Abstract
BackgroundVitamin B12 is a fascinating molecule which acts as a co-factor in the metabolism of many organisms, especially affecting DNA synthesis and regulation, fatty acid synthesis and energy production. The synthesis of vitamin B12 is limited to a few of bacteria and archaea. Therefore, industrial microbial fermentation is used to meet annual demands worldwide of vitamin B12 and as an alternative method to the chemical synthesis which requires at least 60 steps that is uneconomical. Bacillus megaterium is one of vitamin B12 producers and an ideal host for many biotechnology applications and being one of the best tools for the industrial production of several enzymes. Therefore, a two-step optimization strategy was established to produce high yield of vitamin B12 by B. megaterium through the provision of the production requirements and the suitable conditions for the biosynthesis of vitamin B12.ResultsWe achieved the optimum conditions for the fermentation process of B. megaterium to produce high yield of vitamin B12 in a practical way based on statistical design and analysis which allowed vitamin B12 production to increase up to 759-fold (204.46 μg/l) as compared with control without parameters (0.26 μg/L). High performance liquid chromatography coupled to variable wavelength detector and mass spectrometry has been used to identify vitamin B12 forms and confirm the results.ConclusionsWe developed the fermentation process of B. megaterium to enhance the production of vitamin B12 by providing the required supplements for the synthesis of vitamin B12 (CoCl2, δ-aminolevulinic acid (ALA) and 5,6-dimethylbenzimidazole (DMB)) and dividing the fermentation process into three stages. In addition, the optimum incubation times of the three fermentation stages were investigated and performed with reducing number of experimental and evaluated multiple parameters and their interactions by using statistical experimental design and analysis. All of these strategies has proven successful in enhancing the production of vitamin B12 up to 204.46 μg/l and demonstrated that B. megaterium could be a good candidate for the industrial production of vitamin B12.
Highlights
Vitamin B12 is a fascinating molecule which acts as a co-factor in the metabolism of many organisms, especially affecting DNA synthesis and regulation, fatty acid synthesis and energy production
Experimental design A two-step optimization strategy was employed to increase the production of vitamin B12 by Bacillus megaterium through the provision of the required supplements for the synthesis of vitamin B12 (CoCl2, δ-aminolevulinic acid (ALA) and 5,6-dimethylbenzimidazole (DMB)) and the division of the fermentation process into three stages
UV–vis spectra data and retention times obtained by HPLC analysis showed that B. megaterium produces two major forms of vitamin B12 cyanocobalamin and adenosylcobalamin due to the transformation efficiency of the cell extraction but the yield was not high enough
Summary
Vitamin B12 is a fascinating molecule which acts as a co-factor in the metabolism of many organisms, especially affecting DNA synthesis and regulation, fatty acid synthesis and energy production. Bacillus megaterium is one of vitamin B12 producers and an ideal host for many biotechnology applications and being one of the best tools for the industrial production of several enzymes. Vitamin B12 is one of the smallest but structurally complex molecule widely recognized in the field of biological sciences [1]. It is a water-soluble vitamin that functions as a cofactor essential in fat biochemical reactions, protein metabolism and hemoglobin synthesis as well as DNA synthesis and regulation. Attempts have been carried out in other study to engineer the bacteria using metabolic engineering tools [12] but the final concentration of vitamin B12 was not high (8 μg/l) probably due to the lack of understanding due to the lack of understanding the pathway of vitamin B12 and production conditions of B. megaterium on vitamin B12
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