A multifaceted study of Propionibacterium freudenreichii, the food-grade producer of active vitamin B12

Abstract

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.B12-vitamiini on rakenteeltaan vitamiineista monimutkaisin, ja sitä saadaan pääasiassa eläinperäisistä tuotteista. Kasvisruokavaliota noudattavilla ja vain vähän eläinperäisiä elintarvikkeita syövillä on riski kärsiä B12-vitamiinin puutoksesta. Kasvisruokavalion suosion kasvaessa voimakkaasti ja samalla eläinperäisten tuotteiden kulutuksen laskiessa yhä suuremmat väestöryhmät voivat altistua liian vähäiselle B12- vitamiinin saannille. Elintarvikekelpoisten, ihmiselle aktiivista B12-vitamiinimuotoa tuottavien mikrobien hyödyntäminen elintarvikkeiden valmistuksessa on lupaava menetelmä B12-vitamiinin rikastamiseksi kasviperäisiin tuotteisiin joissa sitä ei tällä hetkellä ole. Tässä tutkimuksessa selvitettiin mikrobiologian ja molekyylibiologian menetelmiä hyödyntäen Propionibacterium freudenreichii -bakteerin kykyä tuottaa ihmiselle aktiivista B12-vitamiinia. Tulokset osoittivat, että P. freudenreichii –bakteeri tuottaa elintarvikeolosuhteissa ihmiselle aktiivista B12-vitamiinimuotoa ja sitä voidaan hyödyntää vitamiinin rikastamisessa elintarvikkeisiin. 18 uuden genomisekvenssin määrittäminen ja paljasti paljon uutta tietoa P. freudenreichii -lajista ja loi hyvän perustan jatkotutkimuksille