B12 Biosynthesis Research Articles

Different Alterations in Gut Microbiota between Bifidobacterium longum and Fecal Microbiota Transplantation Treatments in Propionic Acid Rat Model of Autism.

Autism spectrum disorders (ASD) consist of a range of neurodevelopmental conditions accompanied by dysbiosis of gut microbiota. Therefore, a number of microbiota manipulation strategies were developed to restore their balance. However, a comprehensive comparison of the various methods on gut microbiota is still lacking. Here, we evaluated the effect of Bifidobacterium (BF) treatment and fecal microbiota transplantation (FT) on gut microbiota in a propionic acid (PPA) rat model of autism using 16S rRNA sequencing. Following PPA treatment, gut microbiota showed depletion of Bacteroidia and Akkermansia accompanied by a concomitant increase of Streptococcus, Lachnospiraceae, and Paraeggerthella. The dysbiosis was predicted to cause increased levels of porphyrin metabolism and impairments of acyl-CoA thioesterase and ubiquinone biosynthesis. On the contrary, BF and FT treatments resulted in a distinct increase of Clostridium, Bifidobacterium, Marvinbryantia, Butyricicoccus, and Dorea. The taxa in BF group positively correlated with vitamin B12 and flagella biosynthesis, while FT mainly enriched flagella biosynthesis. In contrast, BF and FT treatments negatively correlated with succinate biosynthesis, pyruvate metabolism, nitrogen metabolism, beta-Lactam resistance, and peptidoglycan biosynthesis. Therefore, the present study demonstrated that BF and FT treatments restored the PPA-induced dysbiosis in a treatment-specific manner.

High Cell Density Culture of Dairy Propionibacterium sp. and Acidipropionibacterium sp.: A Review for Food Industry Applications.

The dairy bacteria Propionibacterium sp. and Acidipropionibacterium sp. are versatile and potentially probiotic microorganisms showing outstanding functionalities for the food industry, such as the production of propionic acid and vitamin B12 biosynthesis. They are the only food grade microorganisms able to produce vitamin B12. However, the fermentation batch process using these bacteria present some bioprocess limitations due to strong end-product inhibition, cells slow-growing rates, low product titer, yields and productivities, which reduces the bioprocess prospects for industrial applications. The high cell density culture (HCDC) bioprocess system is known as an efficient approach to overcome most of those problems. The main techniques applied to achieve HCDC of dairy Propionibacterium are the fed-batch cultivation, cell recycling, perfusion, extractive fermentation, and immobilization. In this review, the techniques available and reported to achieve HCDC of Propionibacterium sp. and Acidipropionibacterium sp. are discussed, and the advantages and drawbacks of this system of cultivation in relation to biomass formation, vitamin B12 biosynthesis, and propionic acid production are evaluated.

Elevated Levels of an Enzyme Involved in Coenzyme B12 Biosynthesis Kills Escherichia coli.

ABSTRACTCobamides are cobalt-containing cyclic tetrapyrroles involved in the metabolism of organisms from all domains of life but produced de novo only by some bacteria and archaea. The pathway is thought to involve up to 30 enzymes, five of which comprise the so-called “late” steps of cobamide biosynthesis. Two of these reactions activate the corrin ring, one activates the nucleobase, a fourth one condenses activated precursors, and a phosphatase yields the final product of the pathway. The penultimate step is catalyzed by a polytopic integral membrane protein, namely, the cobamide (5′-phosphate) synthase, also known as cobamide synthase. At present, the reason for the association of all putative and bona fide cobamide synthases to cell membranes is unclear and intriguing. Here, we show that, in Escherichia coli, elevated levels of cobamide synthase kill the cell by dissipating the proton motive force and compromising membrane stability. We also show that overproduction of the phosphatase that catalyzes the last step of the pathway or phage shock protein A prevents cell death when the gene encoding cobamide synthase is overexpressed. We propose that in E. coli, and probably all cobamide producers, cobamide synthase anchors a multienzyme complex responsible for the assembly of vitamin B12 and other cobamides.

Guardian of cobamide diversity: Probing the role of CobT in lower ligand activation in the biosynthesis of vitamin B12 and other cobamide cofactors.

Enzymes catalyze a wide variety of reactions with exquisite precision under crowded conditions within cellular environments. When encountered with a choice of small molecules in their vicinity, even though most enzymes continue to be specific about the substrate they pick, some others are able to accept a range of substrates and subsequently produce a variety of products. The biosynthesis of Vitamin B12, an essential nutrient required by humans involves a multi-substrate α-phosphoribosyltransferase enzyme CobT that activates the lower ligand of B12. Vitamin B12 is a member of the cobamide family of cofactors which share a common tetrapyrrolic corrin scaffold with a centrally coordinated cobalt ion, and an upper and a lower ligand. The structural difference between B12 and other cobamides mainly arises from variations in the lower ligand, which is attached to the activated corrin ring by CobT and other downstream enzymes. In this chapter, we describe the steps involved in identifying and reconstituting the activity of new CobT homologs by deriving lessons from those previously characterized. We then highlight biochemical techniques to study the unique properties of these homologs. Finally, we describe a pairwise substrate competition assay to rank CobT substrate preference, a general method that can be applied for the study of other multi-substrate enzymes. Overall, the analysis with CobT provides insights into the range of cobamides that can be synthesized by an organism or a community, complementing efforts to predict cobamide diversity from complex metagenomic data.

The Effects of Non-Fiber Carbohydrate Content and Forage Type on Rumen Microbiome of Dairy Cows.

Simple SummaryFor sustainable development in the dairy industry, crop by-products play an important role, especially in areas that lack pasture and are not suitable for intensive cereal-adapted production (i.e., diets containing high amounts of cereal grains). However, feeding crop by-products usually lowers milk production in dairy cows due to their poor nutrient quality. In a previous study, we have demonstrated that non-fiber carbohydrate content (NFC) is a major limiting factor for the utilization of diets based on corn stover (CS). Although the higher abundance of easily fermentable energy in NFC and forage type can influence the synthesis of VFAs and MCP in the rumen and higher NFC content or high quality forages normally have a positive influence on the lactation performance of dairy cows, the underlying microbial mechanisms and potential effects on milk production are under-investigated to date. Here, we examined microbial composition and predicted the metabolism from cows fed CS-based diets with either high levels of NFC (H-NFC), or low levels of NFC (L-NFC). Control cows were fed an alfalfa-based diet (AH). Our results show that, supplementation of the CS-based diet with additional NFC increased amino acid biosynthesis in rumen microbiota in dairy cattle, and thus resulted in better nitrogen conversion. However, lower levels of fibrolytic capacity may limit dry matter intake of cows fed H-NFC and may prevent increased milk yield.The main objective of our current study was evaluating the effects of NFC supplementation and forage type on rumen microbiota and metabolism, by comparing microbial structures and composition among samples collected from cows fed AH (alfalfa-based diet), H-NFC (CS-based diet with high NFC) and L-NFC (CS-based diet with low NFC) diets. Our results show that microbial communities were structurally different but functionally similar among groups. When compared with L-HFC, NFC increased the population of Treponema, Ruminobacter, Selenomonas and Succinimonas that were negatively correlated with ruminal NH3-N, and urea nitrogen in blood, milk and urine, as well as significantly increasing the number of genes involved in amino acid biosynthesis. However, when compared to the AH group, H-NFC showed a higher abundance of bacteria relating to starch degradation and lactate production, but a lower abundance of bacteria utilizing pectin and other soluble fibers. This may lead to a slower proliferation of lignocellulose bacteria, such as Ruminococcus, Marvinbryantia and Syntrophococcus. Lower fibrolytic capacity in the rumen may reduce rumen rotation rate and may limit dry matter intake and milk yield in cows fed H-NFC. The enzyme activity assays further confirmed that cellulase and xylanase activity in AH were significantly higher than H-NFC. In addition, the lower cobalt content in Gramineae plants compared to legumes, might have led to the significantly down-regulated microbial genes involved in vitamin B12 biosynthesis in H-NFC compared to AH. A lower dietary supply with vitamin B12 may restrict the synthesis of milk lactose, one of the key factors influencing milk yield. In conclusion, supplementation of a CS-based diet with additional NFC was beneficial for nitrogen conversion by increasing the activity of amino acid biosynthesis in rumen microbiota in dairy cattle. However, lower levels of fibrolytic capacity may limit dry matter intake of cows fed H-NFC and may prevent increased milk yield.

Sex-specific relationships of the infant microbiome and early-childhood behavioral outcomes.

A link between the gut microbiome and behavior is hypothesized, but most previous studies are cross-sectional or in animal models. The modifying role of host sex is poorly characterized. We aimed to identify sex-specific prospective associations between the early-life gut microbiome and preschool-age neurobehavior. In a prospective cohort, gut microbiome diversity and taxa were estimated with 16S rRNA sequencing at 6 weeks, 1 year, and 2 years. Species and gene pathways were inferred from metagenomic sequencing at 6 weeks and 1 year. When subjects were 3 years old, parents completed the Behavioral Assessment System for Children, second edition (BASC-2). A total of 260 children contributed 523 16S rRNA and 234 metagenomics samples to analysis. Models adjusted for sociodemographic characteristics. Higher diversity at 6 weeks was associated with better internalizing problems among boys, but not girls [βBoys = -1.86 points/SD Shannon diversity; 95% CI (-3.29, -0.42), pBoys = 0.01, βGirls = 0.22 (-1.43, 1.87), pGirls = 0.8, pinteraction = 0.06]. Among other taxa-specific associations, Bifidobacterium at 6 weeks was associated with Adaptive Skills scores in a sex-specific manner. We observed relationships between functional features and BASC-2 scores, including vitamin B6 biosynthesis pathways and better Depression scores. This study advances our understanding of microbe-host interactions with implications for childhood behavioral health. This is one of the first studies to examine the early-life microbiome and neurobehavior, and the first to examine prospective sex-specific associations. Infant and early-childhood microbiomes relate to neurobehavior including anxiety, depression, hyperactivity, and social behaviors in a time- and sex-specific manner. Our findings suggest future studies should evaluate whether host sex impacts the relationship between the gut microbiome and behavioral health outcomes.

Recombinant Expression, Purification and Characterization of Pyridoxal 5'-phosphate Synthase from Geobacillus sp. H6a, Thermophilic Bacterium Producing Extracellular Vitamin B6.

Background: Pyridoxal 5ʹ -phosphate synthase (PLPS) is present in deoxyxylose 5ʹ-phosphate-independent of the de novo vitamin B6 biosynthesis pathway. This enzyme complex consists of PdxS and PdxT, which function as synthase and glutamine amidotranferase respectively to produce PLP.Objectives: This study aimed to clone, express, and purify PLPS of Geobacillus sp. H6a, followed by its characterization.Material and Methods: The PdxS and PdxT genes were amplified from Geobacillus (Gh) sp. H6a. Recombinant vectors pET28a-GhpdxS and pET28a-GhpdxT were constructed and the resulting His-tagged proteins were expressed in E. coli BL21(DE3). The soluble rGhpdxS and rGhpdxT were purified via nickel-affinity chromatography and cation-exchange chromatography. The mixture of rGhpdxS and rGhpdxT was further characterized.Results: The molecular weights of rGhpdxS and rGhpdxT were estimated to be 35 and 23 kDa by SDS-PAGE, respectively. The native form of rGhpdxS showed hexamer and dodecamer, whereas those of rGhpdxT were a monomer upon detection with non-denaturing gel electrophoresis and gel filtration. A molar ratio of 1:1 of rGhpdxS:rGhpdxT showed the highest PLP synthesis activity (4.16 U.mg-1) and was used for analyzing the biochemical properties. The kinetic values were obtained by using glyceraldehyde 3-phosphate, ribose 5-phosphate, and glutamine as the substrates. The rGhPLPS showed pentose phosphate isomerization without triose phosphate isomerase activity. The metal ions affected PLP synthesis activity. The optimum pH and optimum temperature of rGhPLPS were 9 and 70 °C, respectively. The rGhPLPS was active over a broad range of temperatures and pH values.Conclusions: These results support the potential of rGhPLPS as a candidate for industrial application.

Cloning and characterization of cobA, one of vitamin B12 biosynthesis pathway genes from Selenomonas ruminantium

Bacterial biosynthesis of vitamin B12 can occur via either aerobic or anaerobic route. While the aerobic pathway has been fully elucidated and understood, less is known about the anaerobic pathway. Selenomonas ruminantium is thought to be the main producer of this vitamin in rumen environment and must use the anaerobic pathway. In our work we found one of the genes of vitamin B12 biosynthetic pathway of S. ruminantium, encoding for the cobalamin adenosyltransferase, enzyme taking part at the last steps of the synthesis process. Deduced amino acid sequence showed the highest similarity to cobalamin adenosyltransferases of other ruminal anaerobic bacteria and that of species Selenomonas. Phylogenetic comparisons of CobA protein sequences of several anaerobic bacteria of Clostridiale order indicate possible horizontal transfer of this gene.

Tight Adherence (Tad) Pilus Genes Indicate Putative Niche Differentiation in Phytoplankton Bloom Associated Rhodobacterales.

The multiple interactions of phytoplankton and bacterioplankton are central for our understanding of aquatic environments. A prominent example of those is the consistent association of diatoms with Alphaproteobacteria of the order Rhodobacterales. These photoheterotrophic bacteria have traditionally been described as generalists that scavenge dissolved organic matter. Many observations suggest that members of this clade are specialized in colonizing the microenvironment of diatom cells, known as the phycosphere. However, the molecular mechanisms that differentiate Rhodobacterales generalists and phycosphere colonizers are poorly understood. We investigated Rhodobacterales in the North Sea during the 2010–2012 spring blooms using a time series of 38 deeply sequenced metagenomes and 10 metaproteomes collected throughout these events. Rhodobacterales metagenome assembled genomes (MAGs) were recurrently abundant. They exhibited the highest gene enrichment and protein expression of small-molecule transporters, such as monosaccharides, thiamine and polyamine transporters, and anaplerotic pathways, such as ethylmalonyl and propanoyl-CoA metabolic pathways, all suggestive of a generalist lifestyle. Metaproteomes indicated that the species represented by these MAGs were the dominant suppliers of vitamin B12 during the blooms, concomitant with a significant enrichment of genes related to vitamin B12 biosynthesis suggestive of association with diatom phycospheres. A closer examination of putative generalists and colonizers showed that putative generalists had persistently higher relative abundance throughout the blooms and thus produced more than 80% of Rhodobacterales transport proteins, suggesting rapid growth. In contrast, putative phycosphere colonizers exhibited large fluctuation in relative abundance across the different blooms and correlated strongly with particular diatom species that were dominant during the blooms each year. The defining feature of putative phycosphere colonizers is the presence of the tight adherence (tad) gene cluster, which is responsible for the assembly of adhesive pili that presumably enable attachment to diatom hosts. In addition, putative phycosphere colonizers possessed higher prevalence of secondary metabolite biosynthetic gene clusters, particularly homoserine lactones, which can regulate bacterial attachment through quorum sensing. Altogether, these findings suggest that while many members of Rhodobacterales are competitive during diatom blooms, only a subset form close associations with diatoms by colonizing their phycospheres.

Distinct Human Gut Microbial Taxonomic Signatures Uncovered With Different Sample Processing and Microbial Cell Disruption Methods for Metaproteomic Analysis.

The use of metaproteomics for studying the human gut microbiota can shed light on the taxonomic profile and the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells, which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing (SSP) and microbial cell disruption methods (CDMs). The combination of a longer disintegration period of the stool sample in a tube rotator with sonication increased the overall number of identified peptides and proteins. Proteobacteria, Bacteroidetes, Planctomycetes, and Euryarchaeota identification was favored by mechanical cell disruption with glass beads. In contrast, the relative abundance of Firmicutes, Actinobacteria, and Fusobacteria was improved when sonication was performed before bead beating. Tenericutes and Apicomplexa identification was enhanced by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads combined with or without sonication. Human protein identifications were affected by sonication. To test the reproducibility of these gut metaproteomic analyses, we examined samples from six healthy individuals using a protocol that had shown a good taxonomic diversity and identification of proteins from Proteobacteria and humans. We also detected proteins involved in microbial functions relevant to the host and related mostly to specific taxa, such as B12 biosynthesis and short chain fatty acid (SCFA) production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. The taxonomic and functional profiles obtained with the different protocols described in this work provides the researcher with valuable information when choosing the most adequate protocol for the study of certain pathologies under suspicion of being related to a specific taxon from the gut microbiota.

Revealing the Promoting Effect of Betaine on Vitamin B12 Biosynthetic Pathway of Pseudomonas denitrificans by Using a Proteomics Analysis.

Our previous comparative metabolomics research revealed that betaine (N,N,Ntrimethylglycine, a typically essential methyl-group donor for vitamin B12 biosynthesis) had powerful promoting effect on the generation of vitamin B12 precursors and intermediates in vitamin B12-producing Pseudomonas denitrificans. However, the integral effect of betaine on the vitamin B12 biosynthetic pathway is still unclear. Considering the vitamin B12 biosynthetic pathway of P. denitrificans as a whole, this work aimed to reveal the biological function of betaine on the vitamin B12 biosynthetic pathway in P. denitrificans, which would sharpen and expand understanding of betaine as the methyl-group donor for vitamin B12 biosynthesis. By using a proteomics method based on the iTRAQ technique, the present study compared and analyzed the differential expression of proteins involved in vitamin B12 biosynthetic pathway under 10 g/L betaine in addition to P. denitrificans fermentation medium. The results showed that betaine could significantly up-regulate the expression of proteins related to the vitamin B12 biosynthetic pathway, which was mainly reflected in the following three aspects: 1) the δ-aminolevulinic acid (ALA) synthase and porphobilinogen synthase that were responsible for the formation of the committed precursors for tetrapyrrole-derived macrocycle in vitamin B12 molecule; 2) the C-methylation-related enzymes (such as precorrin-4 C(11)-methyltransferase, precorrin-2 C(20)- methyltransferase, precorrin-8X methylmutase, and precorrin-6Y C5,15-methyltransferase) and methionine synthase that were crucial to the C-methylation reactions for vitamin B12 biosynthesis; 3) the latestage key enzymes (Cobaltochelatase, and Cob(I)yrinic acid a,c-diamide adenosyltransferase) that were related to cobalt chelation of vitamin B12 molecule. The present study demonstrated clearly that betaine could significantly promote the expression of the integral enzymes involved in the vitamin B12 biosynthetic pathway of P. denitrificans, thus promoting vitamin B12 biosynthesis.

Altered Gut Microbiota in a Fragile X Syndrome Mouse Model.

The human gut microbiome is the ecosystem of microorganisms that live in the human digestive system. Several studies have related gut microbiome variants to metabolic, immune and nervous system disorders. Fragile X syndrome (FXS) is a neurodevelopmental disorder considered the most common cause of inherited intellectual disability and the leading monogenetic cause of autism. The role of the gut microbiome in FXS remains largely unexplored. Here, we report the results of a gut microbiome analysis using a FXS mouse model and 16S ribosomal RNA gene sequencing. We identified alterations in the fmr1 KO2 gut microbiome associated with different bacterial species, including those in the genera Akkermansia, Sutterella, Allobaculum, Bifidobacterium, Odoribacter, Turicibacter, Flexispira, Bacteroides, and Oscillospira. Several gut bacterial metabolic pathways were significantly altered in fmr1 KO2 mice, including menaquinone degradation, catechol degradation, vitamin B6 biosynthesis, fatty acid biosynthesis, and nucleotide metabolism. Several of these metabolic pathways, including catechol degradation, nucleotide metabolism and fatty acid biosynthesis, were previously reported to be altered in children and adults with autism. The present study reports a potential association of the gut microbiome with FXS, thereby opening new possibilities for exploring reliable treatments and non-invasive biomarkers.

The Effect of Arabinogalactans on the Gut Microbiome: A Randomized, Double‐blind, Placebo‐Controlled, Crossover Trial in Healthy Adults

Promising evidence suggests beneficial health effects of arabinogalactan but little is known on the effect of this non-digestible carbohydrate on the gut microbiota, a crucial mediator of human health. The objective of this study was to investigate the effect of an arabinogalactan product (ResistAid®) on the fecal microbiome, short chain fatty acids (SCFAs) and gastrointestinal tolerance in healthy adults in a randomized, double-blind, cross-over trial. Thirty adults were randomly assigned to consume 15 g/d maltodextrin (control) or ResistAid for 6 weeks. At Week 6, and compared to placebo, ResistAid supplementation led to a significant decrease in the ratio of fecal Firmicutes to Bacteriodetes (F/B) driven by an increase in Bacteriodetes and a decrease in Firmicutes. Moreover, the relative abundance of Bifidobacterium tended to increase with ResistAid supplementation. Additionally, ResistAid significantly decreased α-diversity of fecal microbiome. Predicted functional abundances based on 16s rRNA sequences showed that ResistAid supplementation increased gene abundance of the gut microbiome for α-l-rhamnosidase, β-fructosidase and levanase, as well as tricarboxylic acid and vitamin B6 biosynthesis pathways. Fecal isovaleric, valeric, and hexanoic acid contents were significantly lower following ResistAid consumption. There were no statistically significant changes in bowel habit, stool consistency, gastrointestinal tolerance symptoms, chemistry profile, metabolic panel, and vitals, suggesting that consumption of 15 g of ResistAid over 6 weeks is safe. These results demonstrate that the gut microbiome composition and predicted functions can be modulated by ResistAid consumption, suggesting perhaps a mechanistic explanation on its reported benefits in metabolic parameters and the immune system.

Molecular and Physiological Adaptations to Low Temperature in Thioalkalivibrio Strains Isolated from Soda Lakes with Different Temperature Regimes.

ABSTRACTThe genus Thioalkalivibrio comprises sulfur-oxidizing bacteria thriving in soda lakes at high pH and salinity. Depending on the geographical location and the season, these lakes can strongly vary in temperature. To obtain a comprehensive understanding of the molecular and physiological adaptations to low temperature, we compared the responses of two Thioalkalivibrio strains to low (10°C) and high (30°C) temperatures. For this, the strains were grown under controlled conditions in chemostats and analyzed for their gene expression (RNA sequencing [RNA-Seq]), membrane lipid composition, and glycine betaine content. The strain Thioalkalivibrio versutus AL2T originated from a soda lake in southeast Siberia that is exposed to strong seasonal temperature differences, including freezing winters, whereas Thioalkalivibrio nitratis ALJ2 was isolated from an East African Rift Valley soda lake with a constant warm temperature the year round. The strain AL2T grew faster than ALJ2 at 10°C, likely due to its 3-fold-higher concentration of the osmolyte glycine betaine. Moreover, significant changes in the membrane lipid composition were observed for both strains, leading to an increase in their unsaturated fatty acid content via the Fab pathway to avoid membrane stiffness. Genes for the transcriptional and translational machinery, as well as for counteracting cold-induced hampering of nucleotides and proteins, were upregulated. Oxidative stress was reduced by induction of vitamin B12 biosynthesis genes, and growth at 10°C provoked downregulation of genes involved in the second half of the sulfur oxidation pathway. Genes for intracellular signal transduction were differentially expressed, and interestingly, AL2T upregulated flagellin expression, whereas ALJ2 downregulated it.IMPORTANCE In addition to their haloalkaline conditions, soda lakes can also harbor a variety of other extreme parameters, to which their microbial communities need to adapt. However, for most of these supplementary stressors, it is not well known yet how haloalkaliphiles adapt and resist. Here, we studied the strategy for adaptation to low temperature in the haloalkaliphilic genus Thioalkalivibrio by using two strains isolated from soda lakes with different temperature regimes. Even though the strains showed a strong difference in growth rate at 10°C, they exhibited similar molecular and physiological adaptation responses. We hypothesize that they take advantage of resistance mechanisms against other stressors commonly found in soda lakes, which are therefore maintained in the bacteria living in the absence of low-temperature pressure. A major difference, however, was detected for their glycine betaine content at 10°C, highlighting the power of this osmolyte to also act as a key compound in cryoprotection.Author Video: An author video summary of this article is available.

Microbial and Genetic Resources for Cobalamin (Vitamin B12) Biosynthesis: From Ecosystems to Industrial Biotechnology.

Many microbial producers of coenzyme B12 family cofactors together with their metabolically interdependent pathways are comprehensively studied and successfully used both in natural ecosystems dominated by auxotrophs, including bacteria and mammals, and in the safe industrial production of vitamin B12. Metabolic reconstruction for genomic and metagenomic data and functional genomics continue to mine the microbial and genetic resources for biosynthesis of the vital vitamin B12. Availability of metabolic engineering techniques and usage of affordable and renewable sources allowed improving bioprocess of vitamins, providing a positive impact on both economics and environment. The commercial production of vitamin B12 is mainly achieved through the use of the two major industrial strains, Propionobacterium shermanii and Pseudomonas denitrificans, that involves about 30 enzymatic steps in the biosynthesis of cobalamin and completely replaces chemical synthesis. However, there are still unresolved issues in cobalamin biosynthesis that need to be elucidated for future bioprocess improvements. In the present work, we review the current state of development and challenges for cobalamin (vitamin B12) biosynthesis, describing the major and novel prospective strains, and the studies of environmental factors and genetic tools effecting on the fermentation process are reported.

Effect of arabinogalactan on the gut microbiome: A randomized, double-blind, placebo-controlled, crossover trial in healthy adults

ObjectivePromising evidence suggests beneficial health effects of arabinogalactan, but little is known about the effect of this non-digestible carbohydrate on the gut microbiota, a crucial mediator of human health. The objective of this study was to investigate the effect of an arabinogalactan product (ResistAid) on the fecal microbiome and short-chain fatty acids and gastrointestinal tolerance in healthy adults in a randomized, double-blind, crossover trial. MethodsThirty adults were randomly assigned to consume 15 g/d maltodextrin (control) or ResistAid for 6 wk. ResultsAt week 6, compared to placebo, ResistAid supplementation led to a significant decrease in the ratio of fecal Firmicutes to Bacteroidetes, driven by an increase in Bacteroidetes and a decrease in Firmicutes. Moreover, the relative abundance of Bifidobacterium tended to increase with ResistAid supplementation. Additionally, ResistAid significantly decreased the α-diversity of the fecal microbiome. Predicted functional abundances based on 16S rRNA sequences showed that ResistAid supplementation increased the gene abundance of the gut microbiome for α-l-rhamnosidase, β-fructosidase, and levanase, as well as tricarboxylic acid and vitamin B6 biosynthesis pathways. Fecal isovaleric, valeric, and hexanoic acids were significantly lower after ResistAid consumption. There were no statistically significant changes in bowel habit, stool consistency, gastrointestinal tolerance symptoms, chemistry profile, metabolic panel, or vitals, suggesting that consumption of 15 g daily ResistAid over 6 wk is safe. ConclusionThese results demonstrate that the gut microbiome composition and predicted functions can be modulated by ResistAid consumption, perhaps suggesting a mechanistic explanation on its reported benefits in metabolic parameters and the immune system.

197 Investigation of vitamin B12 sharing within the skin microbiome

The skin microbiome has a key role in supporting necessary functions that promote skin health, including protection from pathogens and immune homeostasis. To more fully understand microbiome function in the context of skin health, it is necessary to characterize the fundamental biological and ecological interactions that occur within the skin microbiome. Using comparative genomics across 71 species of the Corynebacterium genus, a dominant skin taxon, we identified that skin-associated species encode for de novo biosynthesis of cobamides (vitamin B12), a cofactor essential for enzymatic reactions across all domains of life. Because a much larger fraction of bacteria require B12 than are able to synthesize it, we hypothesize that B12 sharing occurs within the skin microbiome and that these interactions mediate microbial community composition. To assess B12 biosynthesis and use by skin microbiota, 906 skin metagenomes were queried for conserved sequences of B12 biosynthetic and dependent enzymes and B12 regulatory elements. Reads mapping to B12 dependent and regulatory sequences were classified to 1462 species, while de novo cobamide biosynthesis was restricted to 23 species. This suggests B12 is utilized throughout the phylogenetic diversity of the skin microbiome while only produced by a small number of taxa. In addition, we analyzed published relative abundance data from 594 skin metagenomes and found that in skin sites such as the occiput and inguinal crease, increased microbiome diversity was associated with a higher abundance of B12-producing Corynebacteria (rho=0.36-0.79, p<0.05). Loss of diversity is associated with dermatological disease, therefore our results suggest that B12 sharing plays a role in mediating microbiome structure and ultimately may promote skin health by promoting diversity. In the future, we will test this with ex vivo live human skin and synthetic skin microbial communities consisting of B12 producers and users to examine the effects on community dynamics when B12 biosynthesis is depleted.

Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis while related proteins prefer ZnII

Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.

Chris Abell 1957–2020

Chris Abell, FRS, FMedSci died suddenly on 26 October 2020. He was Professor of Biological Chemistry and Pro-Vice Chancellor (PVC) for Research at the University of Cambridge.Chris was the son of a farmer in Yorkshire. He studied at Selby Grammar School and gained a place to read Natural Sciences at St John’s College, Cambridge, starting in October 1976. I (FJL) met him within days of his arrival as I was his first supervisor (tutor) in chemistry (and he was my first ever supervisee). Chris graduated in chemistry in 1979 and then stayed at Cambridge for his PhD with Jim Staunton, studying the biosynthesis of polyketide natural products. He pioneered the use of dual-isotope labelling using both deuterium and 13C. From the start he displayed the energy and enthusiasm for science that would characterize his whole career. During his PhD, Chris met his wife, Katherine, who was doing her PhD in the same lab with Tony Kirby. He went on to do post-doctoral research with Professor David Cane at Brown University, Providence, Rhode Island, studying biosynthesis of terpenes, including the antibiotic pentalenolactone.In 1984, Chris returned to Cambridge to take up a lectureship and brought the newly developed techniques of gene-cloning into the Chemistry Department. In his own independent research, he studied various enzyme mechanisms, including enzymes of shikimic acid and coenzyme A biosynthesis. He also assisted Professor Alan Battersby in his research, which led to these genetic techniques being applied to the biosynthesis of tetrapyrroles. Without this, Alan’s subsequent discoveries in the biosynthesis of porphyrins and vitamin B12 would not have been possible.Gradually, however, Chris’s interest shifted from the study of enzyme mechanisms to the development of enzyme inhibitors, initially to aid the mechanistic studies but then for the purpose of drug discovery. He collaborated with Professor Sir Tom Blundell in biochemistry and together they promoted fragment-based drug-discovery. This proved to be much more efficient than the high-throughput screening of drug-like molecules, which most pharmaceutical companies were pursuing at that time. On the back of these results, Chris, Tom and Harren Jhoti founded Astex Pharmaceuticals in 1999, which continues to be very successful, with three drugs already on the market. This technique has transformed pharmaceutical research worldwide.Another major area of research was on microdroplets. These picolitre aqueous droplets can be produced in their millions, allowing massively high-throughput testing of different conditions for enzyme assays or growth of micro-organisms. Two companies were spun out from this research: Sphere Fluidics develop instruments that use microdroplets, whereas Aqdot produces microcapsules that are involved in molecule release and capture.In 2013 Chris began to move into University administration, becoming Director of the Office of Postdoctoral Affairs. In 2016 he became PVC for Research, in which role he spearheaded the University’s preparations for Brexit and the upcoming Research Excellence Framework (REF) assessment. He was also a major influence in setting up the Cambridge Zero initiative and the University’s COVID testing facilities.Through his time as PVC, research on fragment-based drug discovery continued in his research group, targeting enzymes associated with diseases such as tuberculosis, cystic fibrosis and cancer. The work on microdroplets is also on-going. Chris was elected FMedSci in 2012, FRS in 2016 and most recently won the 2020 RS.C. Interdisciplinary Prize. He has published over 350 papers and mentored over 150 MPhil, PhD and postdoctoral researchers during his career. They all found him incredibly kind, supportive and keen to enable them to do the best they can.

Early-life exposure to toxic and nutrient elements in relation to the infant microbiome as a possible mediator or modifier of autism-related social behaviors

Background/Aim: In our previous analysis in the New Hampshire Birth Cohort Study, we identified associations between nutrient-toxic element mixtures and the early-life microbiome. The role of nutrient toxic mixtures and microbiome changes are being examined for their associations with autism-related social behaviors at age three years. However, there is limited information on the extent to which the microbiome mediates or modifies the association between a broader range environmental elements and childhood behaviors.Methods: In a subset of infants from a prospective birth cohort, six-week-old toenail clippings were analyzed for eleven elements (manganese, iron, nickel, copper, zinc, arsenic, selenium, cadmium, tin, mercury, and lead) using inductively coupled plasma mass spectrometry. Stool samples were collected from diapers at six weeks and one year of age, metagenomic sequencing was performed, and taxa/gene functions were inferred following established pipelines. At three years, parents completed the Social Responsiveness Scale, 2nd Edition (SRS-2), which assesses the frequency of autism-related social behaviors (higher scores indicate worse behavior). Covariates were determined a priori, and a complete case analysis was conducted.Results: Both nutrient and toxic elements were associated with microbial taxa in six-week-old and one-year-old stools, but none of these taxa was also associated with social behaviors. Arsenic and tin were associated with decreased relative abundance of vitamin B6 biosynthesis genes (PWY0-845) in one-year-old stools, increased abundance of which was found to be negatively associated with SRS-2 total T-scores. In contrast, lead was associated with increased relative abundance of PWY0-845 in six-week-old stools, which was also found to be negatively associated with SRS-2 total T-scores. Formal mediation/modification analyses based on these preliminary findings is underway.Conclusions: Investigation of the potential mediating or modifying role of microbiome may help to elucidate the mechanisms by which environmental exposures influence autism-related social behaviors.