All chemicals, reagents and media were correspondingly purchased from SRL-India, Sigma Aldrich, TCI chemicals and Himedia. Yeast alcohol dehydrogenase (yADH) was purchased from Sigma-Aldrich. Ectopseudomonas alcaliphila MSJ19 was isolated in our previous study, and its 16S rRNA sequence has been deposited in GenBank (ID: PX397011). Plasmid pDK7 (p15a)/pddCDE, gdrAB was developed by amplification of pddCDE genes from genomic DNA isolated from Klebsiella pneumoniae 109 and subsequently cloned into KpnI and HindIII restriction sites of pDK7 (p15a)/dhaB123, gdrAB plasmid. The plasmids were transformed into appropriate hosts following the protocol adopted from (Zhou et al., 2013). All strains, plasmids and primers used in this study are listed in Table 1.

Overnight lysogeny broth (LB) EaM culture was pre-cultured in LB medium until mid-late log phase of growth. Subsequently, 0.1 OD₆₀₀ of exponentially grown cells was reinoculated appropriately in LB production medium containing precursors: CoCl₂ (5 mg/L), DMBI (75 mg/L), and Betaine (1 g/L) and incubated under aerobic conditions at 37 °C, 200 rpm. Wild-type E. coli W were cultivated under similar conditions to serve as a negative control wherever appropriate in this study. Cell growth was measured at regular intervals by a UV–Vis spectrophotometer, and after 18 h, cells were harvested for B₁₂ extraction (4,500 rpm, 15 min). One OD₆₀₀ corresponds to 0.33 g (±0.05 g) of dried cell mass per liter (Arasu et al., 2013).

Cells were washed twice with 100 mM potassium phosphate buffer (pH 7.0) and resuspended in the same buffer for B₁₂ extraction under ice with minimal light exposure. Cell concentration was measured before and after lysis. Cells were lysed by ultrasonication at 30% amplitude for 6 min with a 10-s ON/OFF cycle (VCX 130, Sonics; 20 kHz), centrifuged (4,500 rpm, 10 min), supernatant filtered through a 0.22 μm syringe filter and used as crude B₁₂ extract for further analysis.

For B₁₂ quantification by bioassay, the protocol mentioned in our previously published study was followed exactly (Venkatesan et al., 2024). To convert natural B₁₂ forms into the more stable CNCbl form, 0.1% w/v NaCN was added to the crude B₁₂ extract, and after 5 min incubation (37°C), the mixture was autoclaved (121 °C, 15 min) and cooled on ice. The samples were centrifuged (4,500 rpm, 20 min) and the supernatant was passed through a 0.22 μm syringe filter prior to LC–MS analysis. The LC–MS analysis was performed for both crude B₁₂ extract and cyano-converted extract, using a Waters TQD LC–MS/MS system equipped with a Kinetex (2.6 μm, XB C18 Column, 2.1 × 100 mm). 20 mM ammonium formate in water (Mobile Phase A) and methanol (Mobile Phase B) were used for sample elution under the following linear gradient: 90% mobile phase A for 0–2 min, 90% mobile phase A for 2–4 min, 10% mobile phase A for 4–5 min, 90% mobile phase A for 5–7 min. The flow rate was maintained at 0.3 mL/min, the column temperature was set to 35 °C, and the injection volume was 10 μL. Mass spectrometry was conducted in positive electrospray ionization (ESI) mode with a source temperature of 140°C, desolvation temperature of 300 °C, cone gas flow of 10 L/h and desolvation gas flow of 1,000 L/h. The capillary voltage was set at 26 V, and the cone voltage was 35 V. The quantification of CNCbl was performed using multiple reaction monitoring (MRM) transitions, monitoring the precursor ion at m/z 678.5 and the product ions at m/z 147.0 and 358.9, with collision energies of 34 eV and 24 eV, respectively (Stumpf et al., 2024; Kahoun et al., 2022). This targeted MRM setting was chosen to achieve high specificity and sensitivity for CNCbl, while avoiding cross-detection of other B₁₂ forms in crude extracts (Reddy KotamReddy et al., 2023). The same LC–MS method was also adopted for crude B₁₂ extracts.

The modified M9 medium used for shake flask studies in EcW GD and EcW DD contained: MgSO₄·7H₂O, 0.5 g/L; NaCl, 1.0 g/L; NH₄Cl, 1.0 g/L; yeast extract, 1 g/L; glycerol, 100 mM; potassium phosphate buffer (pH 7.0), 100 mM; kanamycin 50 mg/L; and chloramphenicol 25 mg/L. Unless stated otherwise, LB medium and the same modified M9 medium with appropriate antibiotics were used for primary inoculum and secondary inoculum, respectively. Shake flask cultivation was carried out with a working volume of 50 mL culture with an inoculum of 0.1 OD₆₀₀ in a 250 mL Erlenmeyer flask at 37°C, 250 rpm under aerobic conditions. For enzyme production, the cultures were induced at 0.6 ± 0.05 OD₆₀₀ with 0.5 mM IPTG. After 6 h incubation, cells were harvested (5,000 rpm, 15 min) and washed once with 20 mM potassium phosphate buffer. Subsequently, cells were resuspended in the same buffer and subjected to ultrasonication under ice at 30% amplitude for 4 min with a 10-s ON/OFF cycle. The obtained lysate was centrifuged (13,000 rpm, 30 min), and the supernatant was collected to measure total protein concentration (by the Bradford method), glycerol dehydratase (GD) and diol dehydratase (DD) activity, respectively.

GD activity was measured by following the protocol developed by Sankaranarayanan et al. (2017), and the same method was employed to measure DD activity. Briefly, the substrate mixture (~1.8 mL), containing 20 mM potassium phosphate buffer (pH 8.0), 3 mM MgCl₂ and 40 mM 1,2-PDO, was placed in a 1-cm path length spectrophotometer cuvette. B₁₂ solution (100 μL) was added to this assay mixture, containing 0.15 mM NADH and 1.5 mM ATP. B₁₂ concentration and type were varied individually to study their effects. Then, the coupling enzyme (40 μL) yADH (12 U/mL) was added using an air-tight gas chromatography syringe, and the cuvette was incubated for 3 min in a water bath at 37 °C. The enzymatic reaction was initiated by injecting 50 μL of crude GD or crude DD enzyme solution, appropriately (typically <0.03 U/mL). The NADH concentration was determined at 340 nm with the extinction coefficient (ε₃₄₀) of 6.22 mM⁻¹ cm⁻¹ on a UV spectrophotometer. One unit of GD or DD activity was defined as the amount of enzyme required to convert 1 μmol of 1,2-PDO to propionaldehyde per minute under given assay conditions.

Molecular docking was performed between each B₁₂ form—AdoCbl, CNCbl, MeCbl and the active sites of GD and DD. High-resolution crystal structures of GD (PDB ID: 1IWP) and DD (PDB ID: 1DIO) were retrieved from the protein data bank (PDB) (Yamanishi et al., 2002; Shibata et al., 1999). The crystal structures were refined by eliminating water molecules and ligands using PyMOL software (version 3.1.3). Refined proteins were subsequently processed using Autodock tools (v 1.5.7) by setting grid parameters for both GD and DD based on reference active site coordinates reported already (Yamanishi et al., 2002; Masuda et al., 2000). 3D structure of all the ligand molecules AdoCbl, CNCbl, and MeCbl were procured from protein structures (PDB ID: 5C8A, 5NP4, 3SC0) co-crystalized with respective ligands. Each B₁₂ ligand was assigned its respective charges and docked into the aforementioned active site grid. Docked conformations exhibiting higher binding and similar interactions with key active site residues were considered for further evaluation.

Shake flask 3-HP production with the respective host was carried out aerobically using the same modified M9 medium (50 mL) with a starting inoculum of 0.1 OD₆₀₀ in a 250-ml Erlenmeyer flask incubated at 37°C, 250 rpm. The cultures were induced at 0.6 ± 0.05 OD₆₀₀ with 0.1 mM IPTG and supplemented with various forms and concentrations of B_12, respectively, at 3, 6, 9, and 12 h of cultivation. The details on B₁₂ form and concentration supplemented for each shake flask experiment were furnished in Supplementary Table 1. Samples were collected periodically to determine the cell mass, residual substrate and metabolites. Briefly, the collected culture samples were centrifuged (10,000 rpm, 10 min), then the supernatant was diluted appropriately and filtered using a 0.22 μm PVDF membrane filter (Millipore). Then the samples were passed through an HPLC system equipped with an Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad, United States) maintained at 65°C. The mobile phase consisted of 2.5 mM H₂SO₄ with a flow rate of 0.5 mL/min, and metabolite concentrations were analyzed using a Refractive Index Detector (RID) and a Photo-diode array detector (PDA) (Ravi and Sankaranarayanan, 2024).

Shake flask cultivation of the EaM_r for 3-HP production was carried out in the same B₁₂ production medium with the addition of Kanamycin (30 mg/L). The cultivation was carried out aerobically at 37°C, 200 rpm, with an initial cell concentration of 0.1 OD₆₀₀. 100 mM of glycerol (carbon source for 3-HP production) was added when the cell concentration reached 0.7 ~ 1 OD₆₀₀. The samples were withdrawn periodically to determine the cell mass, glycerol, 3-HP and other metabolites.

Consistent with our previous study, the B₁₂ levels of Ectopseudomonas alcaliphila MSJ19 (EaM) quantified by bioassay were 7.18 μg/g cdw (Venkatesan et al., 2024). To validate B₁₂ forms, LC–MS analysis was performed for: (a) crude B₁₂ extract, (b) cyano-converted B₁₂ extract, and (c) crude B₁₂ extract spiked with 0.5 μM each of standard AdoCbl and MeCbl (Figure 1). A distinct peak at RT 4.32 min was observed for cyano-converted B₁₂ extract corresponding to MRM transitions m/z 147.0 and 358.9, matching precisely with the standard CNCbl profile. These transitions were selected based on their high sensitivity and specificity for CNCbl quantification. The concentration of B₁₂ was quantified as 6.93 μg/g cdw. In contrast, no corresponding peaks were observed at RT 4.3 min for either the crude extract or the spiked crude extract, indicating that CNCbl was not natively present in the bacterial extract. These results collectively support that EaM produces only the natural (coenzyme) forms of B₁₂—namely, MeCbl and AdoCbl—which are not detected in this LC–MS method due to their distinct transition requirements (such as m/z 685.6 and 665.6, respectively) (Heal et al., 2014). By providing a clear distinction between the presence of natural and non-natural forms of B₁₂, the present strategy offers a streamlined and scalable framework for both qualitative and quantitative assessment of B₁₂ production in industrially relevant microbial strains.

Glycerol dehydratase (GD) and diol dehydratase (DD) are isofunctional, coenzyme B₁₂-dependent enzymes, whose characteristics and in vitro assays have been well studied (Toraya et al., 2022; Nasir et al., 2020). These enzymes are known to be catalytically active only in the presence of AdoCbl with varying degrees of sensitivity (Marsh and Meléndez, 2012; Toraya et al., 1979). While other B₁₂ forms, such as MeCbl and CNCbl, are often reported as competitive inhibitors (Poppe and Rétey, 1997; Toraya and Ishida, 1991). These features make GD and DD valuable in vitro tools for evaluating the functional bioactivity of B₁₂ from bacterial extracts.

Generally, activity assays for these isofunctional enzymes are performed at a saturated coenzyme B₁₂ concentration of around 10–20 μM (Kumar et al., 2016; Wei et al., 2014). However, due to the low concentration of crude B₁₂ used for this study (0.35 nM), a preliminary investigation was carried out to study the effect of AdoCbl concentration on GD and DD activity. Maximal activities were observed at 15 μM AdoCbl, yielding 14.32 U/mg for GD and 6.79 U/mg for DD. At 0.35 nM, the enzyme activity dropped to 0.32 U/mg for GD, while no significant activity was observed for DD (Figure 2A). The difference in activities between GD and DD correlates with their known kinetic parameters, specifically the reported K_m values of GD (~ 8 nM to 20 nM) and DD (~ 0.7 μM) from Klebsiella sp. (Yamanishi et al., 2002; Wang et al., 2007). According to previous reports, GD attained 95% of its maximum activity and DD only 4% at 120 nM AdoCbl (Yamada et al., 2004). Relatively, the current study shows that GD and DD attained 81% and 7% of their respective maximum activities at 100 nM AdoCbl, confirming the accuracy of the assay and reinforcing AdoCbl sensitivity among the enzymes. As anticipated, no significant enzyme activity was observed when the assay was performed with CNCbl and MeCbl, even at 15 μM, the saturated concentration used for AdoCbl (Figure 2B). The missing 5′-deoxyadenosyl radical upon binding of CNCbl and MeCbl to the enzyme is expected to be the sole reason for their inability to support GD and DD activity (Toraya, 2000; Bucher et al., 2012). Previous reports support this by showing that MeCbl and CNCbl act as competitive inhibitors for DD (K_i of 0.73 μM and 1.8 μM, respectively) (Toraya et al., 1977) and CNCbl for GD (K_i = 21.6 nM) (Poppe and Rétey, 1997). Notably, with 0.35 nM of crude B₁₂ extract, the GD activity measured was 0.21 U/mg, which was slightly lower than 0.32 U/mg obtained with standard AdoCbl at the same concentration. While this suggests that the crude B₁₂ extract predominantly contains AdoCbl, it confirms that EaM has dominantly produced AdoCbl; the lower activity could likely be due to the presence of some MeCbl, which may exert competitive inhibition. However, further studies are required to confirm this hypothesis. These results collectively demonstrate that the B₁₂ produced by Ectopseudomonas alcaliphila MSJ19 is functionally bioactive, with in vitro-based assays providing indirect but reliable confirmation of AdoCbl as the dominant form in the crude extract.

To complement the differential catalytic activity of B₁₂ forms on a structural basis, molecular docking was performed between three B₁₂ ligands—AdoCbl, CNCbl, and MeCbl—and the known crystal structures of GD and DD. A total of six docking combinations were generated, and a complete summary of interactions and H-bond distances for each docking conformation is provided in Supplementary Table 2. Based on previous crystallographic studies, 12 key active site residues were defined for GD (Yamanishi et al., 2002) and 7 for DD (Masuda et al., 2000) to assess the binding capability of ligands within the functionally active site.

In GD, AdoCbl exhibited the most favorable binding conformation for catalytic function, forming hydrogen bonds with five active site residues (SER122, THR104, SER225, THR173, LYS102) and a binding energy of −3.93 kcal/mol (Figures 3A1,D1). These interactions span both the corrin ring and adenosyl moiety, positioning the ligand in a favorable conformation for Co-C homolysis and radical exchange. CNCbl exhibited a significantly lower binding energy (−14.5 kcal/mol) and formed four interactions with active site residues (SER122, ASP235, ALA124, THR173) (Figures 3B1,E1). While such tight binding reflects higher affinity, the absence of the adenine moiety blocks its catalytic ability and complements its role as a competitive inhibitor. MeCbl also interacted only with one active residue (SER122) and an intermediate binding energy (−5.81 kcal/mol) (Figures 3C1,F1), further reflecting its non-catalytic but potentially competitive inhibitory role.

In DD, AdoCbl again exhibited the highest number of active site interactions, yet fewer than in GD. Only two out of six interactions matched with key active side residues (THR172, SER301) following an intermediate binding energy (−7.23 kcal/mol) (Figures 3A2,D2). This observation aligns with the in vitro enzyme activity assay, where DD activity expressed a higher Km than GD, thus justifying the lower specificity of AdoCbl with DD. CNCbl had only one matching residue (THR172) among five interactions with a lower binding energy (−12.9 kcal/mol) (Figures 3B2,E2). MeCbl had only one matching residue (SER224) among its two interactions with a higher binding energy (−6.99 kcal/mol) among all 3 B₁₂ forms with DD (Figures 3C2,F2). These binding predictions reflect the inferiority of B₁₂-driven catalysis with DD as compared to GD.

Importantly, these findings elucidate the conserved structural and functional preference of GD and DD for AdoCbl. Evidently, comparison of available crystal structures and docking combinations of this study has shown that CNCbl and MeCbl are also capable of binding within the active site, but they lack the adenine moiety necessary to trigger Co-C bond homolysis and substrate rearrangements (Shibata et al., 2018). Hence, the adenine moiety not only acts as a radical initiator, but also participates in key interactions to position the cofactor in an appropriate spatial conformation for catalytic activity. Therefore, the in-silico findings support the in vitro enzymatic assay, confirming that only AdoCbl positions itself in a catalytically active conformation in both GD and DD in a conserved manner. Meanwhile, CNCbl and MeCbl are capable of competitive inhibition due to their catalytically inactive binding conformation.

In the two-step catalytic pathway for 3-HP production, Coenzyme B₁₂ (AdoCbl) serves as an essential cofactor for glycerol dehydratase (Kumar et al., 2012). Therefore, 3-HP production can act as a reliable qualitative metric for assessment of B₁₂ bioactivity, offering a more meaningful output than conventional microbiological assay. To evaluate this, EcW GD and EcW DD were supplemented individually with AdoCbl, MeCbl, and CNCbl for 3-HP production. Among these, AdoCbl yielded the highest 3-HP production in EcW GD, confirming it as the most effective cofactor for GD activity. Whereas MeCbl resulted in only 63% of this maximum, and CNCbl only 36%. A similar trend was observed for EcW DD, yet its maximum 3-HP titre was only 52% of that achieved with EcW GD. Such a low 3-HP titre of DD in this expression system is obvious due to the following well-documented reasons: (i) 1,2-PDO is the preferred substrate for DD over glycerol (Sauvageot et al., 2002), (ii) absence of diol dehydratase reactivase in this expression system, making DD prone to suicide inactivation like GD in the presence of glycerol (Bilić et al., 2019), (iii) gdrAB is known to be ineffective in reactivating DD (Kajiura et al., 2007), and (iv) less B₁₂ specificity of DD as observed in enzyme activity analysis (Poppe and Rétey, 1997). These results further demonstrate a substantial decline in GD and DD activity with synthetic B₁₂ forms and justify the functional superiority of the natural B₁₂ forms.

Notably, the modest 3-HP production with CNCbl suggests that E. coli may possess intrinsic metabolic mechanisms to convert CNCbl into biologically active forms, analogous to human metabolic pathways (Kelly, 1997). However, the relatively low 3-HP titer (70% lower than AdoCbl) indicates that this intracellular conversion is likely rate-limiting. The difference in 3-HP production between MeCbl and CNCbl also reflects the metabolic complexity of their respective conversion process, as CNCbl conversion is mediated by a four-step enzymatic process, while MeCbl requires only a single step (Rizzo et al., 2016).

While earlier studies typically employed 2000 nM AdoCbl for optimal 3-HP production in E. coli (Nguyen-Vo et al., 2019), the concentration of crude B₁₂ extract used is comparatively less (0.35 nM). Therefore, this study also evaluated the effect of B₁₂ concentration on 3-HP production across a wide range (0.35–2,000 nM) for each B₁₂ form (Figures 4A,B). Interestingly, B₁₂ concentration had a significant effect on 3-HP production, similar to the enzyme activity. Remarkably, the highest 3-HP titer of 50.2 mM was observed at 500 nM AdoCbl for EcW GD, beyond which no significant increase in titre could be observed. The optimal 3-HP production at 500 nM reflects seamlessly with the V_max of GD, embarking on the critical impact of B₁₂ on the rate-limiting step of the 3-HP catalytic pathway. The 3-HP production titer (41.5 mM) with 2 μM AdoCbl was also consistent with previous reports (Sankaranarayanan et al., 2017). Similar trends were observed for other B₁₂ forms. Owing to the higher K_m of DD, 3-HP production was maximal (26.2 mM) only at 2,000 nM and suggesting that further increase in B₁₂ may still enhance activity (typically close to the V_max of DD (>7 μM)).

Of particular interest, crude B₁₂ extract at 0.35 nM supported a 3-HP titre of 5.9 mM in EcW GD—closely matching the 7.8 mM titer at 0.35 nM AdoCbl. This confirms the presence of active B₁₂ forms in the extract. The marginal difference could be attributed to the presence of some MeCbl in the extract, as only the total B₁₂ concentration was quantified. Consistently, MeCbl at 0.35 nM attained a lower 3-HP titer of 4.2 mM. In contrast, no measurable 3-HP production was observed at 0.35 nM cyano-converted extract, despite a very low 3-HP titer of 1.1 mM at 0.35 nM CNCbl. This suggests potential interference from matrix effects during conversion (Nakos et al., 2017) or simply the titre falling to the limit of detection (LOD = 0.8–1.0 mM). As expected, no 3-HP production was observed in the negative control with E. coli W extract, thus justifying that any potential impurities in crude bacterial extracts do not affect 3-HP production. These findings reinforce the presence of a biologically active form of B₁₂ in crude extract, and the chemical conversion process has led to a non-natural/synthetic B₁₂ form, which obviously has led to a decrease in or no 3-HP production. Collectively, these results strongly establish the utility of 3-HP production as an in vivo functional assay for B₁₂ bioactivity. Building on these findings, the next section validates the in vivo B₁₂ bioactivity using recombinant Ectopseudomonas alcaliphila MSJ19 itself.

To evaluate the in vivo bioactivity of endogenously produced B₁₂, Ectopseudomonas alcaliphila MSJ19 was engineered to express the 3-HP biosynthetic pathway via plasmid pUCPK harboring dhaB123, gdrAB, and KGSADH. Shake flask cultivation was performed with and without an exogenous supply of 2 μM AdoCbl, thereby ensuring that any observed 3-HP production is solely dependent on the host’s innate B₁₂ biosynthesis capability. Correspondingly, EaM_r produced a maximum 3-HP titre of 3.2 mM without external B₁₂, indicating the endogenous production of coenzyme B₁₂ was sufficient to activate GD and enable 3-HP biosynthesis (Figure 5A). As expected, no 3-HP production was observed in control flasks without glycerol supplementation (data not shown), confirming that 3-HP originated exclusively from glycerol metabolism and not from medium components or endogenous carbon sources. 3-HP production was improved (9.5 mM) under B₁₂ supplementation, indicating that 3-HP flux can be further enhanced through B₁₂ supplementation (Figure 5B). In addition, glycerol consumption and 3-HP production were relatively low in either case compared to EcW GD. This could be presumed due to intrinsic regulatory barriers, such as the presence of transcriptional repressors in the host’s native glycerol catabolic pathway and/or limited compatibility between the heterologous plasmid system and the host transcriptional or translational machinery (Thi Nguyen et al., 2021; Prieto-de Lima et al., 2024). Although elucidating these factors was beyond the scope of this study, the results clearly establish the functional bioavailability of naturally synthesized B₁₂ in EaM_r. These findings not only validate Ectopseudomonas alcaliphila MSJ19 as a biologically competent B₁₂ producer but also highlight its potential as a versatile microbial chassis for value-added chemical production beyond vitamin B₁₂.