Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum.

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualized by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities.

Vitamin B12 Auxotrophy in Isolates from the Deep Subsurface of the Iberian Pyrite Belt.

Vitamin B12 is an enzymatic cofactor that is essential for both eukaryotes and prokaryotes. The development of life in extreme environments depends on cofactors such as vitamin B12 as well. The genomes of twelve microorganisms isolated from the deep subsurface of the Iberian Pyrite Belt have been analyzed in search of enzymatic activities that require vitamin B12 or are involved in its synthesis and import. Results have revealed that vitamin B12 is needed by these microorganisms for several essential enzymes such as ribonucleotide reductase, methionine synthase and epoxyqueosine reductase. Isolate Desulfosporosinus sp. DEEP is the only analyzed genome that holds a set core of proteins that could lead to the production of vitamin B12. The rest are dependent on obtaining it from the subsurface oligotrophic environment in which they grow. Sought proteins involved in the import of vitamin B12 are not widespread in the sample. The dependence found in the genomes of these microorganisms is supported by the production of vitamin B12 by microorganisms such as Desulfosporosinus sp. DEEP, showing that the operation of deep subsurface biogeochemical cycles is dependent on cofactors such as vitamin B12.

HCFC1 variants in the proteolysis domain are associated with X-linked idiopathic partial epilepsy: Exploring the underlying mechanism.

BACKGROUND: HCFC1 encodes transcriptional co-regulator HCF-1, which undergoes an unusual proteolytic maturation at a centrally located proteolysis domain. HCFC1 variants were associated with X-linked cobalamin metabolism disorders and mental retardation-3. This study aimed to explore the role of HCFC1 variants in common epilepsy and the mechanism underlying phenotype heterogeneity. METHODS: Whole-exome sequencing was performed in a cohort of 313 patients with idiopathic partial (focal) epilepsy. Functional studies determined the effects of the variants on the proteolytic maturation of HCF-1, cell proliferation and MMACHC expression. The role of HCFC1 variants in partial epilepsy was validated in another cohort from multiple centers. RESULTS: We identified seven hemizygous HCFC1 variants in 11 cases and confirmed the finding in the validation cohort with additional 13 cases and six more hemizygous variants. All patients showed partial epilepsies with favorable outcome. None of them had cobalamin disorders. Functional studies demonstrated that the variants in the proteolysis domain impaired the maturation by disrupting the cleavage process with loss of inhibition of cell growth but did not affect MMACHC expression that was associated with cobalamin disorder. The degree of functional impairment was correlated with the severity of phenotype. Further analysis demonstrated that variants within the proteolysis domain were associated with common and mild partial epilepsy, whereas those in the kelch domain were associated with cobalamin disorder featured by severe and even fatal epileptic encephalopathy, and those in the basic and acidic domains were associated with mainly intellectual disability. CONCLUSION: HCFC1 is potentially a candidate gene for common partial epilepsy with distinct underlying mechanism of proteolysis dysfunction. The HCF-1 domains played distinct functional roles and were associated with different clinical phenotypes, suggesting a sub-molecular effect. The distinct difference between cobalamin disorders and idiopathic partial epilepsy in phenotype and pathogenic mechanism, implied a clinical significance in early diagnosis and management.

Abnormal chondrocyte development in a zebrafish model of cblC syndrome restored by an MMACHC cobalamin binding mutant.

Variants in the MMACHC gene cause combined methylmalonic acidemia and homocystinuria cblC type, the most common inborn error of intracellular cobalamin (vitamin B12) metabolism. cblC is associated with neurodevelopmental, hematological, ocular, and biochemical abnormalities. In a subset of patients, mild craniofacial dysmorphia has also been described. Mouse models of Mmachc deletion are embryonic lethal but cause severe craniofacial phenotypes such as facial clefts. MMACHC encodes an enzyme required for cobalamin processing and variants in this gene result in the accumulation of two metabolites: methylmalonic acid (MMA) and homocysteine (HC). Interestingly, other inborn errors of cobalamin metabolism, such as cblX syndrome, are associated with mild facial phenotypes. However, the presence and severity of MMA and HC accumulation in cblX syndrome is not consistent with the presence or absence of facial phenotypes. Thus, the mechanisms by which mutations in MMACHC cause craniofacial defects are yet to be completely elucidated. Here we have characterized the craniofacial phenotypes in a zebrafish model of cblC (hg13) and performed restoration experiments with either a wildtype or a cobalamin binding deficient MMACHC protein. Homozygous mutants did not display gross morphological defects in facial development but did have abnormal chondrocyte nuclear organization and an increase in the average number of neighboring cell contacts, both phenotypes were fully penetrant. Abnormal chondrocyte nuclear organization was not associated with defects in the localization of neural crest specific markers, sox10 (RFP transgene) or barx1. Both nuclear angles and the number of neighboring cell contacts were fully restored by wildtype MMACHC and a cobalamin binding deficient variant of the MMACHC protein. Collectively, these data suggest that mutation of MMACHC causes mild to moderate craniofacial phenotypes that are independent of cobalamin binding.

Identification of vitamin B12 producing bacteria based on the presence of bluB/cobT2 homologues.

OBJECTIVES: The objective of the study was to develop a strategy for the identification of new vitamin B12-producing species and to characterize their production capability using a fast and sensitive LC-MS/MS method developed in this study. RESULTS: Searching for homologues of the bluB/cobT2 fusion gene known to be responsible for the production of the active vitamin B12 form in P. freudenreichii was shown to be a successful strategy for the identification of new vitamin B12-producing strains. The analysis of the identified strains via LC-MS/MS showed the ability of Terrabacter sp. DSM102553, Yimella lutea DSM19828 and Calidifontibacter indicus DSM22967 to produce the active form of vitamin B12. Further analysis of vitamin B12 production capability of Terrabacter sp. DSM102553 in M9 minimal medium and peptone-based media revealed that the highest yield of 2.65 µg of vitamin B12 per g dry cell weight was obtained in M9 medium. CONCLUSIONS: The proposed strategy enabled identification of Terrabacter sp. DSM102553, whose relatively high yields obtained in the minimal medium open new perspectives for the possible application of the strain for biotechnological vitamin B12 production.

Altered vitamin B12 metabolism in the central nervous system is associated with the modification of ribosomal gene expression: new insights from comparative RNA dataset analysis.

Recent studies have confirmed the direct role of vitamin B12 (VitB12) in the central nervous system (CNS) homeostasis; nevertheless, the detailed mechanisms are poorly understood. By analyzing RNA-Seq and microarray datasets obtained from databanks, this study aims to identify possible basic mechanisms, related to the brain, involved in altering the gene expression under VitB12 deficiency mimicking conditions. The database inquiry returned datasets generated from distinctly heterogeneous experimental sets and considering the quality and relevance requirements, two datasets from mouse and one from rat models were selected. The analyses of individual datasets highlighted a change in ribosomal gene expression in VitB12 deficiency mimicking conditions within each system. Specifically, a divergent regulation was observed depending on the animal model: mice showed a down regulation of the ribosomal gene expression, while rats an upregulation. Interestingly, E2f1 was significantly upregulated under VitB12 deficiency mimicking conditions in the animal models, with a greater upregulation in rats. The rat model also revealed putative E2F1 Transcription Factor Binding Sites (TFBSs) in the promoter of the differently regulated genes involved in ribosomal gene expression. This suggested the possibility that E2F1, being greater expressed in rats, could activate the ribosomal genes having E2F1 TFBSs, thus giving a plausible explication to the divergent regulation observed in animal models. Despite the great diversity of the experimental sets used to generate the datasets considered, a common alteration of the ribosomes exists, thereby indicating a possible basic and conserved response to VitB12 deficiency. Moreover, these findings could provide new insights on E2F1 and its association with CNS homeostasis and VitB12 deficiency.

[Genetic variant analysis and prenatal diagnosis for Chinese pedigrees affected with cblC methylmalonic acidemia].

OBJECTIVE: To detect variants of the MMACHC gene among 110 ethnic Han Chinese pedigrees affected with metabolic deficiency methylmalonic acidemia (MMA) of cobalamin C (cblC). METHODS: Peripheral blood samples were collected from the probands and their parents. Following DNA extraction, the coding regions of the MMACHC gene were subjected to PCR amplification, Sanger sequencing and quantitative PCR assaying. For 48 pedigrees, chorionic villus samples were taken for prenatal genetic diagnosis. RESULTS: Thirty five types of variants were detected among the 110 pedigrees, which included missense, nonsense, frameshifting, splicing variants and exonic deletions. Most variants have occurred in exons 4 (73.18%). The detection rate for c.609G>A (p.Trp203Ter) variant was the highest (33.64%), followed by c.658_660delAAG (12.27%), c.567dupT (9.09%) and c.80A>G (6.82%). Two variants, namely c.57_58insT (p.Gly20Trpfs*14) and c.505_506delAT (p.Ile169Argfs*12), were unreported previously and both were of frameshifting types. For the 48 pedigrees undergoing prenatal diagnosis, 14 fetuses were found to be normal, 24 have carried heterozygous variants, the remaining 10 have carried compound heterozygous or homozygous variants. CONCLUSION: The discovery of the two novel variants has expanded the spectrum of the MMACHC gene variants among ethnic Han population. Above finding has provide a basis for the prenatal diagnosis and genetic counseling for the affected pedigrees.

HIF1 and DROSHA are involved in MMACHC repression in hypoxia.

The MMACHC gene encodes for an enzyme involved in intracellular vitamin B12 metabolism, and autosomal recessive defects in MMACHC represent the most common disorder of intracellular vitamin B12 metabolism. Recent studies have identified increased levels of reactive oxygen species in cells and tissues with MMACHC dysfunction, suggesting a role for oxidative stress in disease. To investigate the link between oxidative stress and MMACHC, we exposed mice as well as human and mouse cells to hypoxia, and found significant repression of MMACHC in all investigated tissues (retina, eyecup, liver, kidney) and cell lines (HeLa, ARPE-19, human and mouse fibroblasts, 661W). Furthermore, in HeLa cells, we found transcriptional repression already at 5% oxygen, which was stable during prolonged hypoxia up to 5 days, and a return of MMACHC transcripts to normal levels only 24 h after reoxygenation. This hypoxia-induced downregulation of MMACHC was not due to altered function of the known MMACHC controlling transcription factor complex HCFC1/THAP11/ZNF143. Using in vitro RNA interference against hypoxia-induced transcription factors (HIF1A, HIF2A and REST) as well as the microRNA transcription machinery (DROSHA), we observed release of hypoxia-dependent downregulation of MMACHC expression by HIF1A and DROSHA knockdowns, whose combined effect was additive. Together, these results strongly indicate that MMACHC is a hypoxia-regulated gene whose downregulation appears to be partially mediated through both hypoxia-induced transcription factor and microRNA machinery. These findings suggest that oxidative stress could impair vitamin B12 metabolism by repression of MMACHC in healthy as well as in diseased individuals.

Exploring the onset of B12 -based mutualisms using a recently evolved Chlamydomonas auxotroph and B12 -producing bacteria.

Cobalamin (vitamin B12 ) is a cofactor for essential metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonize the algal phycosphere, forming stable communities that gain preferential access to photosynthate and in return provide compounds such as B12 . Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B12 -dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co-culture was slower than the B12 -independent wild-type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co-cultures. We hypothesize that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12 -dependent algae.

Cobalamin cbiP mutant shows decreased tolerance to low temperature and copper stress in Listeria monocytogenes.

BACKGROUND: Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans. This pathogen activates multiple regulatory mechanisms in response to stress, and cobalamin biosynthesis might have a potential role in bacterial protection. Low temperature is a strategy used in the food industry to control bacteria proliferation; however, L. monocytogenes can grow in cold temperatures and overcome different stress conditions. In this study we selected L. monocytogenes List2-2, a strain with high tolerance to the combination of low temperature + copper, to understand whether the cobalamin biosynthesis pathway is part of the tolerance mechanism to this stress condition. For this, we characterized the transcription level of three cobalamin biosynthesis-related genes (cbiP, cbiB, and cysG) and the eutV gene, a transcriptional regulator encoding gene involved in ethanolamine metabolism, in L. monocytogenes strain List2-2 growing simultaneously under two environmental stressors: low temperature (8 °C) + copper (0.5 mM of CuSO4 × 5H2O). In addition, the gene cbiP, which encodes an essential cobyric acid synthase required in the cobalamin pathway, was deleted by homologous recombination to evaluate the impact of this gene in L. monocytogenes tolerance to a low temperature (8 °C) + different copper concentrations. RESULTS: By analyzing the KEGG pathway database, twenty-two genes were involved in the cobalamin biosynthesis pathway in L. monocytogenes List2-2. The expression of genes cbiP, cbiB, and cysG, and eutV increased 6 h after the exposure to low temperature + copper. The cobalamin cbiP mutant strain List2-2ΔcbiP showed less tolerance to low temperature + copper (3 mM) than the wild-type L. monocytogenes List2-2. The addition of cyanocobalamin (5 nM) to the medium reverted the phenotype observed in List2-2ΔcbiP. CONCLUSION: These results indicate that cobalamin biosynthesis is necessary for L. monocytogenes growth under stress and that the cbiP gene may play a role in the survival and growth of L. monocytogenes List2-2 at low temperature + copper.

Decreased cortical Nrf2 gene expression in autism and its relationship to thiol and cobalamin status.

Nuclear factor erythroid 2-related factor 2 (Nrf2) promotes expression of a large number of antioxidant genes and multiple studies have described oxidative stress and impaired methylation in autism spectrum disorder (ASD), including decreased brain levels of methylcobalamin(III) (MeCbl). Here we report decreased expression of the Nrf2 gene (NFE2L2) in frontal cortex of ASD subjects, as well as differences in other genes involved in redox homeostasis. In pooled control and ASD correlation analyses, hydroxocobalamin(III) (OHCbl) was inversely correlated with NFE2L2 expression, while MeCbl and total cobalamin abundance were positively correlated with NFE2L2 expression. Levels of methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and cystathionine were positively correlated with NFE2L2 expression, while homocysteine (HCY) was negatively correlated. The relationship between Nrf2 activity and cobalamin was further supported by a bioinformatics-based comparison of cobalamin levels in different tissues with expression of a panel of 40 Nrf2-regulated genes, which yielded a strong correlation. Lastly, Nrf2-regulated gene expression was also correlated with expression of intracellular cobalamin trafficking and processing genes, such as MMADHC and MTRR. These findings highlight a previously unrecognized relationship between the antioxidant-promoting role of Nrf2 and cobalamin status, which is dysfunctional in ASD.

Causes and consequences of impaired methionine synthase activity in acquired and inherited disorders of vitamin B12 metabolism.

Methyl-Cobalamin (Cbl) derives from dietary vitamin B12 and acts as a cofactor of methionine synthase (MS) in mammals. MS encoded by MTR catalyzes the remethylation of homocysteine to generate methionine and tetrahydrofolate, which fuel methionine and cytoplasmic folate cycles, respectively. Methionine is the precursor of S-adenosyl methionine (SAM), the universal methyl donor of transmethylation reactions. Impaired MS activity results from inadequate dietary intake or malabsorption of B12 and inborn errors of Cbl metabolism (IECM). The mechanisms at the origin of the high variability of clinical presentation of impaired MS activity are classically considered as the consequence of the disruption of the folate cycle and related synthesis of purines and pyrimidines and the decreased synthesis of endogenous methionine and SAM. For one decade, data on cellular and animal models of B12 deficiency and IECM have highlighted other key pathomechanisms, including altered interactome of MS with methionine synthase reductase, MMACHC, and MMADHC, endoplasmic reticulum stress, altered cell signaling, and genomic/epigenomic dysregulations. Decreased MS activity increases catalytic protein phosphatase 2A (PP2A) and produces imbalanced phosphorylation/methylation of nucleocytoplasmic RNA binding proteins, including ELAVL1/HuR protein, with subsequent nuclear sequestration of mRNAs and dramatic alteration of gene expression, including SIRT1. Decreased SAM and SIRT1 activity induce ER stress through impaired SIRT1-deacetylation of HSF1 and hypomethylation/hyperacetylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), which deactivate nuclear receptors and lead to impaired energy metabolism and neuroplasticity. The reversibility of these pathomechanisms by SIRT1 agonists opens promising perspectives in the treatment of IECM outcomes resistant to conventional supplementation therapies.

Cobalamin cbiP mutant shows decreased tolerance to low temperature and copper stress in Listeria monocytogenes

BACKGROUND: Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans. This pathogen activates multiple regulatory mechanisms in response to stress, and cobalamin biosynthesis might have a potential role in bacterial protection. Low temperature is a strategy used in the food industry to control bacteria proliferation; however, L. monocytogenes can grow in cold temperatures and overcome different stress conditions. In this study we selected L. monocytogenes List2-2, a strain with high tolerance to the combination of low temperature +copper, to understand whether the cobalamin biosynthesis pathway is part of the tolerance mechanism to this stress condition. For this, we characterized the transcription level of three cobalamin biosynthesis related genes ( cbiP , cbiB, and cysG ) and the eutV gene, a transcriptional regulator encoding gene involved in ethanolamine metabolism, in L. monocytogenes strain List2-2 growing simultaneously under two environmental stressors: low temperature (8 °C) +copper (0.5 mM of CuSO4 ×5H2O). In addition, the gene cbiP , which encodes an essential cobyric acid synthase required in the cobalamin pathway, was deleted by homologous recombination to evaluate the impact of this gene in L. monocytogenes tolerance to a low temperature (8 °C) +different copper concentrations. RESULTS: By analyzing the KEGG pathway database, twenty-two genes were involved in the cobalamin biosynthesis pathway in L. monocytogenes List2-2. The expression of genes cbiP , cbiB, and cysG, and eutV increased 6 h after the exposure to low temperature +copper. The cobalamin cbiP mutant strain List2-2Δ cbiP showed less tolerance to low temperature +copper (3 mM) than the wild type L. monocytogenes List2-2. The addition of cyanocobalamin (5 nM) to the medium reverted the phenotype observed in List2-2Δ cbiP . CONCLUSION: These results indicate that cobalamin biosynthesis is necessary for L. monocytogenes growth under stress and that the cbiP gene may play a role in the survival and growth of L. monocytogenes List2-2 at low temperature +copper.

Prenatal diagnosis of combined methylmalonic acidemia and homocystinuria cobalamin C type using clinical exome sequencing and targeted gene analysis.

BACKGROUND: Combined methylmalonic acidemia and homocystinuria is a rare inherited disorder of intracellular cobalamin metabolism caused by biallelic variants in one of the following genes: MMACHC (cblC), MMADHC (cblD), LMBRD1 (cblF), ABCD4 (cblJ), THAP11 (cblX-like), and ZNF143 (cblX-like), or a hemizygous variant in HCFC1 (cblX). Prenatal diagnosis of combined methylmalonic acidemia with homocystinuria is crucial for high-risk couples since the disorder can be life-threatening for offspring. We would like to describe two infant deaths both of which are likely attributable to cblC despite not having a genetic confirmation, and subsequent pregnancy and prenatal genetic testing. METHODS: Parental clinical exome sequencing and targeted Sanger sequencing of MMACHC gene in amniotic fluid was performed to check the carrier status of the fetus. RESULTS: Parental clinical exome sequencing revealed a heterozygous pathogenic variant [NM_015506.2:c.217C>T (p.Arg73*)] in the MMACHC gene of the mother and [NM_015506.2:c.609G>A (p.Trp203*)] in the MMACHC gene of the father. Targeted Sanger sequencing of MMACHC gene in amniotic fluid revealed that the fetus carried only one nonsense variant [NM_015506.2:c.609G>A (p.Trp203*)], which was inherited from the father. The mother delivered a healthy baby and the neonate did not show any symptoms or signs of combined methylmalonic acidemia and homocystinuria after birth. CONCLUSION: We present a case of prenatal diagnosis with parental exome sequencing, which successfully diagnosed the carrier status of the fetus and parents in a combined methylmalonic acidemia and homocystinuria family.

Investigating the causality of metabolites involved in one-carbon metabolism with the risk and age at onset of Parkinson's disease: A two-sample mendelian randomization study.

With the aging population and increasing life expectancy, Parkinson's disease (PD), a neurological disorder rapidly increasing in morbidity and mortality, is causing a huge burden on society and the economy. Several studies have suggested that one-carbon metabolites, including homocysteine, vitamin B6, vitamin B12 and folate acid, are associated with PD risk. However, the results remain inconsistent and controversial. Thus, we performed a two-sample Mendelian randomization (MR) study to detect the causality between one-carbon metabolites and PD susceptibility as well as age at PD onset. We collected several genetic variants as instrumental variables from large genome-wide association studies of one-carbon metabolites (homocysteine: N = 14, vitamin B6: N = 1, vitamin B12: N = 10, folate acid: N = 2). We then conducted MR analyses using the inverse variance-weighted (IVW) approach and additional MR-Egger regression, weighted median and MR-pleiotropy residual sum and outlier (MR-PRESSO) methods to further test causality. The results showed no causal association between circulating homocysteine levels and PD risk (p = 0.868) or age at PD onset (p = 0.222) with the IVW method. Meanwhile, similar results were obtained by three complementary analyses. In addition, we did not observe any evidence that the circulating levels of vitamin B6, vitamin B12 and folate acid affected the risk of PD or age at onset of PD. Our findings implied that lowering homocysteine levels through vitamin B6, vitamin B12 or folate acid supplementation may not be clinically helpful in preventing PD or delaying the age at PD onset.

Molecular Pathways and Pigments Underlying the Colors of the Pearl Oyster Pinctada margaritifera var. cumingii (Linnaeus 1758).

The shell color of the Mollusca has attracted naturalists and collectors for hundreds of years, while the molecular pathways regulating pigment production and the pigments themselves remain poorly described. In this study, our aim was to identify the main pigments and their molecular pathways in the pearl oyster Pinctada margaritifera-the species displaying the broadest range of colors. Three inner shell colors were investigated-red, yellow, and green. To maximize phenotypic homogeneity, a controlled population approach combined with common garden conditioning was used. Comparative analysis of transcriptomes (RNA-seq) of P. margaritifera with different shell colors revealed the central role of the heme pathway, which is involved in the production of red (uroporphyrin and derivates), yellow (bilirubin), and green (biliverdin and cobalamin forms) pigments. In addition, the Raper-Mason, and purine metabolism pathways were shown to produce yellow pigments (pheomelanin and xanthine) and the black pigment eumelanin. The presence of these pigments in pigmented shell was validated by Raman spectroscopy. This method also highlighted that all the identified pathways and pigments are expressed ubiquitously and that the dominant color of the shell is due to the preferential expression of one pathway compared with another. These pathways could likely be extrapolated to many other organisms presenting broad chromatic variation.

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.

Genetically predicted circulating B vitamins in relation to digestive system cancers.

BACKGROUND: Folate, vitamin B6 and vitamin B12 have been associated with digestive system cancers. We conducted a two-sample Mendelian randomisation study to assess the causality of these associations. METHODS: Two, one and 14 independent single nucleotide polymorphisms associated with serum folate, vitamin B6 and vitamin B12 at the genome-wide significance threshold were selected as genetic instruments. Summary-level data for the associations of the vitamin-associated genetic variants with cancer were obtained from the UK Biobank study including 367,561 individuals and FinnGen consortium comprising up to 176,899 participants. RESULTS: Genetically predicted folate and vitamin B6 concentrations were not associated with overall cancer, overall digestive system cancer or oesophageal, gastric, colorectal or pancreatic cancer. Genetically predicted vitamin B12 concentrations were positively associated with overall digestive system cancer (ORSD, 1.12; 95% CI 1.04, 1.21, p = 0.003) and colorectal cancer (ORSD 1.16; 95% CI 1.06, 1.26, p = 0.001) in UK Biobank. Results for colorectal cancer were consistent in FinnGen and the combined ORSD was 1.16 (95% CI 1.08, 1.25, p < 0.001). There was no association of genetically predicted vitamin B12 with any other site-specific digestive system cancers or overall cancer. CONCLUSIONS: These results provide evidence to suggest that elevated serum vitamin B12 concentrations are associated with colorectal cancer.

Cobalamin J disease detected on newborn screening: Novel variant and normal neurodevelopmental course.

Cobalamin J disease (CblJ) is an ultra-rare autosomal recessive disorder of intracellular cobalamin metabolism associated with combined methylmalonic acidemia and homocystinuria. It is caused by pathogenic variants in ABCD4, which encodes an ATP-binding cassette (ABC) transporter that affects the lysosomal release of cobalamin (Cbl) into the cytoplasm. Only six cases of CblJ have been reported in the literature. Described clinical features include feeding difficulties, failure to thrive, hypotonia, seizures, developmental delay, and hematological abnormalities. Information on clinical outcomes is extremely limited, and no cases of presymptomatic diagnosis have been reported. We describe a now 17-month-old male with CblJ detected by newborn screening and confirmed by biochemical, molecular, and complementation studies. With early detection and initiation of treatment, this patient has remained asymptomatic with normal growth parameters and neurodevelopmental function. To the best of our knowledge, this report represents the first asymptomatic and neurotypical patient with CblJ.

Novel exon-skipping variant disrupting the basic domain of HCFC1 causes intellectual disability without metabolic abnormalities in both male and female patients.

HCFC1, a global transcriptional regulator, has been shown to associate with MMACHC expression. Pathogenic variants in HCFC1 cause X-linked combined methylmalonic acidemia and hyperhomocysteinemia, CblX type (MIM# 309541). Recent studies showed that certain variants in HCFC1 are associated with X-linked intellectual disability with mild or absent metabolic abnormalities. Here, we report five subjects (three males, two females) from the same family with a novel predicted loss of function HCFC1 variant. All five patients exhibit developmental delay or intellectual disability/learning difficulty and some dysmorphic features; findings were milder in the female as compared to male subjects. Biochemical studies in all patients did not show methylmalonic acidemia or hyperhomocysteinemia but revealed elevated vitamin B12 levels. Trio exome sequencing of the proband and his parents revealed a maternally inherited novel variant in HCFC1 designated as c.1781_1803 + 3del26insCA (NM_005334). Targeted testing confirmed the presence of the same variant in two half-siblings and maternal great uncle. In silico analysis showed that the variant is expected to reduce the quality of the splice donor site in intron 10 and causes abnormal splicing. Sequencing of proband's cDNA revealed exon 10 skipping. Further molecular studies in the two manifesting females revealed moderate and high skewing of X inactivation. Our results support previous observation that HCFC1 variants located outside the Kelch domain exhibit dissociation of the clinical and biochemical phenotype and cause milder or no metabolic changes. We also show that this novel variant can be associated with a phenotype in females, although with milder severity, but further studies are needed to understand the role of skewed X inactivation among females in this rare disorder. Our work expands the genotypes and phenotypes associated with HCFC1-related disorder.