Vitamin B12 Production Research Articles

Screening and isolation of vitamin B12 producing Pseudomonas sp. from different natural sources

Vitamin B12 is an essential vitamin playing a vital role in red blood cell formation, maintenance of central nervous system (CNS) and involved in metabolism of every cell in body. It is widely used as dietary supplement worldwide and used to treat anemia. The present study focuses on isolation and extraction of vitamin B12 producing Pseudomonas sp. from rhizosphere, non-rhizosphere soil and fish gut (Tilapia sp.). The vitamin B12 producers and non-producers were enumerated from the samples. Vitamin B12 producing Pseudomonas sp. from fish gut was analyzed using auxonography method. Further, the vitamin B12 from all the isolated bacterial samples were purified and quantified using high performance liquid chromatography technique (HPLC). Rhizosphere soil (68.4%) showed highest population of vitamin B12 producers than non-rhizosphere soil (40%) and fish gut (60%). Pseudomonas sp. from rhizosphere soil (R6) showed higher vitamin B12 production (11.54 mg/l) whereas non-rhizosphere soil and fish gut showed 5.25 mg/l and 6.68 mg/l. Control strain, Propionibacterium freudenreichii - 1950 had production of 8.1 mg/l which lower than the strain-R6. These wild type organisms when optimized or genetically modified can synthesis high amounts of vitamin B12 which supports to overcome the vitamin B12 deficiency worldwide.

Metabolic profiling analysis of the vitamin B12 producer Propionibacterium freudenreichii.

Vitamin B12 (VB12) is an indispensable cofactor of metabolic enzymes and has been widely used in the food and pharmaceutical industries. In this study, the effects of medium composition on VB12 production by Propionibacterium freudenreichii were evaluated and optimized based on statistical experiments. The results showed that glucose, yeast extract, KH2PO4, and glycine have significant effects on VB12 production. The final titer of VB12 reached 8.32 ± 0.02 mg/L, representing a 120% increase over the non‐optimized culture medium. We employed a metabolomics approach to analyze the differences of metabolite concentrations in P. freudenreichii cells cultivated in the original medium and optimized fermentation medium. Using multivariate data analysis, we identified a range of correlated metabolites, illustrating how metabolomics can be used to explain VB12 production changes by corresponding differences in the overall cellular metabolism. The concentrations of many metabolic intermediates of glycolysis, the Wood–Werkman cycle, the TCA cycle, and amino acid metabolism were increased, which contributed to the synthesis of propionic acid and VB12 due to an improved supply of energy and precursors.

Cholesterol Reduction and Vitamin B12 Production Study on Enterococcus faecium and Lactobacillus pentosus Isolated from Yoghurt

The present study was aimed to test cholesterol reduction and vitamin B12 production abilities of the isolated lactic acid bacteria (LAB). Three LAB isolates, namely, Enterococcus faecium (EF), Enterococcus faecium (Chole1), and Lactobacillus pentosus (7MP), having probiotic potential, were isolated from yoghurt. These isolates were screened for bile salt hydrolase (BSH) activity, cholesterol reduction property in MRS broth, and the production of vitamin B12. The present study revealed that the isolate 7MP possesses the highest potential of (48%) cholesterol reduction compared to the other isolates. The isolates EF and Chole1 produced a good amount of (1 ng/mL) vitamin B12. These isolates were identified by 16S rRNA gene sequencing and confirmed by MALD_TOF analysis. Thus, the use of these LAB isolates for yoghurt-making can offer the value addition of lowering cholesterol and vitamin B12 fortification in fermented food.

Metabolic Flux Analysis of Simultaneous Production of Vitamin B12 and Propionic Acid in a Coupled Fermentation Process by Propionibacterium freudenreichii.

The metabolic processes involved in simultaneous production of vitamin B12 and propionic acid by Propionibacterium freudenreichii are very complicated. To further investigate the regulatory mechanism of this metabolism, a simplified metabolic network was established. The effects of glucose feeding, propionic acid removal, and 5,6-dimethylbenzimidazole (DMB) addition on the metabolic flux distribution were investigated. The results showed that synthesis of propionic acid can be increased by increasing the metabolic flux through the oxaloacetate and methylmalonyl-CoA branches in the early and middle stages of the coupled fermentation. After DMB addition, the synthesis of vitamin B12 was significantly enhanced via increased metabolic flux through the δ-aminolevulinate branch, which promoted the synthesis of uroporphyrinogen III, a precursor of vitamin B12. Therefore, the analysis of metabolic flux at key nodes can provide theoretical guidance for the optimization of P. freudenreichii fermentation processes. In an experimental coupled fermentation process, the concentrations of vitamin B12 and propionic acid reached 21.6 and 50.12 g/L respectively, increased by 105.71% and 73.91% compared with batch fermentation, which provides a new strategy for industrial production.

Bacterial Microcompartment-Dependent 1,2-Propanediol Utilization of Propionibacterium freudenreichii.

Bacterial microcompartments (BMCs) are proteinaceous prokaryotic organelles that enable the utilization of substrates such as 1,2-propanediol and ethanolamine. BMCs are mostly linked to the survival of particular pathogenic bacteria by providing a growth advantage through utilization of 1,2-propanediol and ethanolamine which are abundantly present in the human gut. Although a 1,2-propanediol utilization cluster was found in the probiotic bacterium Propionibacterium freudenreichii, BMC-mediated metabolism of 1,2-propanediol has not been demonstrated experimentally in P. freudenreichii. In this study we show that P. freudenreichii DSM 20271 metabolizes 1,2-propanediol in anaerobic conditions to propionate and 1-propanol. Furthermore, 1,2-propanediol induced the formation of BMCs, which were visualized by transmission electron microscopy and resembled BMCs found in other bacteria. Proteomic analysis of 1,2-propanediol grown cells compared to L-lactate grown cells showed significant upregulation of proteins involved in propanediol-utilization (pdu-cluster), DNA repair mechanisms and BMC shell proteins while proteins involved in oxidative phosphorylation were down-regulated. 1,2-Propanediol utilizing cells actively produced vitamin B12 (cobalamin) in similar amounts as cells growing on L-lactate. The ability to metabolize 1,2-propanediol may have implications for human gut colonization and modulation, and can potentially aid in delivering propionate and vitamin B12 in situ.

ISOLATION, CHARACTERIZATION AND IDENTIFICATION OF Lactobacillus fermentum EIPW5A ISOLATED FROM WHEY

The probiotic organisms are now used widely for different clinical indications. In an attempt to isolate a good probiotic strain for therapeutic applications, we have screened several isolates having probiotic attributes. The essential probiotic characters such as lactic acid production, antimicrobial activity, acid and bile tolerance, vitamin B12 production and antibiotic resistance pattern were considered as parameters for screening of probiotic bacteria from its natural habitats. Considering the said probiotic properties the strain EIPW5A was selected for the present study. The organism was identified as Lactobacillus fermentum based on its morphological, biochemical, physiological characters and 16S rRNA gene sequencing results.

Microbial and Genetic Resources for Cobalamin (Vitamin B12) Biosynthesis: From Ecosystems to Industrial Biotechnology.

Many microbial producers of coenzyme B12 family cofactors together with their metabolically interdependent pathways are comprehensively studied and successfully used both in natural ecosystems dominated by auxotrophs, including bacteria and mammals, and in the safe industrial production of vitamin B12. Metabolic reconstruction for genomic and metagenomic data and functional genomics continue to mine the microbial and genetic resources for biosynthesis of the vital vitamin B12. Availability of metabolic engineering techniques and usage of affordable and renewable sources allowed improving bioprocess of vitamins, providing a positive impact on both economics and environment. The commercial production of vitamin B12 is mainly achieved through the use of the two major industrial strains, Propionobacterium shermanii and Pseudomonas denitrificans, that involves about 30 enzymatic steps in the biosynthesis of cobalamin and completely replaces chemical synthesis. However, there are still unresolved issues in cobalamin biosynthesis that need to be elucidated for future bioprocess improvements. In the present work, we review the current state of development and challenges for cobalamin (vitamin B12) biosynthesis, describing the major and novel prospective strains, and the studies of environmental factors and genetic tools effecting on the fermentation process are reported.

DNA methylation profile of genes involved in inflammation and autoimmunity correlates with vascular function in morbidly obese adults

ABSTRACT Obesity is a major risk factor for cardiovascular disease. Blood-detected epigenetic profiles may serve as non-invasive clinically relevant biomarkers. Therefore, we investigated DNA methylation of genes involved in inflammation in peripheral blood of obese subjects and lean controls and their correlation with cardiometabolic measurements. We obtained blood and adipose tissue (AT) samples from bariatric patients (n = 24) and control adults (n = 24). AT-isolated arterioles were tested for flow-induced dilation (FID) and production of nitric oxide (NO) and reactive oxygen species (ROS). Brachial artery flow-mediated dilation (FMD) was measured via doppler ultrasound. Promoter methylation of 94 genes involved in inflammation and autoimmunity were analysed in whole-blood DNA in relation to vascular function and cardiometabolic risk factors. 77 genes had ahigher methylated fraction in the controls compare obese subjects and 28 proinflammatory genes were significantly hypomethylated in the obese individuals; on top of these genes are CXCL1, CXCL12, CXCL6, IGF2BP2, HDAC4, IL12A, and IL17RA. Fifteen of these genes had significantly higher mRNA in obese subjects compared to controls; on top of these genes are CXCL6, TLR5, IL6ST, EGR1, IL15RA, and HDAC4. Methylation % inversely correlated with BMI, total fat %, visceral fat%, blood pressure, fasting plasma insulin, serum IL6 and C-reactive protein, arteriolar ROS, and alcohol consumption and positive correlations with lean %, HDL, plasma folate and vitamin B12, arteriolar FID and NO production, and brachial FMD. Our results suggest that vascular dysfunction in obese adults may be attributed to asystemic hypomethylation and over expression of the immune-related genes.

Abstract 15715: Dna Methylation of Genes Involved in Inflammation Correlates With Cardiometabolic Function in Morbidly Obese Adults

Obesity is a major risk factor for cardiovascular disease. We previously demonstrated impaired vascular function that correlated with global DNA hypomethylation in adipose tissue (AT) isolated from obese adults (OB). Blood-detected epigenetic profiles may serve as non-invasive clinically relevant biomarkers and stand as an unexploited precision medicine reserve. Hypothesis: We hypothesized a contributing role of DNA methylation to systemic inflammation in OB subjects compared to lean controls (CON). We also explored the correlation between these methylation profiles and cardiometabolic measurements. Methods: We obtained blood and AT samples from bariatric patients (n=24; age: 36±7 yrs; BMI: 50.7±8.7 kg/m2) and CON adults (n=24; age: 36±2 yrs; BMI: 25.8±1 kg/m2). AT-isolated arterioles were tested for flow-induced dilation (FID), nitric oxide (NO) and reactive oxygen species (ROS) production. Brachial artery flow-mediated dilation (FMD) was measured via Doppler ultrasound. Promoter methylation of 94 genes involved in inflammation and autoimmunity (EpiTect Methyl II PCR Arrays) were analyzed in whole-blood DNA in relation to vascular function and cardiometabolic risk factors. Results: 70% of genes had a higher methylated fraction in CON compare to only 28% in OB subjects. After correction for multiple testing, 28 genes were significantly hypermethylated in CON compared to OB; on top of these genes are CXCL1, CXCL12, CXCL6, EGR1, HDAC4, IGF2BP2, IL12A, IL12B, and IL17RA. Ten of these genes had significantly higher mRNA in OB compared to CON indicating the functional impact of such signals on gene transcription; on top of these genes are CXCL6, TLR5, IL6ST, IL15RA, and HDAC4. Methylation % of differentially methylated genes inversely correlated with BMI, total fat %, visceral fat%, blood pressure, fasting plasma insulin, serum IL6 and CRP (C-reactive protein), arteriolar ROS, and alcohol consumption and positive correlations with lean %, HDL, plasma folate and vitamin B12, arteriolar FID and NO production, and brachial FMD. Conclusions: Our results suggest that vascular dysfunction in OB adults may be attributed to aberrant DNA methylation. A downstream target for this pathway could reside in endothelial cells resulting in vascular dysfunction

Fermentation of cereal, pseudo-cereal and legume materials with Propionibacterium freudenreichii and Levilactobacillus brevis for vitamin B12 fortification

The present study investigated the in situ production of vitamin B12 in eleven cereal, pseudo-cereal and legume materials by fermentation with Propionibacterium freudenreichii DSM 20271 and Levilactobacillus brevis (formerly Lactobacillus brevis) ATCC 14869. P. freudenreichii was used as the vitamin producer and L. brevis was selected to improve the consistency and microbial safety of the process. The study showed that more than 300 ng/g dw of vitamin B12 (daily requirement: 2.4 μg) were produced during fermentation in most of the studied brans and legumes. The highest vitamin B12 production was observed in the fermentation of the rice bran (ca. 742 ng/g dw), followed by the fermentation of buckwheat bran (ca. 631 ng/g dw). Furthermore, partial least squares (PLS) regression analysis suggested that the production of vitamin B12 was greatly influenced by the nutrient composition of the fermented raw materials. Meanwhile, L. brevis was found to effectively inhibit the growth of Enterobacteriaceae during fermentation. These results demonstrated that fermentation of cereal, pseudo-cereal and legume materials with P. freudenreichii and L. brevis is effective in fortifying plant-based food with vitamin B12.

Identification of a xylose‐inducible promoter and its application for improving vitamin B12 production in Sinorhizobium meliloti

Sinorhizobium meliloti 320 is a vitamin B12 (VB12 ) high-producing strain that has been isolated and identified in our previous study. Because the regulatory toolbox for S.meliloti is limited, we searched for new genetic components and identified the two xylose-inducible promoters PA and PB based on a promoter-probe vector with a green fluorescent protein (GFP) as reporter. Compared with the ParaA promoter from S.meliloti, both promoters exhibited higher induced expression and lower basal expression. Subsequently, the influence of glucose or sucrose on the expression of GFP driven by these three promoters was assayed. Glucose repressed all three promoters, and the expression of ParaA was the lowest in the presence of glucose. Although sucrose repressed the expression of PA by 35% and improved the expression of ParaA by 16%, the expression level of PA was the highest and was 13% higher than that of ParaA . Lastly, we overexpressed the hemA gene in the C4 pathway using the PA promoter in S.meliloti 320, and the VB12 production of the engineered strain increased by 11%. The VB12 production was further increased by 11% by adding 0.1% sodium succinate to the culture medium.

Robust high-dimensional semiparametric regression using optimized differencing method applied to the vitamin B2 production data

Background and purpose: By evolving science, knowledge, and technology, we deal with high-dimensional data in which the number of predictors may considerably exceed the sample size. The main problems with high-dimensional data are the estimation of the coefficients and interpretation. For high-dimension problems, classical methods are not reliable because of a large number of predictor variables. In addition, classical methods are affected by the presence of outliers and collinearity.Methods: Nowadays, many real-world data sets carry structures of high-dimensional problems. To handle this problem, we used the least absolute shrinkage and selection operator (LASSO). Also, due to the flexibility and applicability of the semiparametric model in medical data, it can be used for modeling the genomic data. Motivated by these, here an improved robust approach in a high-dimensional data set was developed for the analysis of gene expression and prediction in the presence of outliers.Results: Among the common problems in regression analysis, there was the problem of outliers. In the regression concept, an outlier is a point that fails to follow the main linear pattern of the data. The ordinary least-squares estimator was found potentially sensitive to the outliers; this fact provided necessary motivations to investigate robust estimations. Generally, the robust regression is among the most popular problems in the statistics community. In the present study, the least trimmed squares (LTS) estimation was applied to overcome the outlier problem.Conclusions: We have proposed an optimization approach for semiparametric models to combat outliers in the data set. Especially, based on a penalization LASSO scheme, we have suggested a nonlinear integer programming problem as the semiparametric model which can be effectively solved by any evolutionary algorithm. We have also studied a real-world application related to the riboflavin production. The results showed that the proposed method was reasonably efficient in contrast to the LTS Method.

Functional Disassociation Between the Protein Domains of MSMEG_4305 of Mycolicibacterium smegmatis (Mycobacterium smegmatis) in vivo.

MSMEG_4305 is a two-domain protein of Mycolicibacterium smegmatis (Mycobacterium smegmatis) (Mycolicibacterium smegmatis). The N-terminal domain of MSMEG_4305 encodes an RNase H type I. The C-terminal domain is a presumed CobC, predicted to be involved in the aerobic synthesis of vitamin B12. Both domains reach their maximum at distinct pH, approximately 8.5 and 4.5, respectively. The presence of the CobC domain influenced RNase activity in vitro in homolog Rv2228c. Here, we analyzed the role of MSMEG_4305 in vitamin B12 synthesis and the functional association between both domains in vivo in M. smegmatis. We used knock-out mutant of M. smegmatis, deficient in MSMEG_4305. Whole-cell lysates of the mutants strain contained a lower concentration of vitamin B12, as it determined with immunoenzimatic assay. We observed growth deficits, related to vitamin B12 production, on media containing sulfamethazine and propionate. Removal of the CobC domain of MSMEG_4305 in ΔrnhA background hardly affected the growth rate of M. smegmatis in vivo. The strain carrying truncation showed no fitness deficit in the competitive assay and it did not show increased level of RNA/DNA hybrids in its genome. We show that homologs of MSMEG_4305 are present only in the Actinomycetales phylogenetic branch (according to the old classification system). The domains of MSMEG_4305 homologs accumulate mutations at a different rate, while the linker region is highly variable. We conclude that MSMEG_4305 is a multidomain protein that most probably was fixed in the phylogenetic tree of life due to genetic drift.

Expression of yeast homolog of the mammal BCRP gene coding for riboflavin efflux protein activates vitamin B2 production in the flavinogenic yeast Candida famata.

Candida famata is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2 ) in response to iron limitation. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The corresponding protein and gene have not been identified in yeasts. At the same time, the corresponding gene BCRP has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in Debaryomyces hansenii. The closest homolog was expressed under the control of D. hansenii TEF1 promoter in the riboflavin overproducing strain of C. famata. Resulted transformants overexpressed the corresponding gene and produced 1.4- to 1.8-fold more riboflavin as compared with the parental strain. They also were characterized by overexpression of RIB1 and RIB6 genes of riboflavin synthesis and exhibited elevated specific activity of GTP-cyclohydrolase II. Membrane localization of the riboflavin excretase was confirmed by fluorescent microscopy.

Effective nitrogen removal of wastewater from vitamin B2 production by a potential anammox process

The wastewater of vitamin B2 (VB2) production treated after anaerobic digesters contains high total nitrogen (TN) and low organic carbon. To efficiently remove nitrogen pollution in the wastewater, one lab-scale anaerobic ammonium oxidation (anammox) bioreactor was designed. Synthetic wastewater and proper conditions were used to startup the anammox process in the bioreactor. The stoichiometric parameters of the bioreactor showed that anammox process was successfully established during operation. Evaluation of the bioreactor with real VB2 wastewater suggested that the bioreactor can efficiently remove nitrogen pollution at low organic carbon situation. The optimal state of total nitrogen loading rate (TNLR) and the highest total nitrogen removal efficiency (TNRE) of the bioreactor were 0.35 kg/(m3·d) and 75%, respectively. The microbiota run at day 112 (named VBM112) was significantly different with the microbiotas of the two inoculation cultures. At the phylum level, the most abundant phylum was Proteobacteria in the inoculation culture, while Bacteroidetes was the most abundant phylum in VBM112. At the genus level, the composition of nitrogen removal related microbes significantly increased in VBM112. Especially, the anammox bacteria Candidatus Kuenenia increased from 0% of the inoculation culture to 2.2% of VBM112. The analyses of running parameters and microbiota profiles confirmed the well establishment of one lab-scale anammox bioreactor, which might be helpful for future full-scale VB2 wastewater treatment startup.

Efficient ex-situ biosynthesis of vitamin B12 by Propionibacterium freudenreichii using membrane separation coupling technology

Propionibacterium freudenreichii has been successfully applied to industrial anaerobic production of vitamin B12. In the traditional fed-batch fermentation process, 5,6-dimethylbenzimidazole (DMB) is used as a precursor. However, DMB can inhibit the synthesis of vitamin B12 metabolic intermediates, which is not conducive to semi-continuous fermentation of P. freudenreichii. To improve the fermentation efficiency, vitamin B12ex-situ biosynthesis was proposed and a membrane separation coupling fermentation technology was designed with adenosylcobinamide (Ado-cbi) as the indicator signal. The results showed that P. freudenreichii cells should be partly separated online at 84 h with 0.22-μm spiral membrane. The fourfold concentrated suspension was suitable for ex-situ biosynthesis of vitamin B12 with the addition of 3.60 mg L−1 DMB at 30 ℃ and pH 7.0. In the semi-continuous coupling fermentation process, vitamin B12 concentration reached 56.76 ± 3.86 mg L−1, an improvement of 161.6 %, while reutilizing fermentation wastewater and improving the anaerobic fermentation efficiency of P. freudenreichii.

Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies

BackgroundHydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B12. The introduction of a heterologous de novo vitamin B12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B12, such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B12 production.ResultsBy designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L−1, respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L−1, representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA.ConclusionsThrough multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L−1 in E. coli. This lays the foundation for high-yield production of vitamin B12 in E. coli and will hopefully accelerate its industrial production.

Guava pulp fermentation and processing to a vitamin B12‐enriched product

Guava (Psidium guajava) pulp was fermented with Lactobacillus plantarum WU-P19 to a product rich in microbially produced vitamin B12 and conjugated linoleic acid. Different initial sucrose levels were tested in the pulp in different fermentations. The 36-hr fermented material had a pH of 3.3–3.4, a lactic acid content of 7.6–8.2 g/L, and a viable L. plantarum count of 108.51–108.56 cells/ml. The fermented pulp was used to make a novel food product, the guava gummy jelly. The guava gummy jelly formulation that was most accepted by a panel of tasters, comprised of 100 g (fresh weight) of fermented guava pulp, 20 g of sucrose, 11 g of glucose syrup, 6 g of gelatin and 0.5 g of citric acid. A 1-g portion of this tasty product could fully meet the daily requirements of vitamin B12, an essential nutrient that is deficient in many diets. Practical applications Vitamin B12 deficiency in certain diets can be alleviated through consumption of the fermented food product produced here. The fermented product offers a high-value application of waste pulp of commercial guava juice production.

Biosynthesis of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 using liquid acid protein residue of soybean as culture medium.

Vitamin B12 deficiency still persists, mainly caused by low intake of animal food products affecting vegetarians, vegans, and populations of underdeveloped countries. In this study, we investigate the biosynthesis of vitamin B12 by potential probiotic bacterium using an agroindustry residue, the liquid acid protein residue of soybean (LAPRS), as a low-cost, animal derivate-free alternative culture medium. Cultures of Propionibacterium freudenreichii subsp. shermanii ATCC 13673 growing in LAPRS for vitamin B12 biosynthesis were studied using the Plackett-Burman experimental approach, followed by a central composite design 22 to optimize the concentration of significant variables. We also performed a proteolytic treatment of LAPRS and evaluated the optimized-hydrolyzed medium influence on the microbial growth and metabolism in shaker flask and bioreactor experiments. In this all-plant source medium, P. freudenreichii subsp. shermanii produced high concentrations of cells and high amounts of vitamin B12 (0.6 mg/g cells) after process optimization. These results suggest the possibility of producing vitamin B12 by a potential probiotic bacterium in a very cheap, animal derivate-free medium to address the needs of specific population groups, at the same time reducing the production costs of this essential vitamin.

Metabolic engineering and optimization of the fermentation medium for vitamin B12 production in Escherichia coli.

Vitamin B12 is a crucial fine chemical that is widely used in the pharmaceutical, food and chemical industries, and its production solely dependents on microbial fermentation. We previously constructed an artificial vitamin B12 biosynthesis pathway in Escherichia coli, but the yield of the engineered strains was low. Here, we removed metabolic bottlenecks of the vitamin B12 biosynthesis pathway in engineered E. coli strains. After screening cobB genes from different sources, optimizing the expression of cobN and customizing the ribosome binding sites of cobS and cobT, the vitamin B12 yield increased to 152.29μg/g dry cell weight (DCW). Optimization of the downstream module, which converts co(II)byrinic acid a,c-diamide into adenosylcobinamide phosphate, elevated the vitamin B12 yield to 249.04μg/g DCW. A comparison of a variety of equivalent components indicated that glucose and corn steep liquor are optimal carbon and nitrogen sources, respectively. Finally, an orthogonal array design was applied to determine the optimal concentrations of glucose and nitrogen sources including corn steep liquor and yeast extract, through which a vitamin B12 yield of 530.29μg/g DCW was obtained. The metabolic modifications and optimization of fermentation conditions achieved in this study offer a basis for further improving vitamin B12 production in E. coli and will hopefully accelerate its industrial application.