Vitamin B12 is the most complex vitamin in existence and one of the most complex non-polymeric molecules occurring in nature. It is predominantly present in animal-derived products, which places vegetarians and people with limited access to animal-derived foods at risk for developing vitamin B12 deficiency. With the current trend of limiting the consumption of foods of animal origin, the deficiency may also affect other populations. In situ fortification of foods through microbial fermentation with food-grade bacteria is a viable method for the introduction of vitamin B12 into foods, if the microorganism is capable of synthesising the active vitamin form. Here, the capability of Propionibacterium freudenreichii to produce active vitamin B12 was explored with the use of a combination of microbiological and molecular approaches. First, the activity of the heterogolously expressed and purified enzyme BluB/CobT2 was investigated. The results showed that the novel fusion enzyme was responsible for biosynthesis of 5,6-dimethylbenzimidazole (DMBI) base and its activation for attachment as the lower ligand of vitamin B12. The enzyme’s inability to activate adenine, the lower ligand of pseudovitamin B12, revealed a mechanism favouring production of active vitamin B12 in P. freudenreichii. The in vivo study showed that formation of DMBI is oxygen dependent as no vitamin B12 was produced under strictly anaerobic atmosphere. Exogenous DMBI was incorporated into the vitamin molecule under both microaerobic and anaerobic conditions, with a clear preference over incorporation of adenine. In the following study, the capability of 27 P. freudenreichii and 3 Acidipropionibacterium acidipropionici strains to produce active vitamin B12 was examined by UHPLC. The yields obtained from growth in whey-based medium enriched in cobalt and supplemented with either DMBI, with the precursors of DMBI- riboflavin and nicotinamide, or without supplementation. A. acidipropionici strains required supplementation of DMBI to produce small amounts of active vitamin B12 (<0.2 µg/mL), while all of the P. freudenreichii strains were able to produce active vitamin B12 in all conditions tested. The yields of active vitamin B12 produced by P. freudenreichii and responses to supplementation were strain dependent and ranged from 0.2 to 5.3 µg/mL. Subsequently, the active vitamin B12 production by the strain P. freudenreichii 2067 without addition of cobalt or DMBI was tested. The experiments were performed in a medium mimicking cheese environment as well as in the whey-based medium. The production of other key metabolites was examined by HPLC, while the global protein production was compared by gel-based proteomics. The results showed that regardless of different effects of the media on the metabolic state of the cells, which was reflected by distinct metabolite and protein production patterns, P. freudenreichii produced nutritionally relevant levels of active vitamin B12. Finally, whole genome sequencing was employed to better characterise the species through a comparative genomics study. The use of PacBio sequencing platform, a PCR-free method producing long reads, resulted in discovery of additional circular elements: two novel, putative conjugative plasmids and three active, lysogenic bacteriophages. The long reads also permitted characterisation and classification of two distinct types of CRISPR-Cas systems. In addition, the use of PacBio sequencing platform allowed for identification of DNA modifications, which led to characterisation of Restriction-Modification systems together with their recognition motifs, many of which were reported for the first time. Genome mining suggested surface piliation in the strain P. freudenreichii JS18, which was confirmed by transmission electron microscopy and assessment of specific mucus binding.
|Award date||21 Jun 2017|
|Place of Publication||Helsinki|
|Publication status||Published - 21 Jun 2017|
|MoE publication type||G5 Doctoral dissertation (article)|
Fields of Science
- 416 Food Science