Methylotrophic bacteria with cobalamin-dependent mutases in primary metabolism as potential strains for vitamin B12 production

Several bacterial species are known for their ability to synthesize vitamin B12 but biotechnological vitamin B12 production today is restricted to Pseudomonas denitrificans and Propionibacterium freudenreichii. Nevertheless, the rising popularity of veganism leads to a growing demand for vitamin B12 and thereby interest in alternative strains which can be used as efficient vitamin B12 sources. In this work, we demonstrate that methylotrophic microorganisms which utilize the ethylmalonyl-CoA pathway containing B12-dependent enzymes are capable of active vitamin B12 production. Several bacteria with an essential function of the pathway were tested for vitamin B12 synthesis. Among the identified strains, Hyphomicrobium sp. DSM3646 demonstrated the highest vitamin B12 levels reaching up to 17.9 ± 5.05 µg per g dry cell weight. These relatively high vitamin B12 concentrations achieved in simple cultivation experiments were performed in a mineral methanol medium, which makes Hyphomicrobium sp. DSM3646 a new promising cobalamin-producing strain. Supplementary Information The online version contains supplementary material available at 10.1007/s10482-022-01795-9.


Introduction
Vitamin B 12 (cobalamin) is a water-soluble vitamin which plays a very important role in various metabolic processes in human body. The recommended daily dose for adults is 2.4 µg per day (Institute of Medicine 1998) and the inadequate intake is one of the reasons for vitamin B 12 deficiency (Green et al. 2017). Several groups are at a particular risk of vitamin B 12 deficiency. Previous studies have reported the prevalence of the deficiency in the risk groups reaching over 20% among elderly people (Andrès et al. 2004), 25% in pregnant women (Sukumar et al. 2016) and up to 86.5% in adult vegans (Pawlak et al. 2014). The cobalamin deficit in these groups is caused by different reasons but they all are susceptible to symptoms of vitamin B 12 deficiency which include megaloblastic anemia, leukopenia, thrombocytosis Abstract Several bacterial species are known for their ability to synthesize vitamin B 12 but biotechnological vitamin B 12 production today is restricted to Pseudomonas denitrificans and Propionibacterium freudenreichii. Nevertheless, the rising popularity of veganism leads to a growing demand for vitamin B 12 and thereby interest in alternative strains which can be used as efficient vitamin B 12 sources. In this work, we demonstrate that methylotrophic microorganisms which utilize the ethylmalonyl-CoA pathway containing B 12 -dependent enzymes are capable of active vitamin B 12 production. Several bacteria with an essential function of the pathway were tested for vitamin B 12 synthesis. Among the identified strains, Hyphomicrobium sp. DSM3646 demonstrated the highest vitamin and many other clinical manifestations (Langan and Goodbred 2017).
The importance of vitamin B 12 for humans has become clear in the 1920s after two American physicians, Minot and Murphy, demonstrated the role of their so-called "extrinsic factor" in the treatment of pernicious anemia (Martens et al. 2002). After years of further research, the complete chemical synthesis of cobalamin was achieved by Woodward andEschenmoser in 1972 (Eschenmoser andWintner 1977). Nevertheless, the described synthesis procedure included about 70 steps, which makes it economically disadvantageous (Martens et al. 2002). Therefore, microbial synthesis is used as the exclusive strategy for industrial vitamin B 12 production nowadays.
Although there are several cobalamin-producing species (Perlman 1959), only few of them are applied for the large scale industrial production of vitamin B 12 (Fang et al. 2017). Propionibacterium freudenreichii is well known for its ability to synthesize the active form of vitamin B 12 (Deptula et al. 2015). Due to this feature, P. freudenreichii has already been investigated for its ability to fortify various food sources in several studies van Wyk et al. 2011). Other investigations described different Lactobacillus reuteri strains, which can be applied for the B 12 fortification of plant-based foods (Molina et al. 2012;Gu et al. 2015). However, Lactobacillus species were shown to produce a vitamin B 12 analogue called pseudovitamin B 12 in other studies (Crofts et al. 2013;Santos et al. 2007). The lower ligand of the active form of vitamin B 12 is represented by 5,6-dimethylbenzimidazole (DMBI), while pseudovitamin B 12 carries adenine as the lower ligand in its structure and is inactive in humans (Watanabe et al. 2013). For this reason, identification of new bacterial strains, which are able to produce the active vitamin B 12 and, therefore, can be useful for food fortification, is of great importance.
Methylotrophs are a versatile group of bacteria associated with plants. Reduced C1-compounds can be used by methylotrophs as energy and carbon source for the growth and respective species were found to produce phytohormones and other plant growth-stimulating factors (Ivanova et al. 2006;Trotsenko et al. 2001). One group of methylotrophs assimilates C1 compounds via the serine cycle elucidated by Quayle and coworkers (Large et al. 1961(Large et al. , 1962aLarge and Quayle 1963) which requires continuous regeneration of glyoxylate. This occurs in many organisms via the widespread glyoxylate cycle, while some serine cycle-methylotrophs lack the isocitrate lyase, an essential enzyme of the glyoxylate pathway (Anthony 1982). It was proposed that methylotrophs operate an alternative pathway for glyoxylate regeneration and after more than 30 years of investigation the ethylmalonyl-CoA pathway (EMCP) could be identified (Erb et al. 2007), while the activity of EMCP upon C1 assimilation in M.extorquens was demonstrated by Smejkalova et al. 2010. This pathway includes two vitamin B 12 -dependent enzymes, the ethylmalonyl-CoA mutase and methylmalonyl-CoA mutase (Erb et al. 2008;Smejkalova et al. 2010). In this context, there have been reports providing evidence for cobalamin production in methylotrophic bacteria, especially in the genus Methylobacterium (Danilova et al. 2004;Trotsenko et al. 2001;Ivanova et al. 2006).
The aim of this work was to identify microbial species with high vitamin B 12 production capabilities due to an essential function of the compound in their primary metabolism. Since Methylorubrum extorquens (formerly Methylobacterium extorquens) is able to grow on methanol as sole C-source without vitamin B 12 supplementation, we proposed that this organism is capable of cobalamin production. Moreover, we searched for additional microorganisms having the key enzymes of the ethylmalonyl-CoA pathway and analyzed them for the production of the active form of vitamin B 12 .

Identification of strains with probably functional EMCP
To identify microorganisms that possess a functional EMCP, we analyzed bacterial genome sequences for the presence of a crotonyl-CoA carboxylase/reductase (Ccr)-encoding gene. To exclude strains, that additionally contain the glyoxylate pathway, we considered only bacteria containing no isocitrate-lyase (Icl)-encoding gene. The genome dataset used as starting point was created in house from all bacterial genomes available at the Microbial Nucleotide Blast homepage (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi? PAGE_ TYPE= Blast Searc h& BLAST_ SPEC= Micro bialG enomes). If sequences similar to the M. extorquens AM1 Ccr protein sequence with e-values lower than e-value of 10 -43 could be identified within a tblastn analysis with the standard tblastn parameter values, we concluded the presence of the EMCP. Sequences with similarity to Icl of Escherichia coli with a boundary e-value of 10 -14 served as marker genes for the glyoxylate shunt.
If the candidate carried crotonyl-CoA carboxylase/reductase and lacked isocitrate lyase, they were included in the further analysis. Subsequent analysis of the literature was performed to identify the strains capable of C1 and C2 substrate utilization and the strains for which growth on C1 and C2 substrates has already been described in the literature before were selected for further investigations.

Bacterial strains and media
M. extorquens AM1 (DSM1338) and all strains identified in this work were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and propagated in the media recommended by DSMZ or previously described in literature for every specific strain. Xanthobacter autotrophicus DSM1618 and X. autotrophicus DSM432 were cultivated in medium 1, Hyphomicrobium sp. DSM3646 in medium 162, Ruegeria pomeroi DSM15171 in medium 974 (Salgado et al. 2014) and Pseudonocardia dioxanivorans DSM44775 in medium 553, respectively. Cultivation of Lactobacillus reuteri DSM20016 was performed in medium 11.

Media and cultivation conditions for vitamin B 12 production
Mineral medium used for the analysis of cobalamin production with the investigated strains was previously described by (Peyraud et al. 2009). Briefly, mineral salts contained 1.62 g/L NH 4 Cl and 0.2 g/L MgSO 4 , buffer was composed of 2.21 g/L K 2 HPO 4 and 1.25 g/L NaH 2 PO 4 × 2H 2 O (pH 7.1), and the following trace elements were used: 15 mg/L Na 2 EDTAx2H 2 O, 4.5 mg/L ZnSO 4 × 7H 2 O, 0.3 mg/L CoCl 2 × 6H 2 O, 1 mg/L MnCl 2 × 4H 2 O, 1 mg/L H 3 BO 3 , 0.4 mg/L Na 2 MoO 4 × 2H 2 O, 3 mg/L FeSO 4 × 7H 2 O and 0.3 mg/L CuSO 4 × 5H 2 ; CaCl 2 was used in the final concentration of 2.5 mg/L. The standard medium was supplemented with 0.5% (v/v) ethanol for R. pomeroyi DSM15171 and 0.5% (v/v) methanol for all other strains as the sole source of carbon and energy, 1.5% (w/v) agar was added if solid medium was used. In the medium optimization experiments with Hyphomicrobium sp. DSM3646 the amount of methanol, CaCl2 and of each component of mineral salts, buffer and trace elements was reduced or increased twice.
The strains were cultivated on agar plates or in 300 mL shaking flasks for 6 days at 30 °C under aeration and shaking at 180 rpm.

Vitamin B 12 extraction and purification
For the cobalamin analysis from the solid media the cells obtained after the incubation were scratched with the spatula from the agar surface and the whole biomass was used for cobalamin extraction. For the cobalamin analysis from the liquid cultures, 25 mL of the broth was harvested by centrifugation at 3.150 × g for 30 min. The cobalamin was extracted in the cyano-form and the cell pellets were resuspended in 10 mL of acetate buffer (4.1 g/L of sodium acetate, pH adjusted to 4.5 with acetic acid) containing 100 µL of 1% KCN. After the incubation in a water bath at 98 °C for 30 min the samples were cooled on ice for 30 min and centrifuged again. Vitamin B 12 was purified from the obtained supernatants using BAK-ERBOND spe™ C18 columns JB7020-03 (J. T. Baker, VWR, Germany) according to the manufacturer's instructions. The extracts were then syringe filtered (0.2 µM), dried at 60 °C under vacuum and resuspended in 100 µL of deionized H 2 O.

Analysis of vitamin B 12 via liquid chromatography-mass spectrometry (LC-MS/MS)
Analysis of the samples was performed with a triple quadrupole LCMS-8045 (Shimadzu, Germany) and the Lab Solutions Analysis Software (Shimadzu, Germany) was used for data acquisition and analysis. A Luna® Omega 3 µm PS C18 100 Å Column (Phenomenex, Germany) was operated with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). All solvents used were of LC/MS grade and (Carl Roth, Germany). The following LC time program was maintained during the method run: 0-3 min 18-32% B, 3-3.1 min 32-95% B, 3.1-4.1 min 95% B, 4.1-4.3 min 95-18% B, 4.3-7 min 18% B. The flow rate was 0.4 mL/min and the column temperature was maintained at 40 °C. The MS analysis was carried out in positive ion mode using electrospray ionization (ESI) under following parameters: nebulizing gas flow 3 L/min, drying gas flow 10 L/min, interface temperature 300 °C, desolvation line temperature 250 °C and heat block temperature 400 °C. The mass spectrometer was run in multiple reaction monitoring (MRM) mode for cobalamin (MRM ( +) m/z 678.40 → m/z 146.95, m/z 678.40 → m/z 359.10) and pseudocobalamin (MRM ( +) m/z 672.75 → m/z 136.05, m/z 672.75 → m/z 348.05) with method parameters (collision energies, dwell times and exact m/z values) optimized with the software for each transition. The injection volume was 1 µL and the quantification of vitamin B 12 in the samples was performed using a calibration curve obtained from a set of cyanocobalamin standards (Merck, Germany). Since pseudovitamin B 12 is commercially not available as analytical standard, the cell extract of L. reuteri containing pseudovitamin (Santos et al. 2007) was used as reference material.

Cultivation of Hyphomicrobium sp. DSM3646 in BioLector microbioreactor
The pre-cultures of Hyphomicrobium sp. DSM3646 were grown in MeOH minimal medium for 48 h and used for the inoculation of 1 mL medium at the starting OD 600 of 0.1. The cultivation was carried out in a BioLector® MB system (m2p-labs, Germany) in MTP-48 FlowerPlates® with pH optodes at 30 °C, 1000 rpm and 95% humidity. The growth of the cultures was monitored online by scattered light signal measurement.

Statistical analysis
If not stated otherwise, all experiments were repeated in biological triplicates. Data are presented as the mean value ± standard deviation. Two-sample t-test was used to determine the significance of the difference between the means. Values were considered significant at p < 0.05. The analyses were performed using OriginPro® data analysis software.

Results
Confirmation of cobalamin production with M. extorquens AM1 by the means of LC-MS/MS Firstly, we aimed to validate our hypothesis about potentially high vitamin B 12 levels in microorganisms with an essential function of cobalamin-dependent mutases in the EMCP as primary metabolism pathway. Therefore, we confirmed cobalamin production in the cells of M. extorquens AM1 upon growth with methanol as sole carbon source. The performed LC-MS/MS analysis of the obtained cell extracts demonstrated characteristic masses of cyanocobalamin (Fig. 1).
The detected parent ion signal with m/z of 678.40 and fragment ion signals with m/z 146.95 and m/z 359.10, which correspond to [DMBI + H] + and [DMBI + sugar + PO 3 + H] + , respectively, provided evidence for vitamin B 12 synthesis in M. extorquens AM1.

Identification of the candidate strains
To identify other microorganisms with a high vitamin B 12 demand and potential for high vitamin B 12 synthesis, we aimed to find bacterial strains with essential function of the cobalamin-dependent mutases in the EMCP. Among all at the time of the search available bacterial genomes, 65 strains could be identified, which contained a Ccr-encoding gene and lacked an Icl-encoding gene according to the presence of sequences with similarity to the used queries (Supplementary Table S1). Analysis of literature data revealed that X. autotrophicus DSM1618 and X. autotrophicus DSM432, Hyphomicrobium sp. DSM3646 and P. dioxanivorans DSM44775 should be able to grow on methanol as sole carbon source (Ginkel and Bont 1986;Harder et al. 1973; Grostern and Alvarez-Cohen 2013), while R. pomeroyi DSM15171 was reported to be capable of ethanol utilization (González et al. 2003). As the EMCP should be only essential during growth with C1 or C2 carbon sources, we focused on these five bacteria for further investigations.

Verification of active vitamin B 12 production in the cells of selected strains
For the analysis of vitamin B 12 production the five selected strains were cultivated on agar plates with minimal medium containing methanol or ethanol as the sole carbon source. In contrast to previously reported data (González et al. 2003), we could not detect growth of R. pomeroyi DSM15171 in the respective medium. X. autotrophicus DSM1618, X. autotrophicus DSM432, Hyphomicrobium sp. DSM3646 and P. dioxanivorans DSM44775 were able to grow on agar plates with solid minimal medium containing 0.5% (v/v) methanol and were analyzed for the ability to produce the active form of vitamin B 12 and pseudocobalamin.
As Fig. 2 shows, due to the differences in their ligand structure, the chromatograms of the active cobalamin and pseudovitamin B 12 demonstrate characteristic masses of parent and fragment ions and also differences in their retention times. Analysis of the cell extracts obtained after the cultivation in the minimal methanol medium demonstrated a peak at 1.77 min showing the parent ion signal with m/z of 678.40 and fragment ion signals with m/z 146.95 and m/z 359.10 present in the chromatograms of the samples from all strains. These values correspond to the retention time and fragmentation pattern of cyanocobalamin and confirm the ability of the identified strains for the synthesis of active vitamin B 12 .
Moreover, active cobalamin was the only form produced by all strains. The presence of pseudovitamin B 12 could be excluded, since its characteristic peak at 1.38 min showing the parent ion signal with m/z of 672.75 and fragment ion signals with m/z 136.05 and m/z 348.05 was not present in the chromatograms of the four strains.
Comparison of vitamin B 12 production by the selected strains In order to identify strains with high vitamin B 12 production capabilities, the cyanocobalamin peak areas obtained from the cell extracts were compared. As Fig. 3 shows, the peak areas varied greatly between the samples from the four strains. Although all bacteria were capable of vitamin B 12 production, the peak area corresponding to the cell extract of Hyphomicrobium sp. DSM3646 was 7.5 times higher than the peak area of P. dioxanivorans DSM44775 cell extract and nearly 30 times higher than those determined for the cell extracts of X. autotrophicus DSM1618 and X. autotrophicus DSM432.
In addition to the highest vitamin B 12 peak areas, only Hyphomicrobium sp. DSM3646 was able to grow in liquid medium, while no growth in liquid medium was observed for P. dioxanivorans DSM44775, X. autotrophicus DSM1618 and X. autotrophicus DSM432. Vitamin B 12 production by Hyphomicrobium sp. DSM3646 and M. extorquens AM1 As a subsequent step, we decided to compare the initial M. extorquens AM1 strain with the newly identified Hyphomicrobium sp. DSM3646 for their ability to synthesize vitamin B 12 in the minimal methanol medium (Fig. 4).
The OD 600 value of 2.97 ± 0.06 measured in the culture of Hyphomicrobium sp. DSM3646 was significantly higher than 2.06 ± 0.06 reached by M. extorquens AM1. Therefore, the amount of cobalamin was expressed per g dry cell weight (DCW) for the relative comparison. The value detected in the culture of Hyphomicrobium sp. DSM3646 was slightly higher than the amounts reached by M. extorquens AM1, nevertheless, these differences were not significant.  Two-sample t-test was used for statistical comparison between the strains, differences considered as significant at p < 0.05 are denoted by an asterisk However, the subsequently performed t-test for the cobalamin concentrations per 100 mL culture showed that the vitamin B 12 amounts varied significantly between the producing strains. The yield of vitamin B 12 achieved by Hyphomicrobium sp. DSM3646 was 2.2 ± 0.17 µg per 100 mL culture, which is 1.7 times higher than 1.29 ± 0.08 µg per 100 mL culture produced by M. extorquens AM1. Due to the higher volume yield of vitamin B 12 , which is probably caused by higher biomass formation, Hyphomicrobium sp. DSM3646 was chosen for further investigation.
Optimization of Hyphomicrobium sp. DSM3646 cultivation medium The components of the minimal medium were evaluated to identify the factors that have effect on biomass accumulation with Hyphomicrobium sp. DSM3646. For this purpose, we altered the concentration of methanol, buffer, CaCl 2 , trace elements and mineral salts in the standard medium and tested media in which the amount of each single component was increased or reduced twice. The standard minimal medium was used as reference and the growth behavior of Hyphomicrobium sp. DSM3646 under the standard and altered conditions was investigated.
The effect of different media components on the growth of the organism is presented in Fig. 5. Among all investigated components, methanol was identified as the factor having the greatest influence on the growth of Hyphomicrobium sp. DSM3646. The results show that both increase and decrease of methanol concentration, had a significant effect on biomass formation. The values observed in the medium with 1% methanol reached above 35 a. u. after 50 h, while 0.25% of methanol in the medium resulted in a scattered light signal of 12 a. u., which was significantly lower than the values achieved in the standard medium with 0.5% methanol. As Fig. 5 shows, the changes in CaCl 2 concentration had no effect on the growth behavior of Hyphomicrobium sp. DSM3646, while only slight biomass increase was observed when buffer, trace elements and mineral salts concentration was increased.
For the determination of the optimal methanol concentration, the growth of Hyphomicrobium sp. DSM3646 in the standard minimal medium containing 0.5% methanol (control) was compared with the Fig. 5 Influence of the methanol mineral medium components on the growth of Hyphomicrobium sp. DSM3646. The cultivations were performed in BioLector microbioreactor, the data points represent the mean values and standard deviations of three biological replicates growth in media containing 1%, 2%, 3% or 4% of methanol (Fig. 6). All changes in methanol concentrations affected the growth resulting in a maximal scattered light signal 1.6 times higher in comparison to the reference medium. The highest scattered light signal was determined in the media with methanol concentrations of 2% and higher, while no further signal increase was detected in the media containing 3% and 4% of methanol.
Despite the positive influence on biomass formation, higher methanol concentration led to elongated lag phases. Hyphomicrobium sp. DSM3646 could reach the maximum scattered light signal after 40 h in the control medium, while it took 45 h, 50 h, 58 h and 70 h in the media with 1%, 2%, 3% and 4% of methanol, respectively. Overall, most efficient biomass formation with only slight growth inhibition was observed in the medium with 2% of methanol. Therefore, it was chosen as starting point for the following experiment with Hyphomicrobium sp. DSM3646.
Since we aimed to avoid the inhibitory effect of methanol in this medium, we decided to inoculate the cells in the medium containing 1% of methanol and add further 1% of methanol in the mid-exponential phase of growth. These growth conditions were then investigated in combination with the doubled amounts of each other medium component (Fig. 7a).
Stepwise methanol addition enabled to achieve the maximum scattered light signal after 35 h, which was comparable with the medium with standard methanol concentration (Fig. 6). Among all tested components, the increased amount of the buffer had the greatest influence on the biomass formation leading to the significant increase of the scattered light signal over 64 a. u. This value was 1.7 times higher than in the medium with the standard buffer concentration and 2% methanol and 2.8 times higher than the signal reached in the medium with standard buffer concentration and 0.5% methanol. We proposed that pH was the growth-limiting factor in the media with the standard buffer concentration and performed pH measurement in all tested media. As. Figure 7b shows, the pH value changes correlated with the growth behavior of the cultures. The pH drop observed in the media with the standard buffer amount led to a pH value of 5 after 35 h, which was the time point when the stationary growth phase was reached. In case of the medium with the increased buffer concentration, the same trend was observed 5 h later, when acidic pH Fig. 6 Growth curves of Hyphomicrobium sp. DSM3646 with increased methanol concentrations of 1-4%, standard medium with 0.5% methanol used as control. The cultivations were performed in BioLector microbioreactor, the data points represent the mean values and standard deviations of three biological replicates and the stationary growth phase were achieved after 40 h of cultivation.
As no significant effect on the growth of the cells was observed for concentration changes of other medium components, the medium with increased buffer amount supplemented with 2% of methanol was taken for the further investigation of vitamin B 12 production.
Production of vitamin B 12 by Hyphomicrobium sp. DSM3646 in the standard and optimized minimal medium The levels of produced active vitamin B 12 were determined for cells in the late stationary phase of growth and the results were compared for the standard medium and for the media with increased methanol and buffer concentrations (Fig. 8).
Although the optimization of the cultivation medium significantly influenced the biomass formation, this did not increase the yield of vitamin B 12 compared to the standard medium. The amounts of cobalamin in 2% methanol medium were slightly lower than those reached in the standard medium, while minor increase of cobalamin content was detected in the 2% methanol medium with the doubled buffer concentration. However, the unpaired t-test revealed that the differences between the values detected in the tested media were not significant.
Since the OD values varied greatly between 3.2 ± 0.17, 6.5 ± 0.2 and 9.93 ± 0.76 for the standard, 2% methanol and 2% methanol medium with the increased buffer, respectively, we calculated the amount of cobalamin per g DCW for the purpose of relative comparison. The highest yield of 17.9 ± 5.05 µg per g DCW was obtained in the standard medium, which was significantly higher than 8.11 ± 1.21 µg per g DCW of vitamin B 12 achieved in the 2% methanol medium with the increased buffer concentration.

Discussion
Although this issue has not been investigated so far to our knowlewdge, one might assume, that organisms with high vitamin B 12 synthesis capability have an essential need for the cofactor itself. The reduction of pyruvate to propionate, a key reaction of P. freudenreichii metabolism, occurs in the Wood-Werkman cycle, which includes coenzyme B 12 -dependent methylmalonyl-CoA mutase (Thierry et al. 2011). The vitamin B 12 -producing plant symbiont Sinorhizobium meliloti (Burton and Lochhead 1952) requires a cobalamin-dependent ribonucleotide reductase to establish the symbiosis with its plant host (Taga and Walker 2011). Similar to these microorganisms, methylotrophs with cobalamin-dependent enzymes in the EMCP seemed to be promising as a new source for vitamin B 12 . Our findings show that investigation of microorganisms with essential functions of cobalamin in their primary metabolism is a successful strategy for the identification of new vitamin B 12 producers.
Although few preliminary reports have already described production of vitamin B 12 in methylotrophic bacteria (Ivanova et al. 2006;Danilova et al. 2004), the total content of corrinoids was measured with Escherichia coli strain 113-3 auxotrophic for vitamin B 12 in those studies. However, this microbiological assay was shown to respond not only to vitamin B 12 but also to its analogues (Ford 1959), which is why a comparison of these previously reported data with the results obtained for the active vitamin B 12 in this study was not performed. Therefore, if the goal is to produce pure cobalamin or fortify food for human nutrition purposes, it is important to determine the type of the cobalamin synthesized by the microorganisms . In our study, we report on the identification of the active vitamin B 12 with a sensitive LC-MS/MS method in the cell extracts of M. extorquens AM1. Moreover, based on these data, we were also able to identify other strains with high vitamin B 12 production capability, which have not been described in literature before. Our analyses show, that the active B 12 is the form exclusively produced by all identified candidates (Fig. 2) with Hyphomicrobium sp. DSM3646 showing the highest content under the conditions tested (Fig. 3). Direct comparison of vitamin B 12 production with M. extorquens AM1 and the newly identified Hyphomicrobium sp. DSM3646 demonstrated that the latter is the most suitable strain for vitamin B 12 production.
We performed Hyphomicrobium sp. DSM3646 growth experiments which identified conditions enabling higher biomass formation compared to the standard medium. Although increased methanol concentration positively influenced biomass formation, the analysis of vitamin B 12 synthesis revealed that the highest cobalamin amounts were produced in the standard medium. This implies that vitamin B 12 production does not directly correlate with biomass formation and makes the standard medium more advantageous for vitamin B 12 production. Moreover, we proposed that acidification can be another factor limiting growth of Hyphomicrobium sp. DSM3646. To examine this issue, we measured pH and detected significant differences in values between the 2% methanol medium with standard or increased buffer concentrations (Fig. 7b). Nevertheless, high biomass accumulation observed in Fig. 8 Comparison of vitamin B 12 production in the standard medium (control), in the medium with 2% methanol and in the medium with 2% methanol and twofold increased buffer concentration. The data are shown as the mean values and standard deviations of three biological replicates. Two-sample t-test was used for statistical comparison between the media, differences considered as significant at p < 0.05 are denoted by an asterisk the latter medium was accompanied with production of lower vitamin B 12 amounts. This observation underlined the finding that the standard medium is the most appropriate for vitamin B 12 production. Cobalt can also be a limiting factor for vitamin B 12 synthesis, since it is necessary for the corrinoid ring formation, which is why it is often supplied during vitamin B 12 production (Chamlagain et al. 2016;Deptula et al. 2015). Since cobalt was added into the medium composition in this study and the used concentrations exceed greatly the amounts of the obtained vitamin B 12 , we propose that there should be another factor limiting vitamin B 12 production in Hyphomicrobium sp. DSM3646.
The achieved cobalamin levels are lower than the concentration range of 45-335 µg per 100 mL culture reported previously for various P. freudenreichii strains (Chamlagain et al. 2016;Deptula et al. 2017). However, it is worth mentioning, that the values described for P. freudenreichii were obtained after optimization of the production process, while the current study describes the first attempts on vitamin B 12 production with Hyphomicrobium sp. DSM3646. Moreover, the completely aerobic cultivation process for vitamin B 12 production with Hyphomicrobium sp. DSM3646 is advantageous in comparison to the two-step cultivation procedure necessary for vitamin B 12 synthesis with P. freudenreichii (Chamlagain et al. 2016Deptula et al. 2017). Since production of active vitamin B 12 with Hyphomicrobium sp. DSM3646 was demonstrated in this work, it makes it attractive for possible enrichment of plant-based foods with vitamin B 12 . For this reason, further investigations on Hyphomicrobium sp. DSM3646 safety should be made for its possible use in food fortification. Finally, the use of low-cost methanol mineral medium for vitamin B 12 production with Hyphomicrobium sp. DSM3646 can contribute to the development of an economically viable process. The economic benefits of such process can be evaluated in future studies after optimization of its productivity against the commonly used strains.
Author contributions DD carried out literature review, performed the experiments, analyzed the data and wrote the manuscript, DH supervised the work and revised the manuscript, MB performed BLAST analysis of genome data, supervised the work and revised the manuscript.
Funding Open Access funding enabled and organized by Projekt DEAL. This study was supported by the LOEWE project AROMAplus of the State of Hessen (Germany).

Data availability
The data generated during the current study are available from the corresponding author on reasonable request.

Declarations
Competing interests The authors declare no competing interests.

Conflict of interest
The authors have no competing interests to declare that are relevant to the content of this article.
Ethical approval This article does not contain any studies with human participants or animals.
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