BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B₁₂ by Propionibacterium freudenreichii.

Risto Kostiainen,Liisa Holm,Kirsi Savijoki,Paulina Deptula,Bhawani Chamlagain,Pekka Varmanen,Petri Kylli,Vieno Piironen

doi:10.1186/s12934-015-0363-9

Abstract

BackgroundPropionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B12. Production of active form of vitamin is a prerequisite for attempts to naturally fortify foods with B12 by microbial fermentation. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Genomic data indicate that P. freudenreichii possesses a fusion gene, bluB/cobT2, coding for a predicted phosphoribosyltransferase/nitroreductase, which is presumably involved in production of vitamin B12. Understanding the mechanisms affecting the synthesis of different vitamin forms is useful for rational strain selection and essential for engineering of strains with improved B12 production properties.ResultsHere, we investigated the activity of heterologously expressed and purified fusion enzyme BluB/CobT2. Our results show that BluB/CoBT2 is responsible for the biosynthesis of the DMBI base and its activation into α-ribazole phosphate, preparing it for attachment as the lower ligand of cobalamin. The fusion enzyme was found to be efficient in metabolite channeling and the enzymes’ inability to react with adenine, a lower ligand present in the pseudovitamin, revealed a mechanism favoring the production of the active form of the vitamin. P. freudenreichii did not produce cobalamin under strictly anaerobic conditions, confirming the requirement of oxygen for DMBI synthesis. In vivo experiments also revealed a clear preference for incorporating DMBI over adenine into cobamide under both microaerobic and anaerobic conditions.ConclusionsThe herein described BluB/CobT2 is responsible for the production and activation of DMBI. Fusing those two activities results in high pressure towards production of the true vitamin B12 by efficiently activating DMBI formed within the same enzymatic complex. This indicates that BluB/CobT2 is the crucial enzyme in the B12 biosynthetic pathway of P. freudenreichii. The GRAS organism status and the preference for synthesizing active vitamin form make P. freudenreichii a unique candidate for the in situ production of vitamin B12 within food products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0363-9) contains supplementary material, which is available to authorized users.

Highlights

Propionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B12
The predicted size for HaloTag-BluB/CobT2 protein is 100.4 kDa (34 kDa HaloTag + 66.4 kDa BluB/CobT2) and a band of corresponding molecular weight was detected in samples from E. coli whole-cell lysates and soluble fraction of the lysates obtained after induction (Fig. 2a)
The described fusion enzyme BluB/CobT2 is responsible for the production and activation of DMBI for attachment as a lower ligand for cobalamin

Summary

Introduction

Propionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B12. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Cobalamins, distinguished by the presence of 5,6-dimethylbenzimidazole (DMBI) as a lower ligand (Fig. 1a), are of special human nutritional value because of their activity of vitamin B12. Vitamin B12 plays an important role in nucleotide synthesis as well as the metabolism of branched amino acids and odd-chain fatty acids. These molecules fulfill their role as cofactors in cytoplasmic methionine synthase and in mitochondrial methylmalonyl-CoA mutase, which are responsible for the methylation of homocysteine to methionine and the conversion of methylmalonyl-CoA to succinyl-CoA, respectively [1]. Due to the complexity and cost of the chemical synthesis of cobalamin, which requires approximately 70 steps, its industrial production is exclusively through microbial fermentation, most commonly by Pseudomonas denitrificans and Propionibacterium freudenreichii [3]

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Conclusion