millipore, Darmstadt, Germany). Protein concentrations were determined by the Bradford assay. Expression levels were estimated by SDS–PAGE densitometry against bovine serum albumin (BSA) standards (Thermo Scientific) and are reported as the combined intensities of the α- and β-subunit bands when both were present. Protein production was analyzed by SDS-PAGE and Western blotting. His 6 -tagged proteins were detected with a mouse monoclonal anti-His 6 antibody and an alkaline phosphatase-conjugated rabbit anti-mouse secondary antibody IgG H&L (Abcam, ab97043) followed by BCIP/NBT Liquid Substrate System (Sigma-Aldrich, B1911-100ML) development. Enzyme activity assay Enzyme activity was measured at 30 o C for 1 min under conditions adapted from a previous study (Jang et al., 2018). For the formate oxidation assay, reactions were performed at pH 7.0 with 30 mM sodium formate, and NADH formation was monitored at 340 nm (ε 340 = 6220 M -1 cm -1 ). All assays were performed in triplicate. One unit (U, µmol min -1 ) of activity was defined as the amount of enzyme that formed 1 μmol of NADH per min under the assay conditions. RT-PCR and metal analysis To quantify fdh1 a and fdh1 b transcripts in E. coli , cells were grown in modified M9 medium and induced at mid-log phase. Pellets were immediately stabilized with RNAprotect TM Bacteria Reagent (Qiagen) and total RNA was isolated with the NucleoSpin RNA isolation kit (Macherey-Nagel, Germany). First-strand cDNA was synthesized with the iScript TM cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed on a StepOne Real-Time PCR system (Applied Biosystems, USA) using SYBR Green chemistry. Transcript levels were normalized to the housekeeping gene rpo D (RNA polymerase sigma factor), and relative mRNA levels were calculated using the ΔΔCt method (Zhou et al., 2015). All assays were performed in duplicate, and no-template controls were included as negative controls. Iron contents of purified protein preparations were measured by HR-ICP-MS as described previously (Ryu et al., 2024, 2025). Purified proteins were digested in nitric acid before analysis. Tungsten content of the α-subunit was measured for selected samples when sufficient material was available. Codon harmonization Codon optimization was performed by codon harmonization, in which host codon usage is adjusted to approximate native translation kinetics (Schmidt et al., 2023) . Codon frequencies were weighted by amino acid abundance in the complete host proteome. The abundance of amino acid A was defined by Equation (1) (see Additional File: Figure S2): where, n( A ) is the number of occurrences of amino acid A , and ∑n( A ) is the total number of amino acids in the proteome. Translation speed for each codon was estimated according to Equation (2) (see Additional File: Figure S2): The harmonized fdh1a and fdh1b sequences for E. coli were deposited in NCBI GenBank under accession numbers PX353735 and PX353736, respectively. The codon-harmonized genes were cloned into pRSF-Duet-1 and pET-Duet-1 (Figure 1) to generate pRSF8 and pET2. In vitro translation To test whether the α-subunit can be synthesized in the absence of cellular proteolysis, cell-free expression was performed with the PURExpress ® In vitro Protein Synthesis Kit (New England Biolabs, USA) supplemented with murine RNase Inhibitor (NEB). PCR fragments containing the promoter, gene, and terminator regions were amplified from pRSF2, pRSF3, pRSF4, and pRSF5 using the primer pairs listed in Table S1 (see Additional File) and were purified before use as templates. Each 25 µL reaction contained 10 μL Solution A, 7.5 μL Solution B, 0.5 μL RNase inhibitor, approximately 0.9–1.0 μg purified PCR template, and nuclease-free water. Reactions were incubated at 37 o C for 4 h and then cooled on ice. Products were concentrated with 10 kDa MWCO centrifugal filter (Amicon ® Ultra-0.5) and analyzed by SDS-PAGE. Because the PURExpress system lacks ATP-dependent proteases and most maturation pathways, it allows detection of proteins that may be unstable in vivo . Results and Discussion Expression of Me-FDH1 in Escherichia coli K-12 Me-FDH1 was first expressed in wild-type E. coli K-12 MG1655 carrying pCM2, the reference plasmid described in Figure 1 and Table 1. Cultures were supplemented with sodium tungstate (30 μM) to support W-bis-MGD biosynthesis. Although SDS-PAGE of crude extracts did not reveal distinct bands at the expected positions of the α- and β-subunits, indicating very low expression in the host background (Figure 2A), Western blot analysis using an anti-His antibody clearly detected the His 6 -tagged α-subunit in both crude extracts and Ni-NTA purified fractions (Figure 2B). After purification, both α- and β-subunits became visible by SDS-PAGE, consistent with co-purification of the β-subunit through association with the His 6 -tagged α-subunit, as reported previously for Me-FDH1 (Ryu et al., 2024, 2025) (Figure 2A, B; Table 2). Formate oxidation activity was not detectable in the crude cell extract, but the purified enzyme showed a specific activity of 8.2 ± 0.5 U mg -1 (Figure 2C; Table 2). Because Me-FDH1 activity requires proper insertion of the tungsten-containing cofactor (W-bis-MGD), this result indicates that E. coli can produce catalytically competent Me-FDH1. However, the activity remained far below that of the same enzyme produced in the homologous M. extorquens system, where purified recombinant Me-FDH1 reached 84.5 U mg -1 under comparable expression conditions (Table 3), consistent with previous reports of approximately 80–100 U mg -1 for the native/homologous enzyme (Park et al., 2024; Ryu et al., 2024). Importance of molybdate/tungstate transport in E. coli strains Because the K-12 result suggested that E. coli can synthesize and insert W-bis-MGD, we next examined Me-FDH1 production across a broader panel of E. coli strains listed in Table 1 and summarized in Table 2. The strain panel was selected to probe four distinct physiological and engineering features that could limit Me-FDH1 production. T7-based high-expression strains, namely BL21 (DE3), BL21 Star (DE3) pLysS, and JM109 (DE3), were used to evaluate transcriptional and translational capacity; JM109 (DE3) has previously supported functional heterologous expression of several formate dehydrogenases (Alissandratos et al., 2014). Rosetta 2 (DE3) supplies rare tRNAs for codons that are infrequent in E. coli (Lipinszki et al., 2018). K-12 Shuffle T7 provides a redox-engineered cytoplasm compatible with disulfide-bond formation (Lobstein et al., 2012). K-12-derived and natural-isolate strains (MG1655, BW25113, W3110, W, B, C, XL-1 Blue, MC1061) span different ModABC uptake statuses and physiological backgrounds, and MC1061 has been used previously for heterologous expression of FDH cofactor-insertion chaperones (Böhmer et al., 2014). This design allowed us to separate the contribution of metal uptake from the contribution of the host expression machinery. As in K-12, none of the strains showed clearly visible Me-FDH1 bands in crude lysates by SDS-PAGE, whereas Western blot analysis detected the α-subunit broadly across recombinant strains (see Additional File: Figure S3). After Ni-affinity purification, active Me-FDH1 was recovered only from a subset of strains, with purified-enzyme specific activities ranging from 3.3 to 13.9 U mg -1 (Table 2). Among these, BW25113 gave the highest activity, followed by K-12 MG1655, JM109, WA, and W3110 (Table 2). A notable common feature of the active strains was the presence of a functional mod ABC molybdate transporter system (Table 2). The Mod ABC transporter is known to transport tungstate as well as molybdate, albeit with lower specificity than dedicated tungstate transporters (Leimkühler et al., 2011; Otrelo-Cardoso et al., 2017). In contrast, strains lacking a functional ModABC system showed no detectable Me-FDH1 activity (Table 2). One exception was E. coli C, which carries mod ABC but still showed little or no activity, indicating that transporter availability is necessary but not sufficient. Because all tested E. coli strains inherently possess the molybdopterin biosynthetic machinery (see Additional File: Figure S3–S4; Table S2), these results suggest that tungsten uptake, rather than the absence of the cofactor-biosynthetic pathway itself, is the primary requirement for active Me-FDH1 formation in E. coli . To test this interpretation directly, the high-affinity tungstate transporter Tup BCA from M. extorquens was introduced on plasmid pAC2 (Figure 1; Table 1) into E. coli BL21 (DE3), which lacks a functional Mod ABC system and did not produce active Me-FDH1 from pCM2 alone (Table 2). In both EcBL/pCM2/pAC2 and EcBL/pRSF2/pAC2, the α-subunit became detectable after purification, and the purified enzyme displayed activity of approximately 2.6 U mg -1 (Table 2; see Additional File: Figure S5). Thus, either native Mod ABC or heterologous Tup BCA can provide the metal-uptake route required for Me-FDH1 maturation in E. coli . At the same time, the Tup BCA-complemented strains still produced only low amounts of weakly active enzyme, indicating that tungsten uptake is necessary for activity but is not the dominant bottleneck for production yield. While tungsten uptake is required for Me-FDH1 maturation, the present data indicate that it is not the dominant limiting factor under the conditions tested. Increasing tungstate supplementation and the expression of TupBCA did not result in proportional increases in enzymatic activity. Furthermore, constructs that differ substantially in α-subunit accumulation show comparable specific activity per unit of recovered protein (Table 2). Together, these observations indicate that the primary limitation arises upstream of cofactor insertion, most likely at the level of α-subunit maturation. Quantitative analysis of intracellular tungsten incorporation (for example by ICP-MS) will be required to refine this conclusion and is an important direction for future work. Effect of gene organization on expression of Me-FDH1 The preceding experiments indicated two major problems when Me-FDH1 is produced in E. coli : (i) low enzyme yield and (ii) low specific activity. To determine whether these problems arise at the transcriptional or post-transcriptional level, we constructed a series of plasmids with altered operon architectures (Figure 1, 3; Table 1). The reference construct pCM2 contains the native fdh1 b– fdh1 a arrangement separated by a 52-nt intergenic region. This region was predicted to form an extensive secondary structure (see Additional File: Figure S1; ModelArchive: ma-ny5ny), which could in principle influence transcript stability or translation efficiency through a riboswitch-like mechanism (Serganov and Nudler, 2013). Accordingly, pCM3, pCM4, and pCM5 were designed to alter gene order, translation signals, or operon structure, whereas pCM6 and pCM7 expressed the β- and α-subunits individually (Figure 1; Table 1). RT-PCR analysis showed that all constructs produced full-length transcripts at comparable levels (Figure 4). Quantitative RT-PCR was normalized to rpo D, using the ΔΔCt approach described previously (Zhou et al., 2015). This result indicates that transcription of fdh1 a and fdh1 b is not the primary bottleneck and that the native 52-nt intergenic region does not cause severe premature transcription termination under the tested conditions. The observed invariance of full-length transcript levels across pCM2 to pCM5 (Figure 4) and the efficient translation of these constructs in the PURExpress cell-free system (see Section “ In vitro translation”) together indicate that mRNA secondary structure or translational coupling, although predicted by RNAfold, does not impose a dominant in vivo constraint. Direct structural probing by SHAPE (Selective 2’-Hydroxyl Acylation analyzed by Primer Extension) or DMS-MaPseq (Dimethyl Sulfate Mutational Profiling with sequencing) was therefore not pursued. Although ribosome profiling was not performed, the efficient translation of both subunits in PURExpress, combined with comparable mRNA levels across constructs (Figure 4), provides direct evidence that translation initiation and elongation are not rate-limiting for Me-FDH1 production in E. coli . In contrast, protein recovery and enzyme performance were strongly affected by plasmid configuration (Figure 3A; Table 3). In E. coli K-12, pCM2 gave the best balance of α-subunit accumulation and enzyme activity, yielding 0.7 mg g -1 CDW purified Me-FDH1 with a specific activity of 8.0 U mg -1 (Table 3). Reversing gene order (pCM3) or introducing a stronger downstream RBS (pCM4) sharply reduced α-subunit recovery and lowered activity to 0.3 and 0.2 U mg -1 , respectively (Table 3). By contrast, pCM5, in which the two genes were separated into individual operons, gave a large increase in recovered protein because the β-subunit accumulated efficiently, but the specific activity remained low (1.3 U mg -1 ), indicating that α-subunit production remained limiting (Table 3). This interpretation is reinforced by the single-subunit constructs: pCM6 yielded a large amount of purified β-subunit (8.7 mg g -1 CDW), whereas pCM7 yielded almost no purified α-subunit (<0.1 mg g -1 CDW) (Table 3). A parallel experiment in the homologous host M. extorquens MeP4 led to the same qualitative conclusion regarding subunit asymmetry (Figure 3B; Table 3). When both subunits were co-expressed, pCM2, pCM3, pCM4, and pCM5 all yielded highly active enzyme preparations (63.7–84.5 U mg -1 ), but expression of the α-subunit alone from pCM7 again resulted in extremely poor recovery (<0.1 mg g -1 CDW) (Table 3). By contrast, the β-subunit alone was readily produced from pCM6 in both hosts (Figure 3A, B; Table 3). Together, these data indicate that the β-subunit is not merely a passive partner in the final αβ complex, but likely contributes to productive α-subunit accumulation, stabilization, and/or maturation. The Fe-content analysis supports this interpretation but should be viewed as construct-dependent rather than as a general defect in all E. coli preparations (Table 3). The theoretical Fe content of Me-FDH1 holoenzyme is 20 mol/mol protein, with 14 Fe atoms assigned to the α-subunit and 6 to the β-subunit (Yoshikawa et al., 2022). The co-expressed holoenzyme from EcK/pCM2 contained 17.6 mol Fe/mol protein, close to the value observed for MeP4/pCM2 (18.2 mol/mol), indicating that near-complete Fe incorporation is possible in E. coli when productive co-expression occurs (Table 3). In contrast, constructs enriched in the β-subunit and poor in the α-subunit, especially pCM5 and pCM6, showed much lower Fe contents in both hosts (Table 3). Notably, β-subunit-only preparations from EcK/pCM6 and MeP4/pCM6 contained only about half of the expected Fe for the β-subunit alone, suggesting incomplete Fe-S cluster incorporation or reduced cluster stability in the absence of coordinated holoenzyme assembly. Thus, the dominant bottleneck is α-subunit accumulation, whereas impaired Fe-S maturation appears to be most evident in constructs where α-subunit co-production is compromised. The parsimonious interpretation of these data is that the β-subunit stabilizes the α-subunit through post-translational shielding. The α-subunit is translated efficiently in the absence of the β-subunit in the PURExpress cell-free experiment (see Section “ In vitro translation”), and is transcribed normally in pCM7, yet does not accumulate in vivo . The β-subunit therefore appears to protect a partially folded α-subunit from quality control recognition, an arrangement that parallels the role of the FdsC/FdsD chaperones in the assembly of the R. capsulatus FDH α-subunit with bis-MGD (Böhmer et al., 2014; Hartmann and Leimkühler, 2013). A decisive test of the timing of β-subunit action will require orthogonal induction of the two subunits, for example by placing β under an arabinose-inducible promoter and α under an IPTG-inducible promoter. If the β-subunit acts co-translationally, simultaneous induction should be necessary for α-subunit accumulation; if it acts by post-translational shielding, prior induction of the β-subunit should be sufficient. SUMO fusion tag and general chaperones did not improve α-subunit in E. coli Because the α-subunit accumulated poorly in E. coli , we next tested whether solubility enhancement or generic folding support could improve its production. SUMO-based constructs were designed using the plasmids shown in Figure 1 and Table 1 and expressed in E. coli K-12 Shuffle ® T7 (EcKT), a T7-compatible strain that retains the native Mod ABC uptake system and therefore minimizes possible confounding effects from tungsten limitation (Table 1; Figure 5A). None of the tested SUMO configurations increased α-subunit accumulation detectably (Figure 5A). Even in the best SUMO-related cases, purified preparations showed only low activities of approximately 3.1–4.0 U mg -1 (Figure 5C), which remained below the activity of the original EcK/pCM2 enzyme (Table 2 and Table 3). Thus, improved solubility via a SUMO fusion was insufficient to overcome the main production barrier. We then examined whether co-expression of molecular chaperones could improve α-subunit folding. Chaperone systems encoded on pG-KJE8 and pG-Tf2 (Table 1) provide GroEL-GroES together with either DnaK-DnaJ-GrpE or trigger factor, respectively (Nishihara et al., 1998, 2000). Although the chaperones themselves were strongly expressed, no improvement in α-subunit recovery was observed. SDS-PAGE still showed only a faint α-subunit band after purification (Figure 5B), and Me-FDH1 activity did not increase beyond the level of the original construct (Figure 5C). These results indicate that the limitation in α-subunit production is not rescued by a generic solubility tag or by broad cytosolic chaperone overexpression. Codon harmonization did not improve α-subunit production in E. coli Because transcription was not limiting (Figure 4) and neither SUMO fusion nor general chaperones improved α-subunit accumulation (Figure 5), we next investigated whether inefficient co-translational folding due to host-specific translation kinetics could be responsible. Translation-rate mismatch is known to affect protein folding and stability in heterologous hosts (Francis and Page, 2010). Therefore, the fdh1 a and fdh1 b genes were redesigned by codon harmonization to better mimic the native translational speed landscape of M. extorquens in E. coli (Schmidt et al., 2023). Comparison of codon usage among M. extorquens , C. necator , and E. coli showed substantial differences between M. extorquens and E. coli , particularly in the α-subunit gene, whereas M. extorquens and C. necator were more similar (see Additional File: Figure S6). After harmonization, the GC contents of the optimized genes decreased markedly, and the predicted correlations between native and host translation speeds improved for both subunits (see Additional File: Figure S6). The optimized sequences also retained only 77.91% and 75.80% nucleotide identity for the α- and β-subunit genes, respectively (see Additional File: Figure S6A, B). The codon-optimized genes were cloned into the T7-based vectors shown in Figure 1 and Table 1. Despite these substantial sequence changes, codon harmonization did not improve α-subunit accumulation. Neither EcKT/pRSF8 nor EcKT/pRSF8/pET2 showed increased α-subunit recovery relative to the corresponding non-optimized controls, and the purified proteins remained only weakly active (Figure 6A). When expressed individually, neither subunit produced detectable FDH activity, and when both were present the purified preparations still showed only low specific activities (2.8–3.9 U mg -1 ; data not shown). Codon harmonization is effective when translation-rate mismatches perturb co-translational folding. Our data indicate that the limiting step is downstream of translation. The α-subunit is translated efficiently in the PURExpress cell-free experiment (see Section “ In vitro translation”), and the harmonized gene does not accumulate better in vivo . For a large multidomain metalloenzyme such as the Me-FDH1 α-subunit, the rate-limiting steps are domain packing, Fe-S cluster incorporation, and W-bis-MGD insertion, all of which occur after the polypeptide leaves the ribosome. Codon-level tuning therefore cannot by itself overcome this limitation. In vitro translation supports post-translation loss of the α-subunit in vivo To distinguish between failure of α-subunit synthesis and loss of the α-subunit after synthesis, we used a PURExpress cell-free transcription/translation system, which lacks ATP-dependent proteases and most cellular folding and maturation pathways (Figure 6B). Templates derived from the constructs listed in Table 1 were used to express both subunits together (pRSF2 and pRSF5) or individually (pRSF3 and pRSF4). In this cell-free system, both α- and β-subunits were produced efficiently, and the α-subunit was clearly visible even when expressed alone (Figure 6B). These results demonstrate that the fdh1 a transcript is fully translatable and that ribosomes can synthesize the large α-polypeptide. Because the PURExpress system lacks tungsten cofactor biosynthesis and Fe-S cluster assembly machinery, the proteins formed in vitro were necessarily apo-proteins. However, their successful synthesis shows that neither transcription nor the basic translational machinery is intrinsically limiting for Me-FDH1 production. A similar accumulation of apo-Me-FDH1 in the absence of tungsten has also been observed previously in M. extorquens and C. necator (Ryu et al., 2024), indicating that cofactor absence does not by itself prevent full-length polypeptide synthesis. The activity gap between E. coli and M. extorquens is therefore attributable to two coupled effects rather than a single cause. Cofactor insertion itself is not the primary limitation, as indicated by the stable specific activity across constructs and the lack of proportional response to tungstate supplementation. Under the most productive co-expression condition (EcK/pCM2), Fe content reaches 17.6 mol per mol protein, close to the theoretical 20 mol per mol holoenzyme and comparable to the native host (MeP4/pCM2, 18.2 mol/mol; Table 3), indicating efficient Fe-S assembly in this regime. The reduced activity primarily reflects the small pool of α-subunit that escapes proteolysis, combined with residual structural imperfections in that pool. Fe-S cluster assembly is impaired only when α- and β-subunits are not co-produced, consistent with a requirement for coordinated holoenzyme assembly. Two mechanisms could in principle explain the post-translational loss of the α-subunit: proteolytic degradation and misfolding-driven aggregation. Three observations are more consistent with a proteolytic mechanism. The insoluble fraction did not contain a detectable α-subunit band, the protein was produced intact in the protease-free PURExpress system, and co-expression of the β-subunit stabilized the α-subunit even though the β-subunit is not expected to participate directly in α-subunit folding. A contribution from misfolding cannot be excluded and would be consistent with recognition of an immature apo-form by the cellular quality control network, as discussed below. The inability of E. coli to functionally produce Me-FDH1 has been reported previously by Park et al. (Park et al., 2024), who could not obtain active enzyme in E. coli and moved to the native M. extorquens host for their structural study. The present study quantifies this limitation and localizes it to post-translational α-subunit loss. The heterologous-to-native activity ratio observed here (approximately 5–15 %) is comparable to that reported for other complex metalloenzymes produced in E. coli , including W-dependent FDHs (Maia et al., 2017; Niks and Hille, 2019), Mo-dependent FDHs (Böhmer et al., 2014; Hartmann and Leimkühler, 2013), and [FeFe]-hydrogenases (Böck et al., 2006; Kuchenreuther et al., 2010). This work therefore advances the field less by improving absolute activity and more by identifying the rate-limiting step, which defines a concrete engineering target for subsequent studies. Taken together, the data are consistent with a model in which the α-subunit is translated efficiently but remains in an immature apo-state in E. coli long enough to be recognized and degraded by ATP-dependent proteases of the host protein quality control network. The β-subunit reduces this window by stabilizing the α-subunit after translation. This maturation-coupled model, rather than a transcriptional or translational defect, is the parsimonious explanation for the observed bottleneck. A direct test of the proteolysis hypothesis will require cell-free expression supplemented with purified ATP-dependent proteases, ideally Lon and ClpXP, together with reconstitution of the complex cofactor environment of Me-FDH1 (W-bis-MGD, FMN, and multiple Fe-S clusters). W-bis-MGD and intact Fe-S clusters are not available as commercial reagents, and their in vitro supply requires dedicated maturation proteins and strictly anaerobic handling (Böhmer et al., 2014; Kuchenreuther et al., 2010). Such experiments, together with tests in protease-deficient E. coli strains, are the most immediate next step following from this work. Conclusions Although this study did not achieve high-level heterologous production of Me-FDH1 in E. coli , it identifies the dominant obstacle with much greater clarity. E. coli produced catalytically competent Me-FDH1 when tungstate uptake was supported by a functional Mod ABC system or by heterologous expression of Tup BCA (Figure 2; Table 2), showing that the host is not fundamentally incompatible with W-bis-MGD-dependent biogenesis. The major limitation instead lies in poor accumulation and incomplete maturation of the large α-subunit. Gene-organization experiments showed that the β-subunit is readily produced on its own, whereas the α-subunit is not efficiently recovered even in the native host unless the β-subunit is present, indicating that the β-subunit contributes to productive α-subunit stabilization and/or maturation (Figure 3–4; Table 3). SUMO fusion, generic chaperone co-expression, and codon harmonization did not rescue this defect (Figure 5 and Figure 6A), whereas cell-free translation readily produced the α-subunit in the absence of ATP-dependent proteases (Figure 6B), strongly supporting a model in which the α-subunit is synthesized in E. coli but is rapidly lost through folding-coupled instability and post-translational degradation. From a practical standpoint, C. necator remains the more suitable heterologous host for Me-FDH1 production at present (Ryu et al., 2024, 2025). Two non-exclusive causes may underlie the prolonged immature state of the α-subunit. The maturation-related factors of E. coli , including components of the W-bis-MGD pathway and the Fe-S assembly machinery, may be insufficient or mistimed relative to the overexpressed α- and β-subunits. Alternatively, these factors may be intrinsically unable to process the heterologous Me-FDH1 α-subunit efficiently. Sequence identity between the E. coli and M. extorquens machineries is low for several pathway steps (Table S2, see Additional File), and in R. capsulatus FDH, the dedicated maturation chaperones FdsC and FdsD are required for activity but have no close E. coli homolog (Böhmer et al., 2014; Hartmann and Leimkühler, 2013). Both mechanisms are consistent with the observed phenotype and motivate the engineering strategies outlined below. Four concrete engineering strategies are suggested by the identified bottleneck. The first is co-expression of M. extorquens maturation-relevant factors, because protein sequence identity of several steps in the W-bis-MGD pathway is low between M. extorquens and E. coli (Table S2, see Additional File). The second is comparative transcriptomic and proteomic profiling of E. coli and M. extorquens under identical cultivation conditions, to identify W-bis-MGD biosynthesis and Fe-S cluster assembly genes that are expressed at insufficient levels in E. coli relative to M. extorquens ; any such genes can then be targeted for controlled upregulation to reproduce the native expression balance. The third is fragment-based dissection of the α-subunit. The α-subunit contains multiple structural domains involved in Fe-S cluster binding and W-bis-MGD coordination, and the domain-level pattern of accumulation across individually expressed fragments can localize the region that is most susceptible to proteolysis, which can then guide rational stabilization. The fourth is controlled reduction of α-subunit expression rate, using weaker promoters or lower inducer concentrations, to balance synthesis against the available maturation capacity. These strategies are not mutually exclusive and can be combined. More broadly, however, this study defines a clear engineering roadmap for adapting E. coli to produce Me-FDH1 and related tungsten enzymes: improve α-subunit stabilization, identify and suppress the proteolytic pathway(s) responsible for α-subunit loss, and reconstruct the folding and maturation environment required for efficient holoenzyme assembly. By pinpointing the dominant bottleneck rather than merely documenting low expression, this work advances the development of robust microbial platforms for sustainable CO 2 -to-formate biocatalysis. Abbreviations ATP: Adenosine triphosphate BSA: Bovine serum albumin CDW: Cell dry weight FDH: Formate dehydrogenase Fe-S cluster: Iron-sulfur cluster [FeFe]-hydrogenases: Unique 6-iron active site [4Fe–4Fe] cluster linked to a [2Fe] subcluster His 6 tag: hexa-histidine tag HR-ICP-MS: High resolution inductively coupled plasma mass spectrometry IPTG: Isopropyl-β-D-thiogalactopyranoside Me-FDH1: Formate dehydrogenase I from Methylorubrum extorquens ModABC: High-affinity molybdate/tungstate ABC-type transporter system Ni-NTA: Nickel-Nitrilotriacetic Acid RBS: Ribosome-binding site RPM: Revolutions per minute RT-PCR: Reverse transcription polymerase chain reaction SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SUMO: Small ubiquitin-like modifier TupBCA: High-affinity tungstate ABC-type transporter system W-bis-MGD: Tungsto-bis(molybdopterin guanine dinucleotide) cofactor Declarations Ethics approval and consent to participate Not applicable. This study did not involve human participants, human data, human tissue, or animals. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. The predicted structural models generated in this study have been deposited in ModelArchive under accession numbers ma-ny5ny, ma-covi8, ma-wmubt, ma-b9uc9, ma-jp91h, and ma-ifuk9. The codon-harmonized fdh1a and fdh1b sequences have been deposited in NCBI GenBank under accession numbers PX353735 and PX353736, respectively. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2020-NR049543). Sunghoon Park acknowledges financial support from the National Research Foundation of Korea. Authors' contributions N.N.M.C. and H.R. contributed equally to this work as co-first authors. N.N.M.C. contributed to conceptualization, methodology, formal analysis, investigation, visualization, writing – original draft, writing – review & editing. H.R. contributed to conceptualization, methodology, formal analysis, investigation, visualization, writing – original draft. J.Y.P. contributed to software, methodology (codon optimization). Y.H.K. contributed to visualization, supervision. S.H.P. contributed to conceptualization, supervision, project administration, funding acquisition, visualization, writing – review & editing. All authors read and approved the final manuscript. Acknowledgements We gratefully acknowledge the UNIST Office of Research Facilities and Training (ResFacT) for providing access to shared HR-ICP-MS instrumentation and technical support. Authors' information (optional) Not applicable. References Alissandratos, A., Kim, H.K., Easton, C.J., 2014. 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Biofuels 8, 1–8. https://doi.org/10.1186/S13068-015-0353-5/TABLES/2 Tables Table 1 Plasmids and strains used in this study Plasmids Description Source pCM2 pCM110-P L/O4 -RBS b -fdh1ba-His 6 -T7 ter , Tet R (Ryu et al., 2024) pCM3 pCM110-P L/O4 - RBS b - fdh1a-His 6 - fdh1b-T7 ter , Tet R This study pCM4 pCM110-P L/O4 - RBS b - fdh1b- RBS b -fdh1a-His 6- T7 ter , Tet R This study pCM5 pCM110-P L/O4 - RBS b -His 6 - fdh1b-T7 ter -P L/O4 - RBS b - fdh1a-His 6 -T7 ter , Tet R This study pCM6 pCM110-P L/O4 - RBS b -His6- fdh1b-T7 ter , Tet R This study pCM7 pCM110-P L/O4 - RBS b - fdh1a-His6-T7 ter , Tet R This study pAC1 pACYC184, p15A ColE1 ori, Cm R , Tet R Addgene pAC2 pAC-P tac -RBS-tupBCA-T7 ter , Cm R This study pRSF1 pRSF-Duet-1, RSF1030 ori, P T7 , Kan R Addgene pRSF2 pRSF-P T7 -fdh1ba-His 6 -T7 ter , Kan R This study pRSF3 pRSF-P T7 - fdh1a-His 6 -T7 ter , Kan R This study pRSF4 pRSF-P T7 - fdh1b-His 6 -T7 ter , Kan R This study pRSF5 pRSF-P T7 - fdh1b-His 6 -T7 ter -P T7 - fdh1a-His 6 -T7 ter , Kan R This study pRSF6 pRSF-P T7 -SUMO- fdh1ba-His 6 -T7 ter , Kan R This study pRSF7 pRSF-P T7 -SUMO- fdh1a-His 6 -T7 ter , Kan R This study pRSF8 pRSF-P T7 - fdh1a codon optimized-His 6 -T7 ter , Kan R This study pET1 pET-Duet-1, pBR322-derived ColE1 ori, P T7 , Am R Addgene pET2 pET-P T7 - fdh1b codon optimized-His 6 -T7 ter , Am R This study pG-KJE8 P araBAD -dnaK-dnaJ-grpE, P lac -groES-groEL, Cm R , p15A ori TaKaRa, Japan pG-Tf2 P lac -tig, P lac -groES-groEL, Cm R , p15A ori TaKaRa, Japan Strains Description Source EcB E. coli B wildtype DSMZ EcBL E. coli BL21 (DE3) wildtype (Nguyen-Vo et al., 2020) EcBLP E. coli BL21 Star™ (DE3) pLysS wildtype New England Biolabs EcBW E. coli BW25113 wildtype (Sundara Sekar et al., 2016) EcC E. coli C ATCC8739 wildtype (Sundara Sekar et al., 2016) E. coli DH5α Cloning host Toyobo, Japan EcJM E. coli JM109 (DE3) wildtype Promega, Korea EcK E. coli K-12 MG1655 wildtype (Nguyen-Vo et al., 2020) EcKT E. coli K-12 Shuffle® T7 wildtype New England Biolabs EcMC E. coli MC1061 wildtype Thermo Fisher Scientific EcWA E. coli W ATCC 9637 wildtype (Nguyen-Vo et al., 2020) EcW E. coli W3110 wildtype ATCC EcXL E. coli XL-1 Blue wildtype New England Biolabs EcR E. coli Rosetta 2 (DE3) wildtype Novagen MeP M. extorquens PA1, fdh1 gene source DSMZ, Germany MeP4 MePΔfdh1Δfdh2Δfdh3Δfdh4 (Ryu et al., 2024) Table 2 Summary of Me-FDH1 expression in various E. coli Host strain for Me-FDH1 production Expression level (% of total soluble protein) a Enzyme activity (U/mg) b Molybdate transporter c EcB/pCM2 ~1 0 - EcBL/pCM2 ~1 0 - BLP/pCM2 ~1 0 - EcBW/pCM2 ~1 13.9 ± 2.1 + (ModABC) EcC/pCM2 ~1 <0.5 + (ModABC) EcJM/pCM2 ~1 6.6 ± 1.9 + (ModABC) EcK/pCM2 ~1 8.2 ± 0.5 + (ModABC) EcKT/pCM2 ~1 4 ± 0.8 + (ModABC) EcMC/pCM2 ~1 <0.5 + (ModABC) EcWA/pCM2 ~1 4.1 ± 0.7 + (ModABC) EcW/pCM2 ~1 3.3 ± 0.3 + (ModABC) EcXL/pCM2 ~1 <0.5 - EcR/pCM2 ~1 <0.5 - EcBL/pRSF2/pAC2 d ~1 2.6 ± 0.6 ++ (TupABC) a Expression level was estimated by SDS-PAGE densitometry of the soluble fraction obtained after cell disruption and centrifugation, and is reported as the percentage of total soluble protein. Values below the detection limit are indicated as <1 %. b Estimated from enzyme assay for formate oxidation reaction with purified samples. c Molybdate transporter system ( mod ABC) is non-specific to molybdate and possible to bind tungstate (Otrelo-Cardoso et al., 2017) d The TupABC transporter from M. extorquens PA1 was expressed for tungstate uptake (Table 1). Table 3 Quantification of Me-FDH1 during Ni-NTA affinity purification, activity of purified enzymes, and Fe content of selected proteins Strain Plasmid Cell mass (mg) Protein in crude cell extract (mg) FDH1 proteins after Ni-NTA Specific production (mg/g CDW) b Specific activity (U/mg protein) b Fe content (mol/mol) Quantity (mg) Purity (%) a EcK pCM2 1380 711.01 4.69 22 0.7 8.0 17.6 pCM3 1560 1044.08 4.03 5.0 < 0.1 0.3 14.3 pCM4 1680 1127.33 3.88 0.8 < 0.1 0.2 12.9 pCM5 1680 1630.42 10.23 72 6.4 1.3 5.7 pCM6 1650 1633.63 22.91 99 8.7 n/a 3.2 pCM7 990 578.56 2.21 0.5 < 0.1 n/a n/a MeP4 pCM2 1215 439.76 4.38 99 3.6 84.5 18.2 pCM3 1097 199.41 1.07 99 1.0 78.7 16.5 pCM4 1207 527.24 1.33 99 1.1 63.7 19.6 pCM5 1284 451.52 6.61 99 5.1 65.9 22.4 pCM6 1359 559.68 2.54 80 1.5 n/a 2.5 pCM7 1174 368.69 0.74 9 < 0.1 n/a n/a a Determined by densitometry on SDS-PAGE b Adjusted for purity. Supplementary Files AdditionalFile.docx Additional file. Supplementary figures, tables, and texts. Word file containing Figures S1–S6, Tables S1–2, and supplementary text supporting the results of this study. Graphicalabstract.png Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 28 Apr, 2026 Reviewers invited by journal 26 Apr, 2026 Editor assigned by journal 24 Apr, 2026 First submitted to journal 23 Apr, 2026 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-9133619","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629959615,"identity":"a41145c6-3f6f-4655-aa69-03aaab397cd6","order_by":0,"name":"Ngoc Minh Chau Nguyen","email":"","orcid":"","institution":"Ulsan National Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ngoc","middleName":"Minh Chau","lastName":"Nguyen","suffix":""},{"id":629959616,"identity":"d0ac0566-b393-40c7-a87a-230c973c0270","order_by":1,"name":"Huichang Ryu","email":"","orcid":"","institution":"Ulsan National Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Huichang","middleName":"","lastName":"Ryu","suffix":""},{"id":629959617,"identity":"b06c9e2d-6193-4814-a2fb-394a9388386b","order_by":2,"name":"Joon Young Park","email":"","orcid":"","institution":"KRICT: Korea Research Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Joon","middleName":"Young","lastName":"Park","suffix":""},{"id":629959618,"identity":"0c091e09-5dc6-4b53-853c-243b8a4de476","order_by":3,"name":"YongHwan Kim","email":"","orcid":"","institution":"Ulsan National Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"YongHwan","middleName":"","lastName":"Kim","suffix":""},{"id":629959619,"identity":"66717cbc-011a-46c7-9520-51ed34b2e125","order_by":4,"name":"Sunghoon Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYHACZgbGBhsQBQESRGpJg1CkaDkMYhCpxZz97GPDnzvOJ25nZ3/+gKHGjkFy9gH8Wix70o2Tec/cTtzZzGPYwHAsmUGaLwG/FoMDacyHGdtuJ244zAN0GNsBBjkeAg4zOP+M+eDPtnNALewPGxj+EaPlRhpzAm/bAaAWBsMGxrYDDNKEtFjOeMZszNuWbAx0mOGMxL5kHskeAlrM+dOYJX+22cluOH/8wYcP3+zkJM4QchgKL4GBgZCz0LWMglEwCkbBKMAGAAT1P4l9gM96AAAAAElFTkSuQmCC","orcid":"","institution":"Ulsan National Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Sunghoon","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2026-03-16 06:20:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9133619/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9133619/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108538911,"identity":"c973e599-d6dd-4b57-8482-d97f41eaaa11","added_by":"auto","created_at":"2026-05-05 17:56:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":315622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArchitecture of plasmids used to express Me-FDH1.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9133619/v1/c34d484ac08ba21609dada31.png"},{"id":108538913,"identity":"a7d7b6a1-e74e-48f7-9099-022f4764347a","added_by":"auto","created_at":"2026-05-05 17:56:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":760883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression and activity of Me-FDH1 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eK-12. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) SDS-PAGE; (\u003cstrong\u003eB\u003c/strong\u003e) Western blot; (\u003cstrong\u003eC\u003c/strong\u003e) Enzyme activity of crude cell lysate and purified enzyme. EcK and EcK/pCM2 represent \u003cem\u003eE. coli \u003c/em\u003eK-12 wildtype and recombinant host harboring pCM2, respectively, MeP4/pCM2 represents recombinant host harboring pCM2. In (\u003cstrong\u003eA\u003c/strong\u003e) and (\u003cstrong\u003eB\u003c/strong\u003e); lane M indicates protein ladder, lane CE indicates crude cell extract, lane PU indicates Ni-affinity purified protein sample. Red arrows indicate the band corresponding to alpha (108 kDa) and beta (62 kDa) subunits. Enzyme activity values were measured based on formate oxidation with triplicate measurements averaged, and the error was less than 5%.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9133619/v1/96bdd7a579caae644baf05f3.png"},{"id":108538915,"identity":"ed3df769-d110-4e82-aa22-d26b9c404dad","added_by":"auto","created_at":"2026-05-05 17:56:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":521573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of gene organization for heterologous expression of Me-FDH1. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) SDS-PAGE analysis shows protein expression in EcK strain; (\u003cstrong\u003eB\u003c/strong\u003e) SDS-PAGE analysis shows protein expression in MeP4 strain. Lane M, protein ladder; Lane CE, crude cell extract; Lane PU, purified protein. Red arrows indicate the band corresponding to alpha (108 kDa) and beta (62 kDa) subunits.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9133619/v1/afe61fad92375eba763d2d19.png"},{"id":108538918,"identity":"d1635b35-ca71-453d-9ed5-5a3f52c67a3e","added_by":"auto","created_at":"2026-05-05 17:56:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":174890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of mRNA synthesis from various gene organizations for heterologous expression of Me-FDH1 by RT-PCR. \u003c/strong\u003eRelative mRNA levels were quantified by RT-PCR and normalized against the housekeeping gene \u003cem\u003erpo\u003c/em\u003eD as a control. The relative mRNA levels were calculated as the average value from triplicate measurements, with the error in each measurement being less than 5%.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9133619/v1/332865db09dd42c9206900aa.png"},{"id":108538916,"identity":"8fa2ac6b-adeb-4ab6-9e87-468c305f9786","added_by":"auto","created_at":"2026-05-05 17:56:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":211037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of SUMO tag and co-expression of chaperones on Me-FDH1 expression in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli. \u003c/strong\u003e\u003c/em\u003e(\u003cstrong\u003eA\u003c/strong\u003e) SDS-PAGE analysis of the effect of SUMO-tagged Me-FDH1 in \u003cem\u003eE. coli\u003c/em\u003e K-12 Shuffle® T7; (\u003cstrong\u003eB\u003c/strong\u003e) SDS-PAGE analysis of the effect of chaperone co-expression in \u003cem\u003eE. coli\u003c/em\u003eK-12 MG1655; (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eEnzyme activity assay for formate oxidation. Lane M, protein ladder; Lane CE, crude cell extract; Lane PU, purified protein. Red arrows indicate the band corresponding to alpha (108 kDa) and beta (62 kDa) subunits. Enzyme activity values were measured based on formate oxidation with triplicate measurements averaged, and the error was less than 5%.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9133619/v1/4ddb0ef3ed2ca076482452d2.png"},{"id":108804408,"identity":"8bc58be3-89e6-4f0e-9429-aae60ff285f6","added_by":"auto","created_at":"2026-05-08 15:20:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":412816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCodon optimization and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e translation of Me-FDH1. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) SDS-PAGE analysis of codon-optimized \u003cem\u003efdh1\u003c/em\u003e expression in \u003cem\u003eE. coli\u003c/em\u003e K-12 Shuffle® T7; (\u003cstrong\u003eB\u003c/strong\u003e) SDS-PAGE analysis of Me-FDH1 produced in an \u003cem\u003ein vitro\u003c/em\u003e expression reaction. Lane M, protein ladder; Lane CE, crude cell extract; Lane PU, purified protein. Red arrows indicate the band corresponding to alpha (108 kDa) and beta (62 kDa) subunits.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9133619/v1/2ff0ae812b10e0ba631daa70.png"},{"id":108811398,"identity":"961a268e-3e24-4a60-8088-01498f99b573","added_by":"auto","created_at":"2026-05-08 16:04:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2877607,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9133619/v1/5a52190d-a619-4c08-b1d5-b9d389818964.pdf"},{"id":108538912,"identity":"99146d2e-7d11-4acb-bd47-a35700988707","added_by":"auto","created_at":"2026-05-05 17:56:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7179600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file. Supplementary figures, tables, and texts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWord file containing Figures S1–S6, Tables S1–2, and supplementary text supporting the results of this study.\u003c/p\u003e","description":"","filename":"AdditionalFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-9133619/v1/a89d0729535e4a1bf39e3d92.docx"},{"id":108804057,"identity":"cd5d4e91-166f-4472-9e6a-ba57e9da54c5","added_by":"auto","created_at":"2026-05-08 15:15:09","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":154110,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-9133619/v1/55b5eccfb456edfad25681f6.png"}],"financialInterests":"","formattedTitle":"Heterologous production of tungsten-dependent formate dehydrogenase I from Methylorubrum extorquens in Escherichia coli reveals α-subunit maturation as the major bottleneck","fulltext":[{"header":"Key Points","content":"\u003cul\u003e\n \u003cli\u003e\u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eproduced active Me-FDH1 when tungstate uptake was enabled.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eModABC or TupBCA supported W-bis-MGD-dependent Me-FDH1 activity.\u003c/li\u003e\n \u003cli\u003eThe \u0026alpha;-subunit was the major bottleneck in Me-FDH1 production.\u003c/li\u003e\n \u003cli\u003eThe \u0026beta;-subunit promoted \u0026alpha;-subunit accumulation and/or maturation.\u003c/li\u003e\n \u003cli\u003eCell-free translation revealed post-translational loss of the \u0026alpha;-subunit.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe enzymatic reduction of carbon dioxide to formate is an attractive route for carbon capture and utilization because formate can serve both as a C1 chemical feedstock and as a liquid hydrogen carrier (Jang et al., 2018). Formate dehydrogenases (FDHs) are therefore important biocatalysts for sustainable CO\u003csub\u003e2\u003c/sub\u003e conversion. Among them, formate dehydrogenase I from \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e (Me-FDH1) is particularly attractive because it efficiently catalyzes the interconversion of CO\u003csub\u003e2\u003c/sub\u003e and formate and also supports direct electron-transfer bioelectrocatalysis, making it relevant to enzymatic and electrochemical carbon-recycling systems (Laukel et al., 2003; Jang et al., 2018; Yoshikawa et al., 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMe-FDH1 is unusual among FDHs in that it contains a tungsten cofactor rather than the more common molybdenum cofactor (Park et al., 2024). The enzyme is a heterodimer composed of an approximately 107 kDa \u0026alpha;-subunit and a 62 kDa \u0026beta;-subunit. The \u0026alpha;-subunit carries a tungsto-bis(molybdopterin guanine dinucleotide) cofactor (W-bis-MGD), whereas multiple Fe-S clusters are distributed across both subunits (Yoshikawa et al., 2022). This complex architecture likely underlies the high catalytic performance of Me-FDH1, but it also makes heterologous production demanding because active holoenzyme formation requires coordinated expression of two large subunits, correct folding, W-bis-MGD biosynthesis and insertion, and proper Fe-S cluster assembly.\u003c/p\u003e\n\u003cp\u003eMe-FDH1 can be produced efficiently in its native host, \u003cem\u003eM. extorquens\u003c/em\u003e, where recombinant expression under a strong promoter yields high activity and high intracellular accumulation (Jang et al., 2018; Ryu et al., 2024). However, homologous production in \u003cem\u003eM. extorquens\u003c/em\u003e is not ideal for large-scale application because the organism grows relatively slowly, is less convenient for high-cell-density cultivation, and relies on less convenient carbon sources such as methanol or succinate (Ryu et al., 2024, 2025). More recently, \u003cem\u003eCupriavidus necator\u003c/em\u003e H16 was shown to be an effective heterologous host, yielding recombinant Me-FDH1 with near-native activity and high volumetric productivity (Ryu et al., 2024, 2025).\u0026nbsp;Nevertheless, recombinant Me-FDH1 still underwent degradation during later cultivation, and deeper host engineering is less straightforward than in \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003e(Park et al., 2024; Ryu et al., 2024, 2025).\u003c/p\u003e\n\u003cp\u003eThese considerations make \u003cem\u003eE. coli\u003c/em\u003e an attractive candidate host. \u003cem\u003eE. coli\u003c/em\u003e grows rapidly, reaches high cell density, and offers unmatched genetic accessibility. The key question, however, is whether \u003cem\u003eE. coli\u003c/em\u003e can be adapted to produce a catalytically competent tungsten-containing metalloenzyme as complex as Me-FDH1. In this study, we systematically evaluated \u003cem\u003eE. coli\u003c/em\u003e as a host for Me-FDH1 production and used this analysis to identify the dominant bottleneck. We tested multiple \u003cem\u003eE. coli\u003c/em\u003e strains with different tungstate/molybdate transport capacities, examined operon architecture using a series of recombinant plasmids, and evaluated SUMO fusion, chaperone co-expression, and codon harmonization as strategies to improve \u0026alpha;-subunit production. We further analyzed transcript levels by RT-PCR and used a cell-free translation system to distinguish between failure of \u0026alpha;-subunit synthesis and loss of the \u0026alpha;-subunit after synthesis. By comparing the results obtained in \u003cem\u003eE. coli\u003c/em\u003e with those from \u003cem\u003eM. extorquens\u003c/em\u003e and a previously developed heterologous host, we aimed to define the host functions that must be engineered to support robust production of Me-FDH1 and related tungsten-dependent enzymes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\n\u003cp\u003eVarious \u003cem\u003eE. coli\u003c/em\u003e strains, including B, BL21 (DE3), BL21 Star\u0026trade; (DE3) pLysS, BW25113, C ATCC8739, JM109 (DE3), K-12 MG1655, K-12 Shuffle\u003csup\u003e\u0026reg;\u003c/sup\u003e T7, MC1061, W, W3110, XL-1 Blue and Rosetta 2 (DE3) and a derivative of \u003cem\u003eM. extorquens\u003c/em\u003e PA1 lacking all four native FDH genes (MeP4; \u0026Delta;\u003cem\u003efdh\u003c/em\u003e1\u0026ndash;4) were used in this study (Table 1). \u003cem\u003eE. coli\u003c/em\u003e DH5\u0026alpha; (Toyobo, Japan) was used for gene cloning. DNA sequencing and primer synthesis were performed by Macrogen (Korea). In-Fusion\u003csup\u003e\u0026reg;\u003c/sup\u003e Snap Assembly Master Mix (TaKaRa, Japan), restriction endonucleases and T4 DNA ligase (New England Biolabs, USA), and Pfu-X DNA polymerase with standard PCR reagents (SolGent, Korea) were used for plasmid construction. Plasmids and genomic DNA were prepared with commercial kits according to the manufacturers\u0026rsquo; instructions. Isopropyl-\u0026beta;-D-thiogalactopyranoside (IPTG; Bio Basic, Canada) was used for protein induction, and all other reagents were of analytical grade unless otherwise noted.\u003c/p\u003e\n\u003ch2\u003ePlasmid construction and strains preparation\u003c/h2\u003e\n\u003cp\u003eGenes encoding the Me-FDH1 \u0026alpha;- and \u0026beta;-subunits (\u003cem\u003efdh1\u003c/em\u003ea and \u003cem\u003efdh1\u003c/em\u003eb) were amplified from \u003cem\u003eM. extorquens\u003c/em\u003e PA1 genomic DNA and cloned under the IPTG-inducible P\u003csub\u003eL/O4\u003c/sub\u003e promoter in the pBBR1-derived broad-host-range vector pCM110, generating pCM2 (Figure 1; Table 1). The vector carries \u003cem\u003elac\u003c/em\u003eI and is compatible with \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eM. extorquens\u003c/em\u003e, and \u003cem\u003eC. necator\u003c/em\u003e. Additional plasmids were constructed to evaluate the effects of gene organization, solubility tags, chaperone co-expression, and codon harmonization on heterologous expression. All plasmid backbones are summarized in Table 1, and all primers are listed in Table S1 (see Additional File).\u003c/p\u003e\n\u003cp\u003eThe reference construct pCM2 contained the native \u003cem\u003efdh1\u003c/em\u003eb\u0026ndash;\u003cem\u003efdh1\u003c/em\u003ea arrangement, including the 52 nucleotides (52-nt) intergenic region, and a C-terminal hexa-histidine tag (His\u003csub\u003e6\u003c/sub\u003e-tag) on the \u0026alpha;-subunit. RNA secondary structure of the 52-nt intergenic region was predicted using RNAfold ViennaRNA package (Lorenz et al., 2011) and the model was deposited in ModelArchive ma-ny5ny (see Additional File: Figure S1). In pCM3, the gene order was reversed and the \u0026beta;-subunit carried an N-terminal His\u003csub\u003e6\u003c/sub\u003e-tag. In pCM4, the native gene order was retained, but a strong synthetic ribosome-binding site (RBS) was introduced upstream of \u003cem\u003efdh1a\u003c/em\u003e. In pCM5, the \u0026alpha;- and \u0026beta;-subunits were expressed from separate promoter-RBS units. Single-subunit plasmids were also constructed: pCM6 encoded His\u003csub\u003e6\u003c/sub\u003e-tagged \u003cem\u003efdh1b\u003c/em\u003e, whereas pCM7 encoded His\u003csub\u003e6\u003c/sub\u003e-tagged \u003cem\u003efdh1a\u003c/em\u003e with a strong RBS (Figure 1; Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlasmids pCM4, pCM6, and pCM7 were derived from pCM2 by restriction digestion, PCR amplification of the desired fragments, and In-Fusion- or ligase-based assembly. pCM3 and pCM5 were subsequently constructed from the pCM6 backbone by insertion of PCR-amplified \u003cem\u003efdh1a\u003c/em\u003e-containing fragments and, for pCM5, an additional promoter-terminator cassette. When appropriate restriction sites were unavailable, fragments were combined by overlap PCR. All constructs were verified by restriction analysis and DNA sequencing.\u003c/p\u003e\n\u003cp\u003eFor specific tungstate transport, the high-affinity \u003cem\u003etup\u003c/em\u003eBCA operon was amplified from \u003cem\u003eM. extorquens\u003c/em\u003e PA1 and cloned into the tac-promoter vector pAC1, generating pAC2 (Figure 1; Table 1). The operon corresponds to \u003cem\u003eMext\u003c/em\u003e_2850\u0026ndash;\u003cem\u003eMext\u003c/em\u003e_2852. This construct was used to complement \u003cem\u003eE. coli\u003c/em\u003e strains lacking an effective endogenous tungstate uptake route.\u003c/p\u003e\n\u003cp\u003eTo examine whether general folding support improves Me-FDH1 production, the chaperone plasmids pG-KJE8 and pG-Tf2 (TaKaRa) were used. pG-KJE8 expresses \u003cem\u003eDnaK\u003c/em\u003e-\u003cem\u003eDna\u003c/em\u003eJ-\u003cem\u003eGrp\u003c/em\u003eE together with \u003cem\u003eGro\u003c/em\u003eEL-\u003cem\u003eGro\u003c/em\u003eES, whereas pG-Tf2 expresses \u003cem\u003eGro\u003c/em\u003eEL-\u003cem\u003eGro\u003c/em\u003eES with trigger factor (Nishihara et al., 1998, 2000).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCulture conditions and production of recombinant Me-FDH1\u003c/h2\u003e\n\u003cp\u003eFor \u003cem\u003eE. coli\u003c/em\u003e, a modified M9 medium supplemented with 1 g/L yeast extract and 10 g/L glucose was used as described previously (Ryu et al., 2024, 2025). To support tungsten cofactor formation, sodium tungstate (Na\u003csub\u003e2\u003c/sub\u003eWO\u003csub\u003e4\u003c/sub\u003e, 30 \u0026mu;M) was added to the medium after autoclaving by filter sterilization (Ryu et al., 2025). Cultures were grown at 30\u003csup\u003eo\u003c/sup\u003eC and 200 rpm to mid-log phase (OD\u003csub\u003e600\u003c/sub\u003e ~0.4\u0026ndash;0.8), induced with 0.5 mM IPTG, and incubated at 30\u003csup\u003eo\u003c/sup\u003eC for 16\u0026ndash;18 h. When chaperone plasmids were used, arabinose (0.5 mg/mL) or tetracycline (10 ng/mL) was added together with IPTG according to the plasmid system. Antibiotics were used at the following concentrations: kanamycin 50 \u0026micro;g/mL, ampicillin 100 \u0026micro;g/mL, and tetracycline 10 \u0026micro;g/mL. Cultivation and induction of \u003cem\u003eM. extorquens\u003c/em\u003e MeP4 carrying pCM plasmids were performed as described previously (Jang et al., 2018; Ryu et al., 2024, 2025)\u003c/p\u003e\n\u003ch2\u003eProtein extraction, purification and expression analysis\u003c/h2\u003e\n\u003cp\u003eCells were harvested after induction, washed, and resuspended in lysis buffer (20 mM MOPS, 200 mM NaCl, 20 mM imidazole). Cell disruption was performed by sonication on ice, and soluble and insoluble fractions were separated by centrifugation (13000 \u003cem\u003eg\u003c/em\u003e, 10\u0026ndash;20 min, 4\u003csup\u003eo\u003c/sup\u003eC) as described previously (Ryu et al., 2024, 2025).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHis\u003csub\u003e6\u003c/sub\u003e-tag proteins were purified from crude extracts under nondenaturing conditions using Nickel-Nitrilotriacetic Acid (Ni-NTA) Bind Resin column (Qiagen). Eluted proteins were concentrated and buffer-exchanged with Amicon\u003csup\u003e\u0026reg;\u003c/sup\u003e Ultra-15 centrifugal filters (30 kDa cutoff; Millipore, Darmstadt, Germany). Protein concentrations were determined by the Bradford assay. Expression levels were estimated by SDS\u0026ndash;PAGE densitometry against bovine serum albumin (BSA) standards (Thermo Scientific) and are reported as the combined intensities of the \u0026alpha;- and \u0026beta;-subunit bands when both were present.\u003c/p\u003e\n\u003cp\u003eProtein production was analyzed by SDS-PAGE and Western blotting. His\u003csub\u003e6\u003c/sub\u003e-tagged proteins were detected with a mouse monoclonal anti-His\u003csub\u003e6\u003c/sub\u003e antibody and an alkaline phosphatase-conjugated rabbit anti-mouse secondary antibody IgG H\u0026amp;L (Abcam, ab97043) followed by BCIP/NBT Liquid Substrate System (Sigma-Aldrich, B1911-100ML) development.\u003c/p\u003e\n\u003ch2\u003eEnzyme activity assay\u003c/h2\u003e\n\u003cp\u003eEnzyme activity was measured at 30\u003csup\u003eo\u003c/sup\u003eC for 1 min under conditions adapted from a previous study (Jang et al., 2018). For the formate oxidation assay, reactions were performed at pH 7.0 with 30 mM sodium formate, and NADH formation was monitored at 340 nm (\u0026epsilon;\u003csub\u003e340\u003c/sub\u003e = 6220 M\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-1\u003c/sup\u003e). All assays were performed in triplicate. One unit (U, \u0026micro;mol min\u003csup\u003e-1\u003c/sup\u003e) of activity was defined as the amount of enzyme that formed 1 \u0026mu;mol of NADH per min under the assay conditions.\u003c/p\u003e\n\u003cp\u003eRT-PCR and metal analysis\u003c/p\u003e\n\u003cp\u003eTo quantify\u003cem\u003e\u0026nbsp;fdh1\u003c/em\u003ea and \u003cem\u003efdh1\u003c/em\u003eb transcripts in \u003cem\u003eE. coli\u003c/em\u003e, cells were grown in modified M9 medium and induced at mid-log phase. Pellets were immediately stabilized with RNAprotect\u003csup\u003eTM\u003c/sup\u003e Bacteria Reagent (Qiagen) and total RNA was isolated with the NucleoSpin RNA isolation kit (Macherey-Nagel, Germany). First-strand cDNA was synthesized with the iScript\u003csup\u003eTM\u003c/sup\u003e cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed on a StepOne Real-Time PCR system (Applied Biosystems, USA) using SYBR Green chemistry. Transcript levels were normalized to the housekeeping gene \u003cem\u003erpo\u003c/em\u003eD (RNA polymerase sigma factor), and relative mRNA levels were calculated using the \u0026Delta;\u0026Delta;Ct method (Zhou et al., 2015). All assays were performed in duplicate, and no-template controls were included as negative controls.\u003c/p\u003e\n\u003cp\u003eIron contents of purified protein preparations were measured by HR-ICP-MS as described previously (Ryu et al., 2024, 2025). Purified proteins were digested in nitric acid before analysis. Tungsten content of the \u0026alpha;-subunit was measured for selected samples when sufficient material was available.\u003c/p\u003e\n\u003ch2\u003eCodon harmonization\u003c/h2\u003e\n\u003cp\u003eCodon optimization was performed by codon harmonization, in which host codon usage is adjusted to approximate native translation kinetics (Schmidt et al., 2023) . Codon frequencies were weighted by amino acid abundance in the complete host proteome. The abundance of amino acid A was defined by Equation (1) (see Additional File: Figure S2):\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"325\" height=\"57\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere, n(\u003cem\u003eA\u003c/em\u003e) is the number of occurrences of amino acid \u003cem\u003eA\u003c/em\u003e, and \u0026sum;n(\u003cem\u003eA\u003c/em\u003e) is the total number of amino acids in the proteome.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTranslation speed for each codon was estimated according to Equation (2) (see Additional File: Figure S2):\u003c/p\u003e\n\u003cp\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"466\" height=\"34\"\u003e\u003c/v:shape\u003e\u003cbr\u003e\n\u003c/p\u003e\n\u003cp\u003eThe harmonized \u003cem\u003efdh1a\u003c/em\u003e and \u003cem\u003efdh1b\u003c/em\u003e sequences for\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e were deposited in NCBI GenBank under accession numbers PX353735 and PX353736, respectively. The codon-harmonized genes were cloned into pRSF-Duet-1 and pET-Duet-1 (Figure 1) to generate pRSF8 and pET2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e translation\u003c/p\u003e\n\u003cp\u003eTo test whether the \u0026alpha;-subunit can be synthesized in the absence of cellular proteolysis, cell-free expression was performed with the PURExpress\u003csup\u003e\u0026reg;\u003c/sup\u003e \u003cem\u003eIn vitro\u003c/em\u003e Protein Synthesis Kit (New England Biolabs, USA) supplemented with murine RNase Inhibitor (NEB). PCR fragments containing the promoter, gene, and terminator regions were amplified from pRSF2, pRSF3, pRSF4, and pRSF5 using the primer pairs listed in Table S1 (see Additional File) and were purified before use as templates.\u003c/p\u003e\n\u003cp\u003eEach 25 \u0026micro;L reaction contained 10 \u0026mu;L Solution A, 7.5 \u0026mu;L Solution B, 0.5 \u0026mu;L RNase inhibitor, approximately 0.9\u0026ndash;1.0 \u0026mu;g purified PCR template, and nuclease-free water. Reactions were incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 4 h and then cooled on ice. Products were concentrated with 10 kDa MWCO centrifugal filter (Amicon\u003csup\u003e\u0026reg;\u003c/sup\u003e Ultra-0.5) and analyzed by SDS-PAGE. Because the PURExpress system lacks ATP-dependent proteases and most maturation pathways, it allows detection of proteins that may be unstable \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eExpression of Me-FDH1 in \u003cem\u003eEscherichia coli\u003c/em\u003e K-12\u003c/p\u003e\n\u003cp\u003eMe-FDH1 was first expressed in wild-type \u003cem\u003eE. coli\u003c/em\u003e K-12 MG1655 carrying pCM2, the reference plasmid described in Figure 1 and Table 1. Cultures were supplemented with sodium tungstate (30 \u0026mu;M) to support W-bis-MGD biosynthesis. Although SDS-PAGE of crude extracts did not reveal distinct bands at the expected positions of the \u0026alpha;- and \u0026beta;-subunits, indicating very low expression in the host background (Figure 2A), Western blot analysis using an anti-His antibody clearly detected the His\u003csub\u003e6\u003c/sub\u003e-tagged \u0026alpha;-subunit in both crude extracts and Ni-NTA purified fractions (Figure 2B).\u0026nbsp;After purification, both \u0026alpha;- and \u0026beta;-subunits became visible by SDS-PAGE, consistent with co-purification of the \u0026beta;-subunit through association with the His\u003csub\u003e6\u003c/sub\u003e-tagged \u0026alpha;-subunit, as reported previously for Me-FDH1\u0026nbsp;(Ryu et al., 2024, 2025)\u0026nbsp;(Figure 2A, B; Table 2).\u003c/p\u003e\n\u003cp\u003eFormate oxidation activity was not detectable in the crude cell extract, but the purified enzyme showed a specific activity of 8.2 \u0026plusmn; 0.5 U mg\u003csup\u003e-1\u003c/sup\u003e (Figure 2C; Table 2). Because Me-FDH1 activity requires proper insertion of the tungsten-containing cofactor (W-bis-MGD), this result indicates that \u003cem\u003eE. coli\u003c/em\u003e can produce catalytically competent Me-FDH1. However, the activity remained far below that of the same enzyme produced in the homologous \u003cem\u003eM. extorquens\u003c/em\u003e system, where purified recombinant Me-FDH1 reached 84.5 U mg\u003csup\u003e-1\u003c/sup\u003e under comparable expression conditions (Table 3), consistent with previous reports of approximately 80\u0026ndash;100 U mg\u003csup\u003e-1\u003c/sup\u003e for the native/homologous enzyme (Park et al., 2024; Ryu et al., 2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImportance of molybdate/tungstate transport in \u003cem\u003eE. coli\u003c/em\u003e strains\u003c/p\u003e\n\u003cp\u003eBecause the K-12 result suggested that \u003cem\u003eE. coli\u003c/em\u003e can synthesize and insert W-bis-MGD, we next examined Me-FDH1 production across a broader panel of \u003cem\u003eE. coli\u003c/em\u003e strains listed in Table 1 and summarized in Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe strain panel was selected to probe four distinct physiological and engineering features that could limit Me-FDH1 production. T7-based high-expression strains, namely BL21 (DE3), BL21 Star (DE3) pLysS, and JM109 (DE3), were used to evaluate transcriptional and translational capacity; JM109 (DE3) has previously supported functional heterologous expression of several formate dehydrogenases (Alissandratos et al., 2014). Rosetta 2 (DE3) supplies rare tRNAs for codons that are infrequent in \u003cem\u003eE. coli\u003c/em\u003e (Lipinszki et al., 2018). K-12 Shuffle T7 provides a redox-engineered cytoplasm compatible with disulfide-bond formation (Lobstein et al., 2012). K-12-derived and natural-isolate strains (MG1655, BW25113, W3110, W, B, C, XL-1 Blue, MC1061) span different ModABC uptake statuses and physiological backgrounds, and MC1061 has been used previously for heterologous expression of FDH cofactor-insertion chaperones (B\u0026ouml;hmer et al., 2014). This design allowed us to separate the contribution of metal uptake from the contribution of the host expression machinery.\u003c/p\u003e\n\u003cp\u003eAs in K-12, none of the strains showed clearly visible Me-FDH1 bands in crude lysates by SDS-PAGE, whereas Western blot analysis detected the \u0026alpha;-subunit broadly across recombinant strains (see Additional File: Figure S3). After Ni-affinity purification, active Me-FDH1 was recovered only from a subset of strains, with purified-enzyme specific activities ranging from 3.3 to 13.9 U mg\u003csup\u003e-1\u003c/sup\u003e (Table 2). Among these, BW25113 gave the highest activity, followed by K-12 MG1655, JM109, WA, and W3110 (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA notable common feature of the active strains was the presence of a functional \u003cem\u003emod\u003c/em\u003eABC molybdate transporter system (Table 2). The \u003cem\u003eMod\u003c/em\u003eABC transporter is known to transport tungstate as well as molybdate, albeit with lower specificity than dedicated tungstate transporters (Leimk\u0026uuml;hler et al., 2011; Otrelo-Cardoso et al., 2017). In contrast, strains lacking a functional ModABC system showed no detectable Me-FDH1 activity (Table 2). One exception was \u003cem\u003eE. coli\u003c/em\u003e C, which carries \u003cem\u003emod\u003c/em\u003eABC but still showed little or no activity, indicating that transporter availability is necessary but not sufficient. Because all tested \u003cem\u003eE. coli\u003c/em\u003e strains inherently possess the molybdopterin biosynthetic machinery (see Additional File: Figure S3\u0026ndash;S4; Table S2), these results suggest that tungsten uptake, rather than the absence of the cofactor-biosynthetic pathway itself, is the primary requirement for active Me-FDH1 formation in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo test this interpretation directly, the high-affinity tungstate transporter \u003cem\u003eTup\u003c/em\u003eBCA from \u003cem\u003eM. extorquens\u003c/em\u003e was introduced on plasmid pAC2 (Figure 1; Table 1) into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3), which lacks a functional \u003cem\u003eMod\u003c/em\u003eABC system and did not produce active Me-FDH1 from pCM2 alone (Table 2). In both EcBL/pCM2/pAC2 and EcBL/pRSF2/pAC2, the \u0026alpha;-subunit became detectable after purification, and the purified enzyme displayed activity of approximately 2.6 U mg\u003csup\u003e-1\u003c/sup\u003e (Table 2; see Additional File: Figure S5). Thus, either native \u003cem\u003eMod\u003c/em\u003eABC or heterologous \u003cem\u003eTup\u003c/em\u003eBCA can provide the metal-uptake route required for Me-FDH1 maturation in \u003cem\u003eE. coli\u003c/em\u003e. At the same time, the \u003cem\u003eTup\u003c/em\u003eBCA-complemented strains still produced only low amounts of weakly active enzyme, indicating that tungsten uptake is necessary for activity but is not the dominant bottleneck for production yield.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile tungsten uptake is required for Me-FDH1 maturation, the present data indicate that it is not the dominant limiting factor under the conditions tested. Increasing tungstate supplementation and the expression of TupBCA did not result in proportional increases in enzymatic activity. Furthermore, constructs that differ substantially in \u0026alpha;-subunit accumulation show comparable specific activity per unit of recovered protein (Table 2). Together, these observations indicate that the primary limitation arises upstream of cofactor insertion, most likely at the level of \u0026alpha;-subunit maturation. Quantitative analysis of intracellular tungsten incorporation (for example by ICP-MS) will be required to refine this conclusion and is an important direction for future work.\u003c/p\u003e\n\u003cp\u003eEffect of gene organization on expression of Me-FDH1\u003c/p\u003e\n\u003cp\u003eThe preceding experiments indicated two major problems when Me-FDH1 is produced in \u003cem\u003eE. coli\u003c/em\u003e: (i) low enzyme yield and (ii) low specific activity. To determine whether these problems arise at the transcriptional or post-transcriptional level, we constructed a series of plasmids with altered operon architectures (Figure 1, 3; Table 1). The reference construct pCM2 contains the native \u003cem\u003efdh1\u003c/em\u003eb\u0026ndash;\u003cem\u003efdh1\u003c/em\u003ea arrangement separated by a 52-nt intergenic region. This region was predicted to form an extensive secondary structure (see Additional File: Figure S1; ModelArchive: ma-ny5ny), which could in principle influence transcript stability or translation efficiency through a riboswitch-like mechanism (Serganov and Nudler, 2013). Accordingly, pCM3, pCM4, and pCM5 were designed to alter gene order, translation signals, or operon structure, whereas pCM6 and pCM7 expressed the \u0026beta;- and \u0026alpha;-subunits individually (Figure 1; Table 1).\u003c/p\u003e\n\u003cp\u003eRT-PCR analysis showed that all constructs produced full-length transcripts at comparable levels (Figure 4). Quantitative RT-PCR was normalized to \u003cem\u003erpo\u003c/em\u003eD, using the \u0026Delta;\u0026Delta;Ct approach described previously (Zhou et al., 2015). This result indicates that transcription of \u003cem\u003efdh1\u003c/em\u003ea and \u003cem\u003efdh1\u003c/em\u003eb is not the primary bottleneck and that the native 52-nt intergenic region does not cause severe premature transcription termination under the tested conditions.\u0026nbsp;The observed invariance of full-length transcript levels across pCM2 to pCM5 (Figure 4) and the efficient translation of these constructs in the PURExpress cell-free system (see Section \u0026ldquo;\u003cem\u003eIn vitro\u003c/em\u003e translation\u0026rdquo;) together indicate that mRNA secondary structure or translational coupling, although predicted by RNAfold, does not impose a dominant \u003cem\u003ein vivo\u003c/em\u003e constraint. Direct structural probing by SHAPE (Selective 2\u0026rsquo;-Hydroxyl Acylation analyzed by Primer Extension) or DMS-MaPseq (Dimethyl Sulfate Mutational Profiling with sequencing) was therefore not pursued. Although ribosome profiling was not performed, the efficient translation of both subunits in PURExpress, combined with comparable mRNA levels across constructs (Figure 4), provides direct evidence that translation initiation and elongation are not rate-limiting for Me-FDH1 production in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn contrast, protein recovery and enzyme performance were strongly affected by plasmid configuration (Figure 3A; Table 3). In \u003cem\u003eE. coli\u003c/em\u003e K-12, pCM2 gave the best balance of \u0026alpha;-subunit accumulation and enzyme activity, yielding 0.7 mg g\u003csup\u003e-1\u003c/sup\u003e CDW purified Me-FDH1 with a specific activity of 8.0 U mg\u003csup\u003e-1\u003c/sup\u003e (Table 3). Reversing gene order (pCM3) or introducing a stronger downstream RBS (pCM4) sharply reduced \u0026alpha;-subunit recovery and lowered activity to 0.3 and 0.2 U mg\u003csup\u003e-1\u003c/sup\u003e, respectively (Table 3). By contrast, pCM5, in which the two genes were separated into individual operons, gave a large increase in recovered protein because the \u0026beta;-subunit accumulated efficiently, but the specific activity remained low (1.3 U mg\u003csup\u003e-1\u003c/sup\u003e), indicating that \u0026alpha;-subunit production remained limiting (Table 3). This interpretation is reinforced by the single-subunit constructs: pCM6 yielded a large amount of purified \u0026beta;-subunit (8.7 mg g\u003csup\u003e-1\u003c/sup\u003e CDW), whereas pCM7 yielded almost no purified \u0026alpha;-subunit (\u0026lt;0.1 mg g\u003csup\u003e-1\u003c/sup\u003e CDW) (Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA parallel experiment in the homologous host \u003cem\u003eM. extorquens\u003c/em\u003e MeP4 led to the same qualitative conclusion regarding subunit asymmetry (Figure 3B; Table 3). When both subunits were co-expressed, pCM2, pCM3, pCM4, and pCM5 all yielded highly active enzyme preparations (63.7\u0026ndash;84.5 U mg\u003csup\u003e-1\u003c/sup\u003e), but expression of the \u0026alpha;-subunit alone from pCM7 again resulted in extremely poor recovery (\u0026lt;0.1 mg g\u003csup\u003e-1\u003c/sup\u003e CDW) (Table 3). By contrast, the \u0026beta;-subunit alone was readily produced from pCM6 in both hosts (Figure 3A, B; Table 3). Together, these data indicate that the \u0026beta;-subunit is not merely a passive partner in the final \u0026alpha;\u0026beta; complex, but likely contributes to productive \u0026alpha;-subunit accumulation, stabilization, and/or maturation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Fe-content analysis supports this interpretation but should be viewed as construct-dependent rather than as a general defect in all \u003cem\u003eE. coli\u003c/em\u003e preparations (Table 3). The theoretical Fe content of Me-FDH1 holoenzyme is 20 mol/mol protein, with 14 Fe atoms assigned to the \u0026alpha;-subunit and 6 to the \u0026beta;-subunit (Yoshikawa et al., 2022). The co-expressed holoenzyme from EcK/pCM2 contained 17.6 mol Fe/mol protein, close to the value observed for MeP4/pCM2 (18.2 mol/mol), indicating that near-complete Fe incorporation is possible in \u003cem\u003eE. coli\u003c/em\u003e when productive co-expression occurs (Table 3). In contrast, constructs enriched in the \u0026beta;-subunit and poor in the \u0026alpha;-subunit, especially pCM5 and pCM6, showed much lower Fe contents in both hosts (Table 3). Notably, \u0026beta;-subunit-only preparations from EcK/pCM6 and MeP4/pCM6 contained only about half of the expected Fe for the \u0026beta;-subunit alone, suggesting incomplete Fe-S cluster incorporation or reduced cluster stability in the absence of coordinated holoenzyme assembly. Thus, the dominant bottleneck is \u0026alpha;-subunit accumulation, whereas impaired Fe-S maturation appears to be most evident in constructs where \u0026alpha;-subunit co-production is compromised. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe parsimonious interpretation of these data is that the \u0026beta;-subunit stabilizes the \u0026alpha;-subunit through post-translational shielding. The \u0026alpha;-subunit is translated efficiently in the absence of the \u0026beta;-subunit in the PURExpress cell-free experiment (see Section \u0026ldquo;\u003cem\u003eIn vitro\u003c/em\u003e translation\u0026rdquo;), and is transcribed normally in pCM7, yet does not accumulate \u003cem\u003ein vivo\u003c/em\u003e. The \u0026beta;-subunit therefore appears to protect a partially folded \u0026alpha;-subunit from quality control recognition, an arrangement that parallels the role of the FdsC/FdsD chaperones in the assembly of the \u003cem\u003eR. capsulatus\u003c/em\u003e FDH \u0026alpha;-subunit with bis-MGD (B\u0026ouml;hmer et al., 2014; Hartmann and Leimk\u0026uuml;hler, 2013).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA decisive test of the timing of \u0026beta;-subunit action will require orthogonal induction of the two subunits, for example by placing \u0026beta; under an arabinose-inducible promoter and \u0026alpha; under an IPTG-inducible promoter. If the \u0026beta;-subunit acts co-translationally, simultaneous induction should be necessary for \u0026alpha;-subunit accumulation; if it acts by post-translational shielding, prior induction of the \u0026beta;-subunit should be sufficient.\u003c/p\u003e\n\u003cp\u003eSUMO fusion tag and general chaperones did not improve \u0026alpha;-subunit in\u003cem\u003e\u0026nbsp;E. coli\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBecause the \u0026alpha;-subunit accumulated poorly in \u003cem\u003eE. coli\u003c/em\u003e, we next tested whether solubility enhancement or generic folding support could improve its production. SUMO-based constructs were designed using the plasmids shown in Figure 1 and Table 1 and expressed in \u003cem\u003eE. coli\u003c/em\u003e K-12 Shuffle\u003csup\u003e\u0026reg;\u003c/sup\u003e T7 (EcKT), a T7-compatible strain that retains the native \u003cem\u003eMod\u003c/em\u003eABC uptake system and therefore minimizes possible confounding effects from tungsten limitation (Table 1; Figure 5A). None of the tested SUMO configurations increased \u0026alpha;-subunit accumulation detectably (Figure 5A). Even in the best SUMO-related cases, purified preparations showed only low activities of approximately 3.1\u0026ndash;4.0 U mg\u003csup\u003e-1\u003c/sup\u003e (Figure 5C), which remained below the activity of the original EcK/pCM2 enzyme (Table 2 and Table 3). Thus, improved solubility via a SUMO fusion was insufficient to overcome the main production barrier.\u003c/p\u003e\n\u003cp\u003eWe then examined whether co-expression of molecular chaperones could improve \u0026alpha;-subunit folding. Chaperone systems encoded on pG-KJE8 and pG-Tf2 (Table 1) provide GroEL-GroES together with either DnaK-DnaJ-GrpE or trigger factor, respectively (Nishihara et al., 1998, 2000). Although the chaperones themselves were strongly expressed, no improvement in \u0026alpha;-subunit recovery was observed. SDS-PAGE still showed only a faint \u0026alpha;-subunit band after purification (Figure 5B), and Me-FDH1 activity did not increase beyond the level of the original construct (Figure 5C). These results indicate that the limitation in \u0026alpha;-subunit production is not rescued by a generic solubility tag or by broad cytosolic chaperone overexpression.\u003c/p\u003e\n\u003ch2\u003eCodon harmonization did not improve \u0026alpha;-subunit production in E. coli\u003c/h2\u003e\n\u003cp\u003eBecause transcription was not limiting (Figure 4) and neither SUMO fusion nor general chaperones improved \u0026alpha;-subunit accumulation (Figure 5), we next investigated whether inefficient co-translational folding due to host-specific translation kinetics could be responsible. Translation-rate mismatch is known to affect protein folding and stability in heterologous hosts (Francis and Page, 2010). Therefore, the \u003cem\u003efdh1\u003c/em\u003ea and \u003cem\u003efdh1\u003c/em\u003eb genes were redesigned by codon harmonization to better mimic the native translational speed landscape of \u003cem\u003eM. extorquens\u003c/em\u003e in \u003cem\u003eE. coli\u003c/em\u003e (Schmidt et al., 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eComparison of codon usage among \u003cem\u003eM. extorquens\u003c/em\u003e, \u003cem\u003eC. necator\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003e showed substantial differences between \u003cem\u003eM. extorquens\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e, particularly in the \u0026alpha;-subunit gene, whereas \u003cem\u003eM. extorquens\u003c/em\u003e and \u003cem\u003eC. necator\u003c/em\u003e were more similar (see Additional File: Figure S6). After harmonization, the GC contents of the optimized genes decreased markedly, and the predicted correlations between native and host translation speeds improved for both subunits (see Additional File: Figure S6). The optimized sequences also retained only 77.91% and 75.80% nucleotide identity for the \u0026alpha;- and \u0026beta;-subunit genes, respectively (see Additional File: Figure S6A, B). The codon-optimized genes were cloned into the T7-based vectors shown in Figure 1 and Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite these substantial sequence changes, codon harmonization did not improve \u0026alpha;-subunit accumulation. Neither EcKT/pRSF8 nor EcKT/pRSF8/pET2 showed increased \u0026alpha;-subunit recovery relative to the corresponding non-optimized controls, and the purified proteins remained only weakly active (Figure 6A). When expressed individually, neither subunit produced detectable FDH activity, and when both were present the purified preparations still showed only low specific activities (2.8\u0026ndash;3.9 U mg\u003csup\u003e-1\u003c/sup\u003e; data not shown). Codon harmonization is effective when translation-rate mismatches perturb co-translational folding. Our data indicate that the limiting step is downstream of translation. The \u0026alpha;-subunit is translated efficiently in the PURExpress cell-free experiment (see Section \u0026ldquo;\u003cem\u003eIn vitro\u003c/em\u003e translation\u0026rdquo;), and the harmonized gene does not accumulate better \u003cem\u003ein vivo\u003c/em\u003e. For a large multidomain metalloenzyme such as the Me-FDH1 \u0026alpha;-subunit, the rate-limiting steps are domain packing, Fe-S cluster incorporation, and W-bis-MGD insertion, all of which occur after the polypeptide leaves the ribosome. Codon-level tuning therefore cannot by itself overcome this limitation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e translation supports post-translation loss of the \u0026alpha;-subunit \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo distinguish between failure of \u0026alpha;-subunit synthesis and loss of the \u0026alpha;-subunit after synthesis, we used a PURExpress cell-free transcription/translation system, which lacks ATP-dependent proteases and most cellular folding and maturation pathways (Figure 6B). Templates derived from the constructs listed in Table 1 were used to express both subunits together (pRSF2 and pRSF5) or individually (pRSF3 and pRSF4). In this cell-free system, both \u0026alpha;- and \u0026beta;-subunits were produced efficiently, and the \u0026alpha;-subunit was clearly visible even when expressed alone (Figure 6B). These results demonstrate that the \u003cem\u003efdh1\u003c/em\u003ea transcript is fully translatable and that ribosomes can synthesize the large \u0026alpha;-polypeptide.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Because the PURExpress system lacks tungsten cofactor biosynthesis and Fe-S cluster assembly machinery, the proteins formed \u003cem\u003ein vitro\u003c/em\u003e were necessarily apo-proteins. However, their successful synthesis shows that neither transcription nor the basic translational machinery is intrinsically limiting for Me-FDH1 production. A similar accumulation of apo-Me-FDH1 in the absence of tungsten has also been observed previously in \u003cem\u003eM. extorquens\u003c/em\u003e and \u003cem\u003eC. necator\u003c/em\u003e (Ryu et al., 2024), indicating that cofactor absence does not by itself prevent full-length polypeptide synthesis. The activity gap between \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eM. extorquens\u003c/em\u003e is therefore attributable to two coupled effects rather than a single cause. Cofactor insertion itself is not the primary limitation, as indicated by the stable specific activity across constructs and the lack of proportional response to tungstate supplementation. Under the most productive co-expression condition (EcK/pCM2), Fe content reaches 17.6 mol per mol protein, close to the theoretical 20 mol per mol holoenzyme and comparable to the native host (MeP4/pCM2, 18.2 mol/mol; Table 3), indicating efficient Fe-S assembly in this regime. The reduced activity primarily reflects the small pool of \u0026alpha;-subunit that escapes proteolysis, combined with residual structural imperfections in that pool. Fe-S cluster assembly is impaired only when \u0026alpha;- and \u0026beta;-subunits are not co-produced, consistent with a requirement for coordinated holoenzyme assembly.\u003c/p\u003e\n\u003cp\u003eTwo mechanisms could in principle explain the post-translational loss of the \u0026alpha;-subunit: proteolytic degradation and misfolding-driven aggregation. Three observations are more consistent with a proteolytic mechanism. The insoluble fraction did not contain a detectable \u0026alpha;-subunit band, the protein was produced intact in the protease-free PURExpress system, and co-expression of the \u0026beta;-subunit stabilized the \u0026alpha;-subunit even though the \u0026beta;-subunit is not expected to participate directly in \u0026alpha;-subunit folding. A contribution from misfolding cannot be excluded and would be consistent with recognition of an immature apo-form by the cellular quality control network, as discussed below.\u003c/p\u003e\n\u003cp\u003eThe inability of \u003cem\u003eE. coli\u003c/em\u003e to functionally produce Me-FDH1 has been reported previously by Park et al. (Park et al., 2024), who could not obtain active enzyme in \u003cem\u003eE. coli\u003c/em\u003e and moved to the native \u003cem\u003eM. extorquens\u003c/em\u003e host for their structural study. The present study quantifies this limitation and localizes it to post-translational \u0026alpha;-subunit loss. The heterologous-to-native activity ratio observed here (approximately 5\u0026ndash;15 %) is comparable to that reported for other complex metalloenzymes produced in \u003cem\u003eE. coli\u003c/em\u003e, including W-dependent FDHs (Maia et al., 2017; Niks and Hille, 2019), Mo-dependent FDHs (B\u0026ouml;hmer et al., 2014; Hartmann and Leimk\u0026uuml;hler, 2013), and [FeFe]-hydrogenases (B\u0026ouml;ck et al., 2006; Kuchenreuther et al., 2010). This work therefore advances the field less by improving absolute activity and more by identifying the rate-limiting step, which defines a concrete engineering target for subsequent studies.\u003c/p\u003e\n\u003cp\u003eTaken together, the data are consistent with a model in which the \u0026alpha;-subunit is translated efficiently but remains in an immature apo-state in \u003cem\u003eE. coli\u003c/em\u003e long enough to be recognized and degraded by ATP-dependent proteases of the host protein quality control network. The \u0026beta;-subunit reduces this window by stabilizing the \u0026alpha;-subunit after translation. This maturation-coupled model, rather than a transcriptional or translational defect, is the parsimonious explanation for the observed bottleneck.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA direct test of the proteolysis hypothesis will require cell-free expression supplemented with purified ATP-dependent proteases, ideally Lon and ClpXP, together with reconstitution of the complex cofactor environment of Me-FDH1 (W-bis-MGD, FMN, and multiple Fe-S clusters). W-bis-MGD and intact Fe-S clusters are not available as commercial reagents, and their \u003cem\u003ein vitro\u003c/em\u003e supply requires dedicated maturation proteins and strictly anaerobic handling (B\u0026ouml;hmer et al., 2014; Kuchenreuther et al., 2010). Such experiments, together with tests in protease-deficient \u003cem\u003eE. coli\u003c/em\u003e strains, are the most immediate next step following from this work.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAlthough this study did not achieve high-level heterologous production of Me-FDH1 in \u003cem\u003eE. coli\u003c/em\u003e, it identifies the dominant obstacle with much greater clarity. \u003cem\u003eE. coli\u003c/em\u003e produced catalytically competent Me-FDH1 when tungstate uptake was supported by a functional \u003cem\u003eMod\u003c/em\u003eABC system or by heterologous expression of \u003cem\u003eTup\u003c/em\u003eBCA (Figure 2; Table 2), showing that the host is not fundamentally incompatible with W-bis-MGD-dependent biogenesis. The major limitation instead lies in poor accumulation and incomplete maturation of the large \u0026alpha;-subunit. Gene-organization experiments showed that the \u0026beta;-subunit is readily produced on its own, whereas the \u0026alpha;-subunit is not efficiently recovered even in the native host unless the \u0026beta;-subunit is present, indicating that the \u0026beta;-subunit contributes to productive \u0026alpha;-subunit stabilization and/or maturation (Figure 3\u0026ndash;4; Table 3). SUMO fusion, generic chaperone co-expression, and codon harmonization did not rescue this defect (Figure 5 and Figure 6A), whereas cell-free translation readily produced the \u0026alpha;-subunit in the absence of ATP-dependent proteases (Figure 6B), strongly supporting a model in which the \u0026alpha;-subunit is synthesized in \u003cem\u003eE. coli\u003c/em\u003e but is rapidly lost through folding-coupled instability and post-translational degradation. From a practical standpoint, \u003cem\u003eC. necator\u003c/em\u003e remains the more suitable heterologous host for Me-FDH1 production at present (Ryu et al., 2024, 2025).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTwo non-exclusive causes may underlie the prolonged immature state of the \u0026alpha;-subunit. The maturation-related factors of \u003cem\u003eE. coli\u003c/em\u003e, including components of the W-bis-MGD pathway and the Fe-S assembly machinery, may be insufficient or mistimed relative to the overexpressed \u0026alpha;- and \u0026beta;-subunits. Alternatively, these factors may be intrinsically unable to process the heterologous Me-FDH1 \u0026alpha;-subunit efficiently. Sequence identity between the \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eM. extorquens\u003c/em\u003e machineries is low for several pathway steps (Table S2, see Additional File), and in \u003cem\u003eR. capsulatus\u003c/em\u003e FDH, the dedicated maturation chaperones FdsC and FdsD are required for activity but have no close \u003cem\u003eE. coli\u003c/em\u003e homolog (B\u0026ouml;hmer et al., 2014; Hartmann and Leimk\u0026uuml;hler, 2013). Both mechanisms are consistent with the observed phenotype and motivate the engineering strategies outlined below.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFour concrete engineering strategies are suggested by the identified bottleneck. The first is co-expression of \u003cem\u003eM. extorquens\u003c/em\u003e maturation-relevant factors, because protein sequence identity of several steps in the W-bis-MGD pathway is low between \u003cem\u003eM. extorquens\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e (Table S2, see Additional File). The second is comparative transcriptomic and proteomic profiling of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eM. extorquens\u003c/em\u003e under identical cultivation conditions, to identify W-bis-MGD biosynthesis and Fe-S cluster assembly genes that are expressed at insufficient levels in \u003cem\u003eE. coli\u003c/em\u003e relative to \u003cem\u003eM. extorquens\u003c/em\u003e; any such genes can then be targeted for controlled upregulation to reproduce the native expression balance. The third is fragment-based dissection of the \u0026alpha;-subunit. The \u0026alpha;-subunit contains multiple structural domains involved in Fe-S cluster binding and W-bis-MGD coordination, and the domain-level pattern of accumulation across individually expressed fragments can localize the region that is most susceptible to proteolysis, which can then guide rational stabilization. The fourth is controlled reduction of \u0026alpha;-subunit expression rate, using weaker promoters or lower inducer concentrations, to balance synthesis against the available maturation capacity. These strategies are not mutually exclusive and can be combined.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMore broadly, however, this study defines a clear engineering roadmap for adapting \u003cem\u003eE. coli\u003c/em\u003e to produce Me-FDH1 and related tungsten enzymes: improve \u0026alpha;-subunit stabilization, identify and suppress the proteolytic pathway(s) responsible for \u0026alpha;-subunit loss, and reconstruct the folding and maturation environment required for efficient holoenzyme assembly. By pinpointing the dominant bottleneck rather than merely documenting low expression, this work advances the development of robust microbial platforms for sustainable CO\u003csub\u003e2\u003c/sub\u003e-to-formate biocatalysis.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eATP:\u0026nbsp;\u003c/strong\u003eAdenosine triphosphate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBSA:\u0026nbsp;\u003c/strong\u003eBovine serum albumin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDW:\u0026nbsp;\u003c/strong\u003eCell dry weight\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFDH:\u003c/strong\u003e Formate dehydrogenase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFe-S cluster:\u003c/strong\u003e Iron-sulfur cluster\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[FeFe]-hydrogenases:\u003c/strong\u003e Unique 6-iron active site [4Fe\u0026ndash;4Fe] cluster linked to a [2Fe] subcluster\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHis\u003csub\u003e6\u003c/sub\u003e tag:\u003c/strong\u003e hexa-histidine tag\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHR-ICP-MS:\u003c/strong\u003e High resolution inductively coupled plasma mass spectrometry\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIPTG:\u003c/strong\u003e Isopropyl-\u0026beta;-D-thiogalactopyranoside\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMe-FDH1:\u003c/strong\u003e Formate dehydrogenase I from \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModABC:\u0026nbsp;\u003c/strong\u003eHigh-affinity molybdate/tungstate ABC-type transporter system\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNi-NTA:\u003c/strong\u003e Nickel-Nitrilotriacetic Acid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRBS:\u003c/strong\u003e Ribosome-binding site\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRPM:\u0026nbsp;\u003c/strong\u003eRevolutions per minute\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-PCR:\u003c/strong\u003e Reverse transcription polymerase chain reaction\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSDS-PAGE:\u003c/strong\u003e Sodium dodecyl sulfate-polyacrylamide gel electrophoresis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSUMO:\u003c/strong\u003e Small ubiquitin-like modifier\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTupBCA:\u003c/strong\u003e High-affinity tungstate ABC-type transporter system\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eW-bis-MGD:\u003c/strong\u003e Tungsto-bis(molybdopterin guanine dinucleotide) cofactor\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. This study did not involve human participants, human data, human tissue, or animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. The predicted structural models generated in this study have been deposited in ModelArchive under accession numbers ma-ny5ny, ma-covi8, ma-wmubt, ma-b9uc9, ma-jp91h, and ma-ifuk9. The codon-harmonized \u003cem\u003efdh1a\u003c/em\u003e and \u003cem\u003efdh1b\u003c/em\u003e sequences have been deposited in NCBI GenBank under accession numbers PX353735 and PX353736, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2020-NR049543).\u0026nbsp;Sunghoon Park acknowledges financial support from the National Research Foundation of Korea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN.N.M.C.\u003c/strong\u003e and \u003cstrong\u003eH.R.\u003c/strong\u003e contributed equally to this work as co-first authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN.N.M.C.\u0026nbsp;\u003c/strong\u003econtributed to conceptualization, methodology, formal analysis, investigation, visualization, writing \u0026ndash; original draft, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eH.R.\u003c/strong\u003e contributed to conceptualization, methodology, formal analysis, investigation, visualization, writing \u0026ndash; original draft. \u003cstrong\u003eJ.Y.P.\u0026nbsp;\u003c/strong\u003econtributed to software, methodology (codon optimization). \u003cstrong\u003eY.H.K.\u0026nbsp;\u003c/strong\u003econtributed to visualization, supervision. \u003cstrong\u003eS.H.P.\u0026nbsp;\u003c/strong\u003econtributed to conceptualization, supervision, project administration, funding acquisition, visualization, writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge the UNIST Office of Research Facilities and Training (ResFacT) for providing access to shared HR-ICP-MS instrumentation and technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information (optional)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlissandratos, A., Kim, H.K., Easton, C.J., 2014. 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Biofuels 8, 1\u0026ndash;8. https://doi.org/10.1186/S13068-015-0353-5/TABLES/2\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e Plasmids and strains used in this study\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlasmids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epCM110-P\u003csub\u003eL/O4\u003c/sub\u003e-RBS\u003csub\u003eb\u003c/sub\u003e-fdh1ba-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e, Tet\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e(Ryu et al., 2024)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epCM3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epCM110-P\u003csub\u003eL/O4\u003c/sub\u003e- RBS\u003csub\u003eb\u003c/sub\u003e-\u0026nbsp;fdh1a-His\u003csub\u003e6\u003c/sub\u003e-\u0026nbsp;fdh1b-T7\u003csub\u003eter\u003c/sub\u003e, Tet\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epCM4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epCM110-P\u003csub\u003eL/O4\u003c/sub\u003e- RBS\u003csub\u003eb\u003c/sub\u003e-\u0026nbsp;fdh1b- RBS\u003csub\u003eb\u003c/sub\u003e-fdh1a-His\u003csub\u003e6-\u003c/sub\u003eT7\u003csub\u003eter\u003c/sub\u003e, Tet\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epCM5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epCM110-P\u003csub\u003eL/O4\u003c/sub\u003e- RBS\u003csub\u003eb\u003c/sub\u003e-His\u003csub\u003e6\u003c/sub\u003e-\u0026nbsp;fdh1b-T7\u003csub\u003eter\u003c/sub\u003e-P\u003csub\u003eL/O4\u003c/sub\u003e- RBS\u003csub\u003eb\u003c/sub\u003e-\u0026nbsp;fdh1a-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e, Tet\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epCM6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epCM110-P\u003csub\u003eL/O4\u003c/sub\u003e- RBS\u003csub\u003eb\u003c/sub\u003e-His6-\u0026nbsp;fdh1b-T7\u003csub\u003eter\u003c/sub\u003e, Tet\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epCM7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epCM110-P\u003csub\u003eL/O4\u003c/sub\u003e- RBS\u003csub\u003eb\u003c/sub\u003e-\u0026nbsp;fdh1a-His6-T7\u003csub\u003eter\u003c/sub\u003e, Tet\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epAC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epACYC184, p15A ColE1 ori, Cm\u003csup\u003eR\u003c/sup\u003e, Tet\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eAddgene\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epAC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epAC-P\u003csub\u003etac\u003c/sub\u003e-RBS-tupBCA-T7\u003csub\u003eter\u003c/sub\u003e, Cm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epRSF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epRSF-Duet-1, RSF1030 ori, P\u003csub\u003eT7\u003c/sub\u003e, Kan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eAddgene\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epRSF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epRSF-P\u003csub\u003eT7\u003c/sub\u003e-fdh1ba-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e, Kan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epRSF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epRSF-P\u003csub\u003eT7\u003c/sub\u003e-\u0026nbsp;fdh1a-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e, Kan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epRSF4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epRSF-P\u003csub\u003eT7\u003c/sub\u003e-\u0026nbsp;fdh1b-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e, Kan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epRSF5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epRSF-P\u003csub\u003eT7\u003c/sub\u003e-\u0026nbsp;fdh1b-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e-P\u003csub\u003eT7\u003c/sub\u003e-\u0026nbsp;fdh1a-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e, Kan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epRSF6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epRSF-P\u003csub\u003eT7\u003c/sub\u003e-SUMO-\u0026nbsp;fdh1ba-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e, Kan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epRSF7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epRSF-P\u003csub\u003eT7\u003c/sub\u003e-SUMO-\u0026nbsp;fdh1a-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e, Kan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epRSF8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epRSF-P\u003csub\u003eT7\u003c/sub\u003e-\u0026nbsp;fdh1a codon optimized-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e, Kan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epET1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epET-Duet-1, pBR322-derived ColE1 ori, P\u003csub\u003eT7\u003c/sub\u003e, Am\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eAddgene\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epET2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003epET-P\u003csub\u003eT7\u003c/sub\u003e-\u0026nbsp;fdh1b codon optimized-His\u003csub\u003e6\u003c/sub\u003e-T7\u003csub\u003eter\u003c/sub\u003e, Am\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epG-KJE8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eP\u003csub\u003earaBAD\u003c/sub\u003e-dnaK-dnaJ-grpE, P\u003csub\u003elac\u003c/sub\u003e-groES-groEL,\u0026nbsp;Cm\u003csup\u003eR\u003c/sup\u003e, p15A ori\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eTaKaRa, Japan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003epG-Tf2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eP\u003csub\u003elac\u003c/sub\u003e-tig, P\u003csub\u003elac\u003c/sub\u003e-groES-groEL, Cm\u003csup\u003eR\u003c/sup\u003e, p15A ori\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eTaKaRa, Japan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;B wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eDSMZ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;BL21 (DE3) wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e(Nguyen-Vo et al., 2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcBLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;BL21 Star\u0026trade; (DE3) pLysS wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eNew England Biolabs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcBW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;BW25113 wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e(Sundara Sekar et al., 2016)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;C ATCC8739 wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e(Sundara Sekar et al., 2016)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;DH5\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eCloning host\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eToyobo, Japan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcJM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;JM109 (DE3) wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003ePromega, Korea\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;K-12 MG1655 wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e(Nguyen-Vo et al., 2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcKT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;K-12 Shuffle\u0026reg; T7 wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eNew England Biolabs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcMC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;MC1061 wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcWA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;W ATCC 9637 wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e(Nguyen-Vo et al., 2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;W3110 wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eATCC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcXL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;XL-1 Blue wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eNew England Biolabs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eEcR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eE. coli\u0026nbsp;Rosetta 2 (DE3) wildtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eNovagen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eMeP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eM. extorquens\u0026nbsp;PA1,\u0026nbsp;fdh1\u0026nbsp;gene source\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eDSMZ, Germany\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109px;\"\u003e\n \u003cp\u003eMeP4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 354px;\"\u003e\n \u003cp\u003eMeP\u0026Delta;fdh1\u0026Delta;fdh2\u0026Delta;fdh3\u0026Delta;fdh4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e(Ryu et al., 2024)\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\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e Summary of Me-FDH1 expression in various E. coli\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\u003eHost strain for Me-FDH1 production\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eExpression level (% of total soluble protein)\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEnzyme activity (U/mg)\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMolybdate transporter\u003csup\u003ec\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcB/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcBL/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBLP/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcBW/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e13.9 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+ (ModABC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcC/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+ (ModABC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcJM/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e6.6 \u0026plusmn; 1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+ (ModABC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcK/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e8.2 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+ (ModABC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcKT/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e4\u0026nbsp;\u0026plusmn;\u0026nbsp;0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+ (ModABC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcMC/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+ (ModABC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcWA/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e4.1 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+ (ModABC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcW/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3.3 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+ (ModABC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcXL/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEcR/pCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eEcBL/pRSF2/pAC2\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e~1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2.6 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e++ (TupABC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eExpression level was estimated by SDS-PAGE densitometry of the soluble fraction obtained after cell disruption and centrifugation, and is reported as the percentage of total soluble protein. Values below the detection limit are indicated as \u0026lt;1 %.\u003cem\u003e\u003cbr\u003e \u003csup\u003eb\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eEstimated from enzyme assay for formate oxidation reaction with purified samples.\u003cem\u003e\u003cbr\u003e \u003csup\u003ec\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eMolybdate transporter system (\u003cem\u003emod\u003c/em\u003eABC) is non-specific to molybdate and possible to bind tungstate (Otrelo-Cardoso et al., 2017)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ed\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eThe TupABC transporter from \u003cem\u003eM. extorquens\u003c/em\u003e PA1 was expressed for tungstate uptake (Table 1).\u003c/p\u003e\n\u003cp\u003eTable 3 Quantification of Me-FDH1 during Ni-NTA affinity purification, activity of purified enzymes, and Fe content of selected proteins\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlasmid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell mass\u003cbr\u003e\u0026nbsp;(mg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein in crude cell extract\u003cbr\u003e\u0026nbsp;(mg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 169px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFDH1 proteins after Ni-NTA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecific production\u003cbr\u003e (mg/g CDW)\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecific activity\u003cbr\u003e (U/mg protein)\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFe content\u003cbr\u003e\u0026nbsp;(mol/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eQuantity\u003cbr\u003e\u0026nbsp;(mg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePurity\u003cbr\u003e (%)\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" style=\"width: 112px;\"\u003e\n \u003cp\u003eEcK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1380\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e711.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e17.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1560\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e1044.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026lt; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e14.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e1127.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026lt; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e12.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e1630.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e10.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e1633.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e22.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e8.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003en/a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 158px;\"\u003e\n \u003cp\u003e578.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026lt; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003en/a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003en/a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" style=\"width: 112px;\"\u003e\n \u003cp\u003eMeP4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1215\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e439.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e84.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e18.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1097\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e199.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e78.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e16.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e527.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e63.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e19.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1284\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e451.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e6.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e65.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e22.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1359\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e559.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003en/a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003epCM7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e368.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026lt; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003en/a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003en/a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Determined by densitometry on SDS-PAGE\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e Adjusted for purity. \u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Methylorubrum extorquens, tungsten-dependent formate dehydrogenase, W-bis-MGD, tungstate transport, α-subunit maturation, post-translational instability","lastPublishedDoi":"10.21203/rs.3.rs-9133619/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9133619/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Methylorubrum extorquens formate dehydrogenase I (Me-FDH1) is a tungsten-dependent heterodimeric enzyme with high activity for CO2/formate interconversion, making it an attractive biocatalyst for carbon capture, formate production, and bioelectrocatalysis. Its broader application, however, is limited by the lack of a heterologous production host for this complex metalloenzyme. Here, we systematically evaluated Escherichia coli as a host for Me-FDH1 production and compared its performance with that of the native host and a previous heterologous host. Across multiple E. coli strains, active Me-FDH1 was obtained only when tungstate uptake was supported by a functional ModABC system or by heterologous expression of the TupBCA transporter, demonstrating that E. coli can synthesize and incorporate the W-bis-MGD cofactor. Nevertheless, expression remained low (\u0026lt;1% of total cellular protein), and the purified enzyme displayed only 4–14 U mg-1 specific activity, far below the 80–100 U.mg-1 observed for the enzyme produced in M. extorquens. Operon redesign, altered gene order, stronger ribosome-binding sites, SUMO fusion, chaperone co-expression, and codon harmonization did not improve α-subunit production. In both E. coli and M. extorquens, the α-subunit was poorly produced in the absence of the β-subunit, indicating that the β-subunit contributes to α-subunit stabilization and/or maturation. Cell-free translation produced both subunits efficiently, showing that the principal barrier in E. coli is not transcription or translation, but post-translational instability and likely proteolytic loss of the α-subunit. These findings define the key bottleneck for Me-FDH1 production in E. coli and provide a roadmap for engineering hosts for tungsten-containing enzymes.","manuscriptTitle":"Heterologous production of tungsten-dependent formate dehydrogenase I from Methylorubrum extorquens in Escherichia coli reveals α-subunit maturation as the major bottleneck","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-05 17:56:49","doi":"10.21203/rs.3.rs-9133619/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-28T14:37:12+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-27T03:29:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-24T11:56:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2026-04-23T21:45:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6a87a479-0cb1-49ae-9954-9bb18115996b","owner":[],"postedDate":"May 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T17:56:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-05 17:56:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9133619","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9133619","identity":"rs-9133619","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}