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

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

Complete Genome Sequences and Methylome Analyses of Cutibacterium acnes subsp. acnes Strains DSM 16379 and DSM 1897T

Cutibacterium acnes is a member of the normal human skin micro-biome. However, it is also associated with skin disorders and persistent infections of orthopedic implants. Here, we announce complete genome sequences and methyl-omes of the C. acnes subsp. acnes strains DSM 1897(T) and DSM 16379 together with their active restriction-modification systems.Peer reviewe

In situ fortification of vitamin B12 in wheat flour and wheat bran by fermentation with Propionibacterium freudenreichii

Vitamin B12 is a micronutrient naturally existing in animal products. A growing interest and need to replace animal protein with plant protein sources have resulted in increased attention to developing vitamin B12-fortified plant-based food. Natural fortification by Propionibacterium freudenreichii is a promising alternative to chemical fortification, as P. freudenreichii can synthesize active vitamin B12. In this work, we studied vitamin B12 production in non-sterile matrices prepared from three raw materials of wheat: durum flour, wholewheat flour and wheat bran. Viable cell counts, pH, total titratable acidity and concentration of acids were determined. After seven days of fermentation, vitamin B12 levels reached 33 +/- 4, 87 +/- 10 and 155 +/- 17 ng/g dry weight in durum flour, wholewheat flour, and wheat bran, respectively. While durum flour supported the growth of P. freudenreichii to higher cell densities and more efficient propionic acid production compared with the other two matrices, wholewheat flour and wheat bran were found to be the most promising of the three matrices for in situ production of vitamin B12. (C) 2018 Elsevier Ltd. All rights reserved.Peer reviewe

Food-Like Growth Conditions Support Production of Active Vitamin B12 by Propionibacterium freudenreichii 2067 without DMBI, the Lower Ligand Base,or Cobalt Supplementation

Propionibacterium freudenreichii is a traditional dairy bacterium and a producer of short chain fatty acids (propionic and acetic acids) as well as vitamin B12. In food applications, it is a promising organism for in situ fortification with B12 vitamin since it is generally recognized as safe (GRAS) and it is able to synthesize biologically active form of the vitamin. In the present study, vitamin B12 and pseudovitamin biosynthesis by P. freudenreichii was monitored by UHPLC as a function of growth in food-like conditions using a medium mimicking cheese environment, without cobalt or 5,6-dimethylbenzimidazole (DMBI) supplementation. Parallel growth experiments were performed in industrial-type medium known to support the biosynthesis of vitamin B12. The production of other key metabolites in the two media were determined by HPLC, while the global protein production was compared by gel-based proteomics to assess the effect of growth conditions on the physiological status of the strain and on the synthesis of different forms of vitamin. The results revealed distinct protein andmetabolite production, which reflected the growth conditions and the potential of P. freudenreichii for synthesizing nutritionally relevant amounts of active vitamin B12 regardless of the metabolic state of the cells.Peer reviewe

Letter to the editor on ‘Enhancing vitamin B12 content in soy-yogurt by Lactobacillus reuteri, IJFM. 206:56–59’

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Red-Brown Pigmentation of Acidipropionibacterium jensenii Is Tied to Haemolytic Activity and cyl-Like Gene Cluster

The novel Acidipropionibacterium genus encompasses species of industrial importance but also those associated with food spoilage. In particular, Acidipropionibacterium acidipropionici, Acidipropionibacterium thoenii, and Acidipropionibacterium jensenii play an important role in food fermentation, as biopreservatives, or as potential probiotics. Notably, A. jensenii and A. thoenii can cause brown spot defects in Swiss-type cheeses, which have been tied to the rhamnolipid pigment granadaene. In the pathogenic bacterium Streptococcus agalactiae, production of granadaene depends on the presence of a cyl gene cluster, an important virulence factor linked with haemolytic activity. Here, we show that the production of granadaene in pigmented Acidipropionibacterium, including A. jensenii, A. thoenii, and Acidipropionibacterium virtanenii, is tied to haemolytic activity and the presence of a cyl-like gene cluster. Furthermore, we propose a PCR-based test, which allows pinpointing acidipropionibacteria with the cyl-like gene cluster. Finally, we present the first two whole genome sequence analyses of the A. jensenii strains as well as testing phenotypic characteristics important for industrial applications. In conclusion, the present study sheds light on potential risks associated with the presence of pigmented Acidipropionibacterium strains in food fermentation. In addition, the results presented here provide ground for development of a quick and simple diagnostic test instrumental in avoiding potential negative effects of Acidipropionibacterium strains with haemolytic activity on food quality

Red-Brown Pigmentation of Acidipropionibacterium jensenii Is Tied to Haemolytic Activity and cyl-Like Gene Cluster

The novel Acidipropionibacterium genus encompasses species of industrial importance but also those associated with food spoilage. In particular, Acidipropionibacterium acidipropionici, Acidipropionibacterium thoenii, and Acidipropionibacterium jensenii play an important role in food fermentation, as biopreservatives, or as potential probiotics. Notably, A. jensenii and A. thoenii can cause brown spot defects in Swiss-type cheeses, which have been tied to the rhamnolipid pigment granadaene. In the pathogenic bacterium Streptococcus agalactiae, production of granadaene depends on the presence of a cyl gene cluster, an important virulence factor linked with haemolytic activity. Here, we show that the production of granadaene in pigmented Acidipropionibacterium, including A. jensenii, A. thoenii, and Acidipropionibacterium virtanenii, is tied to haemolytic activity and the presence of a cyl-like gene cluster. Furthermore, we propose a PCR-based test, which allows pinpointing acidipropionibacteria with the cyl-like gene cluster. Finally, we present the first two whole genome sequence analyses of the A. jensenii strains as well as testing phenotypic characteristics important for industrial applications. In conclusion, the present study sheds light on potential risks associated with the presence of pigmented Acidipropionibacterium strains in food fermentation. In addition, the results presented here provide ground for development of a quick and simple diagnostic test instrumental in avoiding potential negative effects of Acidipropionibacterium strains with haemolytic activity on food quality

BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B 12 by Propionibacterium freudenreichii

Background 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 B 12 . Production of active form of vitamin is a prerequisite for attempts to naturally fortify foods with B 12 by microbial fermentation. Active vitamin B 12 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 B 12 . Understanding the mechanisms affecting the synthesis of different vitamin forms is useful for rational strain selection and essential for engineering of strains with improved B 12 production properties. Results Here, 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. Conclusions The 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 B 12 by efficiently activating DMBI formed within the same enzymatic complex. This indicates that BluB/CobT2 is the crucial enzyme in the B 12 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 B 12 within food products. Keywords: Propionibacterium freudenreichii ; Cobalamin; B12; DMBI; α-Ribazole; Phosphoribozyltransferase; Nitroreductase; Fusion enzyme; BluB; CobTPeer reviewe

De novo assembly of genomes from long sequence reads reveals uncharted territories of Propionibacterium freudenreichii

Background: Propionibacterium freudenreichii is an industrially important bacterium granted the Generally Recognized as Safe (the GRAS) status, due to its long safe use in food bioprocesses. Despite the recognized role in the food industry and in the production of vitamin B12, as well as its documented health-promoting potential, P. freudenreichii remained poorly characterised at the genomic level. At present, only three complete genome sequences are available for the species. Results: We used the PacBio RS II sequencing platform to generate complete genomes of 20 P. freudenreichii strains and compared them in detail. Comparative analyses revealed both sequence conservation and genome organisational diversity among the strains. Assembly from long reads resulted in the discovery of additional circular elements: two putative conjugative plasmids and three active, lysogenic bacteriophages. It also permitted characterisation of the CRISPR-Cas systems. The use of the PacBio sequencing platform allowed identification of DNA modifications, which in turn allowed characterisation of the restriction-modification systems together with their recognition motifs. The observed genomic differences suggested strain variation in surface piliation and specific mucus binding, which were validated by experimental studies. The phenotypic characterisation displayed large diversity between the strains in ability to utilise a range of carbohydrates, to grow at unfavourable conditions and to form a biofilm. Conclusion: The complete genome sequencing allowed detailed characterisation of the industrially important species, P. freudenreichii by facilitating the discovery of previously unknown features. The results presented here lay a solid foundation for future genetic and functional genomic investigations of this actinobacterial species.Peer reviewe

Effect of the lower ligand precursors on vitamin B12 production by food-grade Propionibacteria

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