B12 Production Research Articles

Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

The only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea. Here, using genetic and metabolic engineering, we generate an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. In vitro and/or in vivo analysis of genes involved in adenosylcobinamide phosphate biosynthesis from Rhodobacter capsulatus suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increase the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg g−1 DCW via metabolic engineering and optimization of fermentation conditions. Beyond our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

Analysis and characterization of coenzyme B12 biosynthetic gene clusters and improvement of B12 biosynthesis in Pseudomonas denitrificans ATCC 13867.

Coenzyme B12 is an essential cofactor for many enzymes such as glycerol dehydratase, methionine synthase and methylmalonyl-CoA mutase. Herein, we revisited the B12 biosynthetic gene clusters (I and II) in Pseudomonas denitrificans, a well-known industrial producer of the coenzyme B12, to understand the regulation of gene expression and improve the production of coenzyme B12. There were eight operons, seven in cluster I and one in cluster II, and four operons were regulated by B12-responsive riboswitches with a switch-off concentration at ∼5 nM coenzyme B12. DNA sequences of the four riboswitches were partially removed, individually or in combination, to destroy the structures of riboswitches, but no improvement was observed. However, when the whole length of riboswitches in cluster I were completely removed and promoters regulated by the riboswitches were replaced with strong constitutive ones, B12 biosynthesis was improved by up to 2-fold. Interestingly, modification of the promoter region for cluster II, where many (>10) late genes of B12 biosynthesis belong, always resulted in a significant, greater than 6-fold reduction in B12 biosynthesis.

The B-Vitamin Mutualism Between the Dinoflagellate Lingulodinium polyedrum and the Bacterium Dinoroseobacter shibae

Recent research has shown that in aquatic systems pairs of prokaryote and eukaryote species exercise symbiotic exchanges of metabolites that are essential for the proliferation of either species. Using dinoflagellate Lingulodinium polyedrum cultures and a factorial design, we examined its growth at different concentrations of vitamin B1 (thiamine) and B12 (cobalamin). When both vitamins were at their lowest concentrations tested, 0.033 pM of B1 and 0.053 pM of B12 the growth was limited. When axenic L. polyedrum was co-cultured with the bacterium Dinoroseobacter shibae, a known B1 and B12 producer, then L. polyedrum grew at the same rate as in culture media supplemented with B1 and B12. In the L. polyedrum vitamin - limited culture (V-L), the abundance of attached and free-living D. shibae was higher than in the vitamin - replete (V-R) culture. In the V-R and V-L co-cultures the measured particulate B12 (pB12) concentration of attached and free-living D. shibae were in the range of 4.7x10-19 to 3x10-18 and 8.4x10-21 to1.2x10-19 (mol cell-1) respectively. Without B12 or B7 (biotin) added to the culture medium of a co-culture of L. polyedrum and D. shibae, the measured dissolved B12 (dB12) concentration was more than 60 pM higher than necessary for un-limited growth rates of L. polyedrum. In the same culture we measured B7 in the L. polyedrum particulate fraction (pB7; 4.7x10-19 to 9.4x10-19 mol cell-1). We suggest that in response to the production of B1 and B12 by D. shibae to supply L. polyedrum requeriments, the latter produced B7, which is required by D. shibae, and in our culture was only produced by L. polyedrum when D. shibae was present. We propose that D. shibae can control L. polyedrum through the release of B1 and B12, and L. polyedrum can control D. shibae through the release of B7. D. shibae is also auxotroph for niacin and 4-amino-benzoic acid, not provided by the culture medium. Therefore, L. polyedrum might affect a similar control through the release of these specific compounds and organic substrate necessary for the growth of D. shibae.

Use of a Potential Probiotic, Lactobacillus casei L4, in the Preparation of Fermented Coconut Water Beverage

Coconut water (CW) is a clear, nutritive liquid found as the coconut endosperm of green coconuts such as Cocos nucifera L., and its widespread consumption owes to its unique composition of sugars, minerals, vitamins, enzymes, and hormones. Probiotic fermentation of CW may facilitate the development of an improved functional beverage with probiotic benefits; therefore, we aimed to produce a fermented CW beverage using the potential probiotic Lactobacillus casei L4. CW was fermented with L. casei L4 for 48 h at 35°C, and the pH, organic acid-production rate, antioxidant activity, antibacterial activity, sugar, mineral, vitamin B12 levels, and total viable bacteria counts were investigated at 24 and 48 h. We demonstrated that the fermentation of CW with probiotic lactobacilli increased the cell viability count. Vitamin B12 production was highest in the extracellular environment at 48 h (11.47 μg/mL), while the total phenolic content was significantly (p < 0.05) higher in the fermented CW at 48 h (72.1 μg/mL gallic acid equivalents) than observed with the other investigated groups or time points. The fermented materials exhibited the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activities at 48 h (58.4 and 69.2%, respectively). The levels of most minerals remained unchanged in the fermented CW, except for calcium, manganese, phosphorus, and sodium. Furthermore, the culture supernatant from fermented CW inhibited the growth of foodborne pathogens such as Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, and Salmonella typhi, although the degree of inhibition varied between the species. Moreover, adding 15% honey and artificial coconut flavor to the fermented CW resulted in a better-tasting product, as demonstrated by a sensory-evaluation test. The obtained results indicated that the CW product fermented by L. casei L4 may be used as a novel functional beverage containing both electrolytes and probiotics, and can serve as a good vehicle for preparing a wider range of novel products.

Enhancing vitamin B12 content in co-fermented soy-milk via a Lotka Volterra model

Abstract Soybean products are popular because of its taste, digestibility, and health benefits. However, soybean lacks vitamin, mainly the low water-soluble vitamin B12. This study investigated the effects of fermentation conditions on the synthesis of vitamin B12, production of metabolites, and growth of Lactobacillus reuteri and Propionibacterium shermainii in fermented soy-milk. A Lotka Volterra model was successfully employed to describe the competition relationship between the two microorganisms under various fermentation conditions. A quadratic function between the ratio of interaction coefficients and vitamin B12 content was found. Higher vitamin B12 in soy-milk can be produced when the ratio of interaction coefficients approach to one. Compared with other fermented soybean products, fermented soy-milk contains more acetate, ethanol, and propionic acid. This study successfully demonstrated a mathematical model to enhance soy-milk vitamin B12 production.

In situ fortification of vitamin B12 in wheat flour and wheat bran by fermentation with Propionibacterium freudenreichii

Vitamin B12 is a micronutrient naturally existing in animal products. A growing interest and need to replace animal protein with plant protein sources have resulted in increased attention to developing vitamin B12-fortified plant-based food. Natural fortification by Propionibacterium freudenreichii is a promising alternative to chemical fortification, as P. freudenreichii can synthesize active vitamin B12. In this work, we studied vitamin B12 production in non-sterile matrices prepared from three raw materials of wheat: durum flour, wholewheat flour and wheat bran. Viable cell counts, pH, total titratable acidity and concentration of acids were determined. After seven days of fermentation, vitamin B12 levels reached 33 ± 4, 87 ± 10 and 155 ± 17 ng/g dry weight in durum flour, wholewheat flour, and wheat bran, respectively. While durum flour supported the growth of P. freudenreichii to higher cell densities and more efficient propionic acid production compared with the other two matrices, wholewheat flour and wheat bran were found to be the most promising of the three matrices for in situ production of vitamin B12.

Transient plasma cobalamin elevation in patients with pneumonia – two case reports

We report two cases of transient significantly elevated plasma cobalamin (B12) in geriatric patients acutely admitted with fever, increased C-reactive protein and X-ray verified pneumonia. Extensive diagnostic workup did not reveal kidney or liver disease, neither any signs of cancer. Furthermore, none of the patients had received therapeutic B12 supplementation prior to admission. In both cases, plasma B12 normalized at an out-patient control few months later. We were not able to identify the reason for the initial B12 elevation in any of the patients, since none of the usually recognized causes were evident. Since both patients had an infection and temporarily elevated B12, we suggest a possible inflammatory response or a vitamin B12 production by the infectious agents as the cause. Both suggestions, however, need further exploration.

Quantification of Vitamin B12-Related Proteins in Marine Microbial Systems Using Selected Reaction Monitoring Mass Spectrometry.

Mass spectrometry-based proteomic approaches to studying microbial systems enable assessment of taxonomically resolved functional capacity. A subset of these proteomic approaches are absolutely quantitative, enabling comparisons of protein expression patterns between different studies and across environments. This chapter outlines a method for applying quantitative assays in marine microbial communities, using proteins involved in vitamin B12 (cobalamin) utilization and production as specific examples. This approach involves identifying important protein targets, determining taxonomic resolution of the required assays, identifying suitable peptides, developing and optimizing liquid chromatography-selected reaction monitoring mass spectrometry assays (LC-SRM-MS), and processing the resulting data. Implementing the method outlined here results in measurements (fmol diagnostic peptide per μg of total bulk protein) that, in this case, define the nutritional status of microbial community members with respect to vitamin B12, and are comparable across and between marine microbial systems.

In situ production of active vitamin B12 in cereal matrices using Propionibacterium freudenreichii.

The in situ production of active vitamin B12 was investigated in aqueous cereal‐based matrices with three strains of food‐grade Propionibacterium freudenreichii. Matrices prepared from malted barley flour (33% w/v; BM), barley flour (6%; BF), and wheat aleurone (15%; AM) were fermented. The effect of cobalt and the lower ligand 5,6‐dimethylbenzimidazole (DMBI) or its natural precursors (riboflavin and nicotinamide) on active B12 production was evaluated. Active B12 production was confirmed by UHPLC–UV–MS analysis. A B12 content of 12–37 μg·kg−1 was produced in BM; this content increased 10‐fold with cobalt and reached 940–1,480 μg·kg−1 with both cobalt and DMBI. With riboflavin and nicotinamide, B12 production in cobalt‐supplemented BM increased to 712 μg·kg−1. Approximately, 10 μg·kg−1 was achieved in BF and AM and was increased to 80 μg·kg−1 in BF and 260 μg·kg−1 in AM with cobalt and DMBI. The UHPLC and microbiological assay (MBA) results agreed when both cobalt and DMBI or riboflavin and nicotinamide were supplemented. However, MBA gave ca. 20%–40% higher results in BM and AM supplemented with cobalt, indicating the presence of human inactive analogues, such as pseudovitamin B12. This study demonstrates that cereal products can be naturally fortified with active B12 to a nutritionally relevant level by fermenting with P. freudenreichii.

Enhanced vitamin B12 production in an innovative lupin tempeh is due to synergic effects of Rhizopus and Propionibacterium in cofermentation

Fermentation represents a valuable and cost-effective approach for food stabilisation and nutritional improvement. Tempeh is an example of soybean solid-state fermentation. In this work, we investigated the possibility of producing a tempeh analogue containing high amounts of vitamin B12 using seeds of three different species of the legume lupin, namely Lupinus albus, L. angustifolius and L. mutabilis, with Rhizopus oligosporus and Propionibacterium freudenreichii cofermentation. Synergic effects of Rhizopus and Propionibacterium in increasing vitamin B12 up to 1230 ng/g dw was observed. These findings indicate that this cofermentation can improve lupin nutritional quality and safety to provide a tempeh analogue with added value for vegan and vegetarian communities and low-income populations. The level of potentially toxic lupin alkaloids was also monitored during the tempeh preparation.

Applicability of ribosome engineering to vitamin B12 production by Propionibacterium shermanii.

Ribosome engineering has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking vitamin B12 production as a representative example. The introduction into Propionibacterium shermanii of mutations conferring resistance to rifampicin, gentamicin, and erythromycin, respectively, increased per cell production (μg/L/OD600) of vitamin B12 5.2-fold, although net production (μg/L) was unchanged, as the cell mass of the mutants was reduced. Real-time qPCR analysis demonstrated that the genes involved in vitamin B12 fermentation by P. shermanii were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of vitamin B12 by the mutants. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites.

Novel vitamin B12-producing Enterococcus spp. and preliminary in vitro evaluation of probiotic potentials.

Vitamin B12 is an essential nutrient required for crucial metabolic processes in humans. Vitamin B12-producing lactic acid bacteria (LAB) have been attracting increased attentions currently because of the generally recognized as safe (GRAS) status. Most of recent studies focused on Lactobacillus, and little is known about B12-producing Enterococcus. In the present study, five Enterococcus strains isolated from infant feces were identified as vitamin B12 producers. Among them, Enterococcus faecium LZ86 had the highest B12 production (499.8±83.7μg/L), and the B12 compound from LZ86 was identified as the biological active adenosylcobalamin, using reversed phase high-performance liquid (RP-HPLC) chromatogram. We examined basic probiotic and safety properties of E. faecium LZ86 and found that it was able to survive harsh environmental conditions (hot temperature, cold temperature, ethanol and osmotic stresses), tolerate gastric acid (pH 2.0, 3h) and bile salts (0.3%), and adhere to Caco-2 cells. We also showed that E. faecium LZ86 is devoid of transferable antibiotic resistance and potential virulence factors. Together, here we report a B12-producing E. faecium strain LZ86 firstly, which has desirable probiotic properties and may serve as a good candidate for vitamin B12 fortification in food industry.

Characterization of extracellular vitamin B12 producing Lactobacillus plantarum strains and assessment of the probiotic potentials

We investigated extracellular vitamin B12-producing Lactobacillus strains and their characteristics in tolerance to environmental stresses, gastric acid and bile salts. Two isolates, Lactobacillus plantarum LZ95 and CY2, showed high extracellular B12 production, 98±15μg/L and 60±9μg/L respectively. Extracellular B12 from LZ95 were identified as adenosylcobalamin and methylcobalamin using a combination of solid phase extraction and reverse-phase HPLC, while that from CY2 was adenosylcobalamin. Both strains grew under environmental stresses, and LZ95 exhibited better tolerance to low temperature and high ethanol concentration. LZ95 also showed good viability when exposed to gastric acid (pH 2.0 and 3.0) and bile salts (0.3%) as well as good adhesion to Caco-2 cells. The viability of CY2 was significantly reduced under low pH and exposure to bile salt. Together, extracellular B12 producer LZ95 with good probiotic properties might be a candidate for in situ B12 fortification in the food industry.

Adaptation of Propionibacterium freudenreichii to long-term survival under gradual nutritional shortage.

Background Propionibacterium freudenreichii is an Actinobacterium widely used in the dairy industry as a ripening culture for Swiss-type cheeses, for vitamin B12 production and some strains display probiotic properties. It is reportedly a hardy bacterium, able to survive the cheese-making process and digestive stresses.ResultsDuring this study, P. freudenreichii CIRM-BIA 138 (alias ITG P9), which has a generation time of five hours in Yeast Extract Lactate medium at 30 °C under microaerophilic conditions, was incubated for 11 days (9 days after entry into stationary phase) in a culture medium, without any adjunct during the incubation. The carbon and free amino acids sources available in the medium, and the organic acids produced by the strain, were monitored throughout growth and survival. Although lactate (the preferred carbon source for P. freudenreichii) was exhausted three days after inoculation, the strain sustained a high population level of 9.3 log10 CFU/mL. Its physiological adaptation was investigated by RNA-seq analysis and revealed a complete disruption of metabolism at the entry into stationary phase as compared to exponential phase.Conclusions P. freudenreichii adapts its metabolism during entry into stationary phase by down-regulating oxidative phosphorylation, glycolysis, and the Wood-Werkman cycle by exploiting new nitrogen (glutamate, glycine, alanine) sources, by down-regulating the transcription, translation and secretion of protein. Utilization of polyphosphates was suggested.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3367-x) contains supplementary material, which is available to authorized users.

Phenotypic and genotypic screening of human-originated lactobacilli for vitamin B12 production potential: process validation by micro-assay and UFLC.

Vitamin B12 (B12) production is a strain specific, rare and hidden functional attribute of lactobacilli and a cogent protocol for selection of such isolates from the herd of lactobacilli is required. The present study included isolation of lactobacilli from human samples (milk and fecal), screening them by a polyphasic (three-phase) methodology for probable B12 production potential and validating the screening protocol by exploring selected strains for in vitro vitamin production (two-phase fermentation) and quantification [micro-assay and ultra fast liquid chromatography (UFLC)]. Fifty-nine Lactobacillus strains were recovered from tested biological samples. Contrary to screening inapplicabilities of first [growth potential (GP) in B12-free medium] and second phases (GP in B12-free and cobalt chloride-supplemented conditions), third phase (cbiK gene detection on genomic DNA) alone was revealed as a validated strategy for selection of two probable B12-producing lactobacilli. Microbiological assay confirmed production and bioavailability of produced vitamin, while UFLC testing validated the results by precisely quantifying the cyanocobalamin (industrially produced bio-available form of B12) in cell extracts of both possible B12 producers [BHM10 (10.91±1.55μg/l) and BCF20 (23.90±1.73μg/l)] and positive standard [Lactobacillus reuteri DSM20016 (20.03±4.17μg/l)]. Moreover, this study generates a novel report for genomic detection, partial amplification and sequencing of cbiK gene in Lactobacillus plantarum species (both BHM10 and BCF20). In conclusion, contrary to first two phases, cbiK gene detection strategy successfully selects B12-producing strains from a group of human-originated lactobacilli and can be used in the future for similar screening studies.

Effect of the lower ligand precursors on vitamin B12 production by food-grade Propionibacteria

Propionibacterium freudenreichii is the only generally recognized safe (GRAS) bacterium known to synthesize active vitamin B12 and offers previously untapped potential for naturally fortifying foods with vitamin B12. Biosynthesis of the lower ligand 5,6-dimethylbenizimidazole (DMBI) is often a key limiting factor in the production of active vitamin B12 in Propionibacteria. Here, we studied the effect of the natural food-grade precursors of DMBI [riboflavin (RF) and nicotinamide (NAM)] on vitamin B12 production by 27 P. freudenreichii and 3 Propionibacterium acidipropionici strains in whey-based medium. We employed sensitive and selective UHPLC and LC–MS/MS to confirm and quantify the synthesized vitamin B12. In 12 P. freudenreichii strains, co-supplementation with RF (40 μM) and NAM (27 mM) increased the volumetric yield of vitamin B12 up to 4-fold compared to the control cultures. For the majority of these strains, the production level with RF and NAM exceeded the yield obtained with DMBI supplementation (100 μM). The significant positive correlation between RF consumption and vitamin B12 production suggests that RF is proportionally directed towards vitamin B12 biosynthesis. This study shows that the availability of RF and NAM enhances the production of active vitamin B12 by P. freudenreichii in a strain-dependent manner.

The Online Morphology Control and Dynamic Studies on Improving Vitamin B12 Production by Pseudomonas denitrificans with Online Capacitance and Specific Oxygen Consumption Rate.

The relationship between the morphological character of Pseudomonas denitrificans and vitamin B12 synthesis based on real-time capacitance measurement and online specific oxygen consumption rate (Q O2) control was established for enhancing vitamin B12 production. Results demonstrated that the threshold Q O2 value lower than 2.0mmol/gDCW/l would greatly stimulate the state transfer from the cell number growth phase to the cell elongation phase and promote rapid vitamin B12 biosynthesis, while the vitamin B12 biosynthesis rate could also be inhibited when the rate of cell's length-to-width ratio (ratio-LW) was higher than 10:1. Furthermore, the optimal morphology controlling strategy was achieved based on online Q O2 control, which increases the appropriate active cell numbers at the former phase, and then control the elongation of ratio-LW no more than 10:1 at the vitamin B12 biosynthesis phase. The maximal vitamin B12 production reached 239.7mg/l at 168h, which was improved by 14.7% compared with the control (208mg/l). This online controlling strategy would be effectively applied for improving industrial vitamin B12 fermentation.

Permanent draft genome sequence of the probiotic strain Propionibacterium freudenreichii CIRM-BIA 129 (ITG P20).

Propionibacterium freudenreichii belongs to the class Actinobacteria (Gram positive with a high GC content). This “Generally Recognized As Safe” (GRAS) species is traditionally used as (i) a starter for Swiss-type cheeses where it is responsible for holes and aroma production, (ii) a vitamin B12 and propionic acid producer in white biotechnologies, and (iii) a probiotic for use in humans and animals because of its bifidogenic and anti-inflammatory properties. Until now, only strain CIRM-BIA1T had been sequenced, annotated and become publicly available. Strain CIRM-BIA129 (commercially available as ITG P20) has considerable anti-inflammatory potential. Its gene content was compared to that of CIRM-BIA1 T. This strain contains 2384 genes including 1 ribosomal operon, 45 tRNA and 30 pseudogenes.

Highly Heterogeneous Probiotic Lactobacillus Species in Healthy Iranians with Low Functional Activities

BackgroundLactic acid bacteria (LAB) have been considered as potentially probiotic organisms due to their potential human health properties. This study aimed to evaluate both in vitro and in vivo, the potential probiotic properties of Lactobacillus species isolated from fecal samples of healthy humans in Iran.Methods and ResultsA total of 470 LAB were initially isolated from 53 healthy individual and characterized to species level. Of these, 88 (86%) were Lactobacillus species. Biochemical and genetic fingerprinting with Phene-Plate system (PhP-LB) and RAPD-PCR showed that the isolates were highly diverse consisted of 67(76.1%) and 75 (85.2%) single types (STs) and a diversity indices of 0.994 and 0.997, respectively. These strains were tested for production of adhesion to Caco-2 cells, antibacterial activity, production of B12, anti-proliferative effect and interleukin-8 induction on gut epithelial cell lines and antibiotic resistance against 9 commonly used antibiotics. Strains showing the characteristics consistent with probiotic strains, were further tested for their anti-inflammatory effect in mouse colitis model. Only one L. brevis; one L. rhamnosus and two L. plantarum were shown to have significant probiotic properties. These strains showed shortening the length of colon compared to dextran sulfate sodium and disease activity index (DAI) was also significantly reduced in mouse.ConclusionLow number of LAB with potential probiotic activity as well as high diversity of lactobacilli species was evident in Iranian population. It also suggest that specific strains of L. plantarum, L. brevis and L. rhamnosus with anti-inflammatory effect in mouse model of colitis could be used as a potential probiotic candidate in inflammatory bowel disease to decrease the disease activity index.

Influence of vitamin B12 availability on oceanic dimethylsulfide and dimethylsulfoniopropionate

Environmental context Cobalamin, or vitamin B12, is receiving increased attention as a critical trace nutrient in the growth and metabolic processes of oceanic phytoplankton and bacterial communities. We present evidence that indicates B12 has a more significant role in the biogeochemical cycling of the climatically important compounds dimethylsulfide and dimethylsulfoniopropionate than previously understood. Several possible mechanisms are examined that link cellular-level processes involving B12 to global-scale biogeochemical processes involving the oceanic cycling of dimethylsulfoniopropionate and dimethylsulfide. Abstract Evidence is presented showing that dissolved dimethylsulfoniopropionate (DMSPd) and dimethylsulfide (DMS) concentrations are influenced by the availability of vitamin B12 in two oceanographically distinct regions with different DMS production capacities, the central equatorial Pacific Ocean and the Ross Sea, Antarctica. In both locations, addition of B12 to incubation experiments resulted in decreases in DMS and, in some cases, DMSPd concentrations relative to unamended controls. In no case did increasing iron availability significantly (α=0.1) alter DMS concentrations relative to controls. The relative decreases in DMS between B12 addition and control experiments were significant (α=0.1) in five of seven experiments conducted at ambient iron levels. Overall, DMS concentrations were on average 33.4% (±15.1%; 1 standard deviation) lower, relative to unamended controls, by the end of incubation experiments when B12 was added. Declines in DMSPd were observed in three of five experiments. Similar trends were observed when B12 was added to iron-supplemented bottle incubation experiments (30.4±10.4% lower final DMS concentrations in +B12Fe treatments relative to +Fe treatments). Several possible molecular-level explanations exist for this link between B12 and DMS production, including potential B12 dependence of methyltransferase enzymes involved in both DMS and DMSP degradation. Although the enzymology of these reactions remains unclear, the relationships described here provide evidence for plausible mechanisms behind the microbial modulation of oceanic DMS.