Vitamin B12 Production Research Articles

Fermentative production of 3-hydroxypropionic acid by using metabolically engineered Klebsiella pneumoniae strains

3-Hydroxypropionic acid (3-HP) is an industrially important platform chemical for super-absorbent or biodegradable polymers. Its production via biological methods is expected to be more competitive than chemical methods. Klebsiella pneumoniae is the most promising host due to its innate capabilities for 3-HP and vitamin-B12 production, ease of culture, and ease of engineering. In this study, step-by-step metabolic engineering and fermentation technologies were used to enhance the production of 3-HP. K. pneumoniae-derived ydcW gene was overexpressed using a plasmid after screening candidate genes. Major competing pathways encoded by dhaT, yqhD, ldhA, glpK, poxB, and pta-ackA were blocked. Additionally, it was demonstrated that simultaneous reinforcement of two native aldehyde dehydrogenase encoded by the ydcW gene preferring NADPH and the puuC gene preferring NADH, synergistically improved 3-HP production. Additional reinforcement of the acs gene to reduce acetate accumulation resulted in 93.7 g/L of 3-HP with a yield of 0.42 g/g·glycerol over a 72-h fed-batch fermentation. This performance is deemed sufficient for industrial applications.

Fermentative production of vitamin B12 by Propionibacterium shermanii and Pseudomonas denitrificans and its promising health benefits: A review

AbstractCobalamin, generally known as vitamin B12, is a crucial component required for humans in several physiological processes. It has been produced from sources that are derived from animals, making it difficult for vegetarians and vegans to consume the recommended amount each day. The importance of vitamin B12 in red blood cell production, DNA synthesis, and brain processes has been highlighted. Recent studies have looked at different methods of producing vitamin B12, such as microbial fermentation. Propionibacterium shermanii and Pseudomonas denitrificans have demonstrated remarkable potential as fermented sources of vitamin B12. Compared to conventional sources, the bioavailability of vitamin B12 produced by P. denitrificans and P. shermanii is more effective in meeting dietary needs. Vitamin B12 can be produced naturally by P. denitrificans. It is equipped with the enzymes and metabolic pathways required to produce this vital vitamin. The fermentation of several dietary substrates by P. shermanii can improve nutrient bioavailability. P. shermanii generates enzymes during fermentation that aid in the breakdown of complex nutrients, facilitating easier absorption and utilization by the body. The motive of the following critical evaluation is to assess the advantages of vitamin B12 for health and the capacity of P. denitrificans and P. shermanii to produce it through fermentation.

Crystal structure of l-threonine-O-3-phosphate decarboxylase CobC from Sinorhizobium meliloti involved in vitamin B12 biosynthesis

Vitamin B12 is involved in many important biochemical reactions for humans, and its deficiency can lead to serious diseases. The industrial production of vitamin B12 is achieved through microbial fermentation. In this work, we determine the crystal structures of the l-threonine-O-3-phosphate (Thr-P) decarboxylase CobC from Sinorhizobium meliloti (SmCobC), an industrial vitamin B12-producing bacterium, in apo form and in complex with a reaction intermediate. Our structures supported the Thr-P decarboxylase activity of SmCobC and revealed that the positively charged substrate-binding pocket between the large and small domains determines its substrate selectivity for Thr-P. Moreover, our results provided evidence for the proposition that the AP-P linker is formed by direct incorporation of AP-P in the biosynthetic pathway of vitamin B12 in S.meliloti.

Metabolite release by nitrifiers facilitates metabolic interactions in the ocean.

Microbial chemoautotroph-heterotroph interactions may play a pivotal role in the cycling of carbon in the deep ocean, reminiscent of phytoplankton-heterotroph associations in surface waters. Nitrifiers are the most abundant chemoautotrophs in the global ocean, yet very little is known about nitrifier metabolite production, release, and transfer to heterotrophic microbial communities. To elucidate which organic compounds are released by nitrifiers and potentially available to heterotrophs, we characterized the exo- and endometabolomes of the ammonia-oxidizing archaeon Nitrosopumilus adriaticus CCS1 and the nitrite-oxidizing bacterium Nitrospina gracilis Nb-211. Nitrifier endometabolome composition was not a good predictor of exometabolite availability, indicating that metabolites were predominately released by mechanisms other than cell death/lysis. Although both nitrifiers released labile organic compounds, N. adriaticus preferentially released amino acids, particularly glycine, suggesting that its cell membranes might be more permeable to small, hydrophobic amino acids. We further initiated co-culture systems between each nitrifier and a heterotrophic alphaproteobacterium, and compared exometabolite and transcript patterns of nitrifiers grown axenically to those in co-culture. In particular, B vitamins exhibited dynamic production and consumption patterns in nitrifier-heterotroph co-cultures. We observed an increased production of vitamin B2 and the vitamin B12 lower ligand dimethylbenzimidazole by N. adriaticus and N. gracilis, respectively. In contrast, the heterotroph likely produced vitamin B5 in co-culture with both nitrifiers and consumed the vitamin B7 precursor dethiobiotin when grown with N. gracilis. Our results indicate that B vitamins and their precursors could play a particularly important role in governing specific metabolic interactions between nitrifiers and heterotrophic microbes in the ocean.

Traditionally produced tempeh harbors more diverse bacteria with more putative health-promoting properties than industrially produced tempeh

In recent years, there has been a significant shift towards industrialization in food production, resulting in the implementation of higher hygiene standards globally. Our study focused on examining the impact of hygiene standards on tempeh, a popular Rhizopus-based fermented soybean product native to Indonesia, and now famous around the world. We observed that tempeh produced with standardized hygiene measures exhibited a microbiome with comparable bacterial abundances but a markedly different community structure and function than traditionally produced tempeh. In detail, we found a decreased bacterial abundance of lactobacilli and enterobacteria, bacterial diversity, different indicator taxa, and significantly changed community structure in industrial tempeh. A similar picture was found for functional analysis: the quantity of bacterial genes was similar but qualitative changes were found for genes associated with human health. The resistome of tempeh varied based on its microbiome composition. The higher number of antimicrobial resistance genes in tempeh produced without standardized hygiene measures mainly belong to multidrug efflux pumps known to occur in plant-based food. Our findings were confirmed by functional insights into genomes and metagenome-assembled genomes from the dominant bacteria, e.g. Leuconostoc, Limosilactobacillus, Lactobacillus, Enterococcus, Paenibacillus, Azotobacter and Enterobacter. They harboured an impressive spectrum of genes important for human health, e.g. for production of vitamin B1, B7, B12, and K, iron and zinc transport systems and short chain fatty acid production. In conclusion, industrially produced tempeh harbours a less diverse microbiome than the traditional one. Although this ensures production at large scales as well as biosafety, in the long-term it can lead to potential effects for human gut health.

MicroRNA maintains nutrient homeostasis in the symbiont-host interaction.

Endosymbionts provide essential nutrients for hosts, promoting growth, development, and reproduction. However, the molecular regulation of nutrient transport from endosymbiont to host is not well understood. Here, we used bioinformatic analysis, RNA-Sequencing, luciferase assays, RNA immunoprecipitation, and in situ hybridization to show that a bacteriocyte-distributed MRP4 gene (multidrug resistance-associated protein 4) is negatively regulated by a host (aphid)-specific microRNA (miR-3024). Targeted metabolomics, microbiome analysis, vitamin B6 (VB6) supplements, 3D modeling/molecular docking, in vitro binding assays (voltage clamp recording and microscale thermophoresis), and functional complementation of Escherichia coli were jointly used to show that the miR-3024/MRP4 axis controls endosymbiont (Serratia)-produced VB6 transport to the host. The supplementation of miR-3024 increased the mortality of aphids, but partial rescue was achieved by providing an external source of VB6. The use of miR-3024 as part of a sustainable aphid pest-control strategy was evaluated by safety assessments in nontarget organisms (pollinators, predators, and entomopathogenic fungi) using virus-induced gene silencing assays and the expression of miR-3024 in transgenic tobacco. The supplementation of miR-3024 suppresses MRP4 expression, restricting the number of membrane channels, inhibiting VB6 transport, and ultimately killing the host. Under aphids facing stress conditions, the endosymbiont titer is decreased, and the VB6 production is also down-regulated, while the aphid's autonomous inhibition of miR-3024 enhances the expression of MRP4 and then increases the VB6 transport which finally ensures the VB6 homeostasis. The results confirm that miR-3024 regulates nutrient transport in the endosymbiont-host system and is a suitable target for sustainable pest control.

Lactic and propionic acid bacteria starter cultures for improved nutritional properties of pea, faba bean and lentil

Increasing plant-based food consumption as a sustainable and health-oriented alternative to meat is pivotal. Pulses are rich in proteins, minerals, and vitamins; however, they also contain antinutritional compounds, impairing their nutritional value. This study addresses this challenge through the development and application of four distinct microbial consortia in pulse-based fermentations, featuring lactic acid bacteria or a combination of lactic and propionic acid bacteria. Microbial starters significantly reduced galacto-oligosaccharides in all pulse materials, concurrently degrading vicine and convicine in faba beans, while the impact on tannins in faba beans and lentil was moderate. Fermentation with lactic acid and propionic acid bacteria consortia exhibited notable vitamin B12 production, and the effect on the content of phenolic compounds of the studied pulses was also evidenced. Additionally, genomic analyses discerned distinctive profiles among the samples, elucidating the microbial community dynamics shaping fermentation outcomes. The results of this study proved how fermentation can advance the development of pulse-based products with improved nutritional and sustainability attributes.

In situ fortification of cereal by-products with vitamin B12: An eco-sustainable approach for food fortification

Vitamin B12 deficiency poses significant health risks, especially among populations with limited access to animal-based foods. This study explores the utilisation of cereal bran by-products, wheat (WB) and oat bran (OB), as substrates for in situ vitamin B12 fortification through solid-state fermentation (SSF) using Propionibacterium freudenreichii. The impact of various precursors addition, including riboflavin, cobalt, nicotinamide and DMBI on vitamin B12 production, along with changes in microbial growth, chemical profiles, and vitamin B12 yields during fermentation was evaluated. Results showed that WB and OB possess favourable constituents for microbial growth and vitamin B12 synthesis. The substrates supplemented with riboflavin, cobalt, and DMBI demonstrated enhanced B12 production. In addition, pH levels are essential in microbial viability and cobalamin biosynthesis. Monosaccharides and organic acids play a crucial role, with maltose showing a strong positive association with B12 production in OB, while in WB, citric acid exhibits significant correlations with various factors.

Source of Vitamin B12 in plants of the Lemnaceae family and its production by duckweed-associated bacteria

While most plants are not known to contain significant levels of the essential corrinoid cobalamin (vitamin B12), considerable levels of vitamin B12 were found in the duckweed clone Wolffia globosa Mankai. Although endophytes were speculated to be the source of this vitamin B12, it has not been tested yet. In this study, we carried out comparative analysis of vitamin B12 content in 15 duckweed accessions across 11 species. Significant but variable levels of vitamin B12 were found in all unsterile duckweed cultures tested. In contrast, disinfected duckweed cultures with lower levels of bacteria, as monitored by PCR-based methods, contained little vitamin B12. Importantly, two strains of duckweed-associated bacteria were shown to produce vitamin B12. This work provides evidence for bacteria as the source of vitamin B12 in duckweeds. Bioinformatics-aided identification of vitamin B12 producers among duckweed-associated bacteria could facilitate their systematic incorporation into duckweed-based foods to support a planetary health diet.

Biofortification, metabolomic profiling and quantitative analysis of vitamin B12 enrichment in guava juice via lactic acid fermentation using Levilactobacillus brevis strain KU15152.

Chemical fortification and dose supplementation of vitamin B12 are widely implemented to combat deficiency symptoms. However, in situ, fortification of vitamin B12 in food matrixes can be a promising alternative to chemical fortification. The present study aimed to produce vitamin B12-rich, probiotic guava juice fermented with Levilactobacillus brevis strain KU15152. Pasteurized fresh guava juice was inoculated with 7.2 log CFU mL-1 L. brevis strain KU15152 and incubated for 72 h at 37 °C anaerobically. The antioxidants, total phenolic compounds, vitamin B12 production, sugars, organic acids, pH and viable count were analyzed at 24, 48 and 72 h of incubation. The fermented juice was stored at 4 °C, and the changes in its functional properties were analyzed at 7-day intervals up to 28 days of storage. During fermentation, the bacteria cell count was increased from 7.01 ± 0.06 to 9.76 ± 0.42 log CFU mL-1 after 72 h of fermentation and was decreased to 6.94 ± 0.34 CFU mL-1 during storage at 4 °C after 28 days. The pH, total soluble solids, crude fiber, citric acid and total sugars decreased, while titratable acidity, total protein, antioxidants, phenolic compounds and lactic acid contents increased during fermentation. The fermented guava juice exhibited higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)) radical scavenging activities (85.97% and 75.97%, respectively) at 48 h of fermentation. The concentration of active vitamin B12 in the sample reached 109.5 μg L-1 at 72 h of fermentation. However, this concentration gradually decreased to 70.2 μg L-1 during the storage period. During storage for 28 days at 4 °C, both the fermented and control guava juices exhibited a decline in antioxidant and phenolic compound concentrations. Furthermore, the addition of 20% honey and guava flavor enhanced the organoleptic properties and acceptability of fermented guava juice. The value-added fermented guava juice could be a novel functional food product to combat vitamin B12 deficiency. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Obtaining Novel Vitamin B12 Production Strains Acetobacter malorum HFD 3141 and Acetobacter orientalis HFD 3031 from Home-Fermented Sourdough

Vitamin B12 is a critical nutrient in vegan and vegetarian lifestyles as plant-based vitamin sources are rare. Traditional fermented foods could be enriched by adding vitamin B12-producing bacteria to offer non-animal vitamin sources. The aim was to isolate a vitamin B12 producer that is capable of producing the human-active vitamin even at low pH values so that it can be used in fruit juice fortification. Therefore, fermented foods (homemade and industrial) and probiotics were screened for vitamin B12 production strains. A modified microbiological vitamin B12 assay based on Lactobacillus delbrueckii subsp. lactis DSM 20355 was used to identify vitamin B12-containing samples and the presence of vitamin B12-producing strains. The screening resulted in isolating several positive strains for vitamin B12 formation derived from sourdough. Mass spectrometry confirmed the biosynthesis of solely the human physiologically active form. Species identification carried out by the German Strain Collection of Microorganisms and Cell Cultures resulted in two species: Acetobacter orientalis and Acetobacter malorum, of which two isolates were further characterised. The potential for cobalamin biosynthesises in food matrixes was demonstrated for A. malorum HFD 3141 and A. orientalis HFD 3031 in apple juice at different pH values (2.85–3.80). The isolates synthesised up to 18.89 µg/L and 7.97 µg/L vitamin B12 at pH 3.80. The results of this study suggest that acetic acid bacteria (AAB) and fermented acetic acid foods are promising resources for vitamin B12 and its producers, which might have been overlooked in the past.

The Obesogenic Gut Microbiota as a Crucial Factor Defining the Depletion of Predicted Enzyme Abundance for Vitamin B12 Synthesis in the Mouse Intestine.

Currently, obesity is a critical global public health burden. Numerous studies have demonstrated the regulation of the pathogenesis of obesity and metabolic abnormalities by the gut microbiota and microbial factors; however, their involvement in the various degrees of obesity is not yet well understood. Previously, obesity has been shown to be associated with decreased levels of vitamin B12. Considering exclusive microbial production of vitamin B12, we hypothesized that a decrease in cobalamin levels in obese individuals may be at least partially caused by its depleted production in the intestinal tract by the commensal microbiota. In the present study, our aim was to estimate the abundance of enzymes and metabolic pathways for vitamin B12 synthesis in the gut microbiota of mouse models of alimentary and genetically determined obesity, to evaluate the contribution of the obesogenic microbiome to vitamin B12 synthesis in the gut. We have defined a significantly lower predicted abundance of enzymes and metabolic pathways for vitamin B12 biosynthesis in obese mice compared to non-obese mice, wherein enzyme depletion was more pronounced in lepr(-/-) (db/db) mice, which developed severe obesity. The predicted abundance of enzymes involved in cobalamin synthesis is strongly correlated with the representation of several microbes in high-fat diet-fed mice, while there were almost no correlations in db/db mice. Therefore, the degree of obesity and the composition of the correspondent microbiota are the main contributors to the representation of genes and pathways for cobalamin biosynthesis in the mouse gut.

Tandem gene duplication selected by activation of horizontally transferred gene in bacteria

Horizontal gene transfer occurs frequently in bacteria, but the mechanism driving activation and optimization of the expression of horizontally transferred genes (HTGs) in new recipient strains is not clear. Our previous study found that spontaneous tandem DNA duplication resulted in rapid activation of HTGs. Here, we took advantage of this finding to develop a novel technique for tandem gene duplication, named tandem gene duplication selected by activation of horizontally transferred gene in bacteria (TDAH), in which tandem duplication was selected by the activation of horizontally transferred selectable marker gene. TDAH construction does not contain any reported functional elements based on homologous or site-specific recombination and DNA amplification. TDAH only contains an essential selectable marker for copy number selection and 9-bp-microhomology border sequences for precise illegitimate recombination. One transformation and 3 days were enough to produce a high-copy strain, so its procedure is simple and fast. Without subsequent knockout of the endogenous recombination system, TDAH could also generate the relatively stable high-copy tandem duplication for plasmid-carried and genome-integrated DNA. TDAH also showed an excellent capacity for increase gene expression and worked well in different industrial bacteria. We also applied TDAH to select the optimal high copy number of ribA for vitamin B2 production in E. coli; the yield was improved by 3.5 times and remained stable even after 12 subcultures. TDAH is a useful tool for recombinant protein production and expression optimization of biosynthetic pathways.Key points• We develop a novel and efficient technique (TDAH) for tandem gene duplication in bacterium. TDAH is based on the mechanism of HTG rapid activation. TDAH does not contain any reported functional elements based on homologous recombination and DNA amplification. TDAH only contains an essential selectable marker for copy number selection, so its construction and procedure are very simple and fast.• TDAH is the first reported selected and stable tandem-gene-duplication technique in which the selected high-copy plasmid-carried and genome-integrated DNA could remain stable without the subsequent knockout of recombination system.• TDAH showed an excellent capacity for regulating gene expression and worked well in different industrial bacteria, indicating it is a useful tool for recombinant protein production and expression optimization of biosynthetic pathways.• TDAH was applied to select the optimal high copy number of ribA for vitamin B2 production in E. coli; the yield was improved by 3.5-fold and remained stable even after 12 subcultures.

Enhancement of vitamin B6 production driven by omics analysis combined with fermentation optimization

BackgroundMicrobial engineering aims to enhance the ability of bacteria to produce valuable products, including vitamin B6 for various applications. Numerous microorganisms naturally produce vitamin B6, yet the metabolic pathways involved are rigorously controlled. This regulation by the accumulation of vitamin B6 poses a challenge in constructing an efficient cell factory.ResultsIn this study, we conducted transcriptome and metabolome analyses to investigate the effects of the accumulation of pyridoxine, which is the major commercial form of vitamin B6, on cellular processes in Escherichia coli. Our omics analysis revealed associations between pyridoxine and amino acids, as well as the tricarboxylic acid (TCA) cycle. Based on these findings, we identified potential targets for fermentation optimization, including succinate, amino acids, and the carbon-to-nitrogen (C/N) ratio. Through targeted modifications, we achieved pyridoxine titers of approximately 514 mg/L in shake flasks and 1.95 g/L in fed-batch fermentation.ConclusionOur results provide insights into pyridoxine biosynthesis within the cellular metabolic network for the first time. Our comprehensive analysis revealed that the fermentation process resulted in a remarkable final yield of 1.95 g/L pyridoxine, the highest reported yield to date. This work lays a foundation for the green industrial production of vitamin B6 in the future.

Whole-Genome Shotgun Metagenomic Sequencing Reveals Shifts in the Skin Microbiome and Bacteriophages of Psoriasis: An Extended Analysis of Published Data

Background Psoriasis is an immune-mediated cutaneous disease that may have shifts in the skin microbiome. Prior research on the skin microbiome in psoriasis has been limited to rRNA based approaches that lack resolution of taxonomic and functional level assessment. Objective To further illuminate strain and sub-strain level analysis of psoriatic lesions using the CosmosID-HUB Microbiome pipeline. Methods A previous study completed by Tett et al recruited patients with psoriasis who had skin microbiome samples taken from psoriatic plaques on the ear and the elbow as well as sites on the skin unaffected by psoriasis. They performed whole genome shotgun sequencing and made their dataset publicly available. We analyzed the dataset using the CosmosID-HUB Microbiome pipeline to evaluate the strain and sub-strain taxonomic analysis as well as functional gene profiling. Results When analyzed with the CosmosID pipeline, both ear and elbow sites in affected areas had decreased alpha diversity compared to unaffected areas. There was an increased relative abundance of Staphylococcus and Corynebacteria at affected sites. We identified distinguishing species and strains of the yeast Malassezia, including M. restricta. that were significantly enriched in healthy elbow samples. Vitamin B12 production genes were not present in psoriatic skin whereas it was present in healthy samples, supporting the notion of relative vitamin B12 deficiency in psoriatic plaques. Phage analysis revealed a greater diversity of Staphylococcus-related phages in unaffected elbow samples. Conclusion A greater diversity of microbial strains and their functional roles identified in this study may help to tailor treatment for psoriasis.

Metabolic relationships between marine red algae and algae-associated bacteria

Mutualistic interactions between marine phototrophs and associated bacteria are an important strategy for their successful survival in the ocean, but little is known about their metabolic relationships. Here, bacterial communities in the algal sphere (AS) and bulk solution (BS) of nine marine red algal cultures were analyzed, and Roseibium and Phycisphaera were identified significantly more abundantly in AS than in BS. The metabolic features of Roseibium RMAR6-6 (isolated and genome-sequenced), Phycisphaera MAG 12 (obtained by metagenomic sequencing), and a marine red alga, Porphyridium purpureum CCMP1328 (from GenBank), were analyzed bioinformatically. RMAR6-6 has the genetic capability to fix nitrogen and produce B vitamins (B1, B2, B5, B6, B9, and B12), bacterioferritin, dimethylsulfoniopropionate (DMSP), and phenylacetate that may enhance algal growth, whereas MAG 12 may have a limited metabolic capability, not producing vitamins B9 and B12, DMSP, phenylacetate, and siderophores, but with the ability to produce bacitracin, possibly modulating algal microbiome. P. purpureum CCMP1328 lacks the genetic capability to fix nitrogen and produce vitamin B12, DMSP, phenylacetate, and siderophore. It was shown that the nitrogen-fixing ability of RMAR6-6 promoted the growth of P. purpureum, and DMSP reduced the oxidative stress of P. purpureum. The metabolic interactions between strain RMAR6-6 and P. purpureum CCMP1328 were also investigated by the transcriptomic analyses of their monoculture and co-culture. Taken together, potential metabolic relationships between Roseibium and P. purpureum were proposed. This study provides a better understanding of the metabolic relationships between marine algae and algae-associated bacteria for successful growth.

Production of vitamin B12 via microbial strains isolated from marine and food sources in Egypt

Background Vitamin B12 is a very important water-soluble vitamin, which was first isolated from the liver as an anti-pernicious anemia factor. The sole source of vitamin B12 is the animal-based food. It has a complicated structure and requires expensive multi-steps to be synthesized chemically. Intriguingly, vitamin B12 can be produced through microbial fermentation by microorganisms in a cheap and more effective manner. Objective This study aims to isolate and characterize microorganisms that have the capability to produce vitamin B12. In addition, the current work aims to optimize the vitamin B12 production conditions by isolating strains using suitable waste materials to obtain a high vitamin B12 yield. Materials and methods Different bacterial and yeast isolates were isolated from marine and food samples using the pour-plate technique. These isolates were screened for vitamin B12 production using a specific growth medium that lacked vitamin B12 and a test indicator bacterium. The content of vitamin B12 was estimated using spectrophotometer measurement and high-performance liquid chromatography (HPLC). The isolates that showed high vitamin B12 productivity were identified using MALDI-TOF technique. The identified strains were implemented for the optimization of vitamin B12 production to reveal the most proper and optimum conditions for the production. Response surface methodology (RSM) was employed to enhance the production of vitamin B12 in a flask scale. Agro-industrial wastes such as molasses were used for vitamin B12 production using the most optimum conditions as determined by RSM. Results and conclusion Eighty-seven actinomycetes, bacterial, and yeast isolates were screened for vitamin B12 production. Out of these isolates, 15 showed high vitamin B12 productivity. We found that bacilli and yeast isolates were the most productive among the tested cocci and actinomycetes isolates. The highly productive Bacillus and yeast isolates were identified using the MALDI-TOF analysis. The isolates were identified as Candida pelliculosa, Geotrichum candidum, Bacillus subtilis and Bacillus sp. One strain of Candida pelliculosa (coded BYI), three strains of Geotrichum candidum (coded as MZYC, MZYD, and MZYG) were selected for studying the effect of sugar type and inoculum size on the yield of vitamin B12 production. Strain MZYD was selected for the statistical modelling using RSM to optimize seven factors for the vitamin B12 production. These factors included temperature, fermentation time, salt concentration, pH, sugar concentration, inoculum size, and aeration. Five factors i.e., temperature, pH, sugar concentration, and inoculum size were shown to significantly improve the vitamin B12 production. A maximum yield of 64.21 μg/100 ml was obtained using the optimized RSM conditions. These optimized conditions were used to produce vitamin B12 using molasses as a raw material for the microbial growth.

Co-culturing Propionibacterium freudenreichii and Bifidobacterium animalis subsp. lactis improves short-chain fatty acids and vitamin B12 contents in soy whey

The lack of vitamin B12 in unprocessed plant-based foods can lead to health problems in strict vegetarians and vegans. The main aim of this study was to investigate the potential synergy of co-culturing Bifidobacterium animalis subsp. lactis and Propionibacterium freudenreichii in improving production of vitamin B12 and short-chain fatty acids in soy whey. Different strategies including mono-, sequential and simultaneous cultures were adopted. Growth, short-chain fatty acids and vitamin B12 were assessed throughout the fermentation while free amino acids, volatiles, and isoflavones were determined on the final day. P. freudenreichii monoculture grew well in soy whey, whereas B. lactis monoculture entered the death phase by day 4. Principal component analysis demonstrates that metabolic changes in both sequential cultures did not show drastic differences to those of P. freudenreichii monoculture. However, simultaneous culturing significantly improved vitamin B12, acetic acid and propionic acid contents (1.3 times, 5 times, 2.5 times, compared to the next highest treatment [sequential cultures]) in fermented soy whey relative to other culturing modes. Hence, co-culturing of P. freudenreichii and B. lactis would provide an alternative method to improve vitamin B12, acetic acid and propionic acid contents in fermented foods.

Engineering and finetuning expression of SerC for balanced metabolic flux in vitamin B6 production

Vitamin B6 plays a crucial role in cellular metabolism and stress response, making it an essential component for growth in all known organisms. However, achieving efficient biosynthesis of vitamin B6 faces the challenge of maintaining a balanced distribution of metabolic flux between growth and production. In this study, our focus is on addressing this challenge through the engineering of phosphoserine aminotransferase (SerC) to resolve its redundancy and promiscuity. The enzyme SerC was semi-designed and screened based on sequences and predicted kcat values, respectively. Mutants and heterologous proteins showing potential were then fine-tuned to optimize the production of vitamin B6. The resulting strain enhances the production of vitamin B6, indicating that different fluxes are distributed to the biosynthesis pathway of serine and vitamin B6. This study presents a promising strategy to address the challenge posed by multifunctional enzymes, with significant implications for enhancing biochemical production through engineering processes.

Gorse (Ulex europeaus) wastes with 5,6-dimethyl benzimidazole supplementation can support growth of vitamin B12 producing commensal gut microbes.

Many commensal gut microbes are recognized for their potential to synthesize vitamin B12, offering a promising avenue to address deficiencies through probiotic supplementation. While bioinformatics tools aid in predicting B12 biosynthetic potential, empirical validation remains crucial to confirm production, identify cobalamin vitamers, and establish biosynthetic yields. This study investigates vitamin B12 production in three human colonic bacterial species: Anaerobutyricum hallii DSM 3353, Roseburia faecis DSM 16840, and Anaerostipes caccae DSM 14662, along with Propionibacterium freudenreichii DSM 4902 as a positive control. These strains were selected for their potential use as probiotics, based on speculated B12 production from prior bioinformatic analyses. Cultures were grown in M2GSC, chemically defined media (CDM), and Gorse extract medium (GEM). The composition of GEM was similar to CDM, except that the carbon and nitrogen sources were replaced with the protein-depleted liquid waste obtained after subjecting Gorse to a leaf protein extraction process. B12 yields were quantified using liquid chromatography with tandem mass spectrometry. The results suggested that the three butyrate-producing strains could indeed produce B12, although the yields were notably low and were detected only in the cell lysates. Furthermore, B12 production was higher in GEM compared to M2GSC medium. The positive control, P. freudenreichii DSM 4902 produced B12 at concentrations ranging from 7 ng mL-1 to 12 ng mL-1. Univariate-scaled Principal Component Analysis (PCA) of data from previous publications investigating B12 production in P. freudenreichii revealed that B12 yields diminished when the carbon source concentration was ≤30 g L-1. In conclusion, the protein-depleted wastes from the leaf protein extraction process from Gorse can be valorised as a viable substrate for culturing B12-producing colonic gut microbes. Furthermore, this is the first report attesting to the ability of A. hallii, R. faecis, and A. caccae to produce B12. However, these microbes seem unsuitable for industrial applications owing to low B12 yields.