One-carbon Transfer Reactions Research Articles

Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions.

Folate (vitamin B9) is involved in one-carbon transfer reactions and plays a significant role in nucleic acid synthesis and control of cellular proliferation, among other key cellular processes. It is now recognized that the role of folates in different stages of carcinogenesis is complex, and more research is needed to understand how folate reactions become dysregulated in cancers and the metabolic consequences that occur as a result. ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism expressed in many tissues, is ubiquitously downregulated in cancers and is not expressed in cancer cell lines. The RT4 cell line (derived from papillary bladder cancer) which expresses high levels of ALDH1L1 represents an exception, providing an opportunity to explore the metabolic consequences of the loss of this enzyme. We have downregulated this protein in RT4 cells (shRNA driven knockdown or CRISPR driven knockout) and compared metabolomes of ALDH1L1-expressing and -deficient cells to determine if metabolic changes linked to the loss of this enzyme might provide proliferative and/or survival advantages for cancer cells. In this study, cell extracts were analyzed using Ultra High Performance Liquid Chromatography High Resolution Mass Spectrometry (UHPLC-HR-MS). A total of 13,339 signals were identified or annotated using an in-house library and public databases. Supervised and unsupervised multivariate analysis revealed metabolic differences between RT4 cells and ALDH1L1-deficient clones. Glycine (8-fold decrease) and metabolites derived from S-adenosylmethionine utilizing pathways were significantly decreased in the ALDH1L1-deficient clones, compared with RT4 cells. Other changes linked to ALDH1L1 downregulation include decreased levels of amino acids, Krebs cycle intermediates, and ribose-5-phosphate, and increased nicotinic acid. While the ALDH1L1-catalyzed reaction is directly linked to glycine biosynthesis and methyl group flux, its overall effect on cellular metabolism extends beyond immediate metabolic pathways controlled by this enzyme.

First report on Vitamin B9 production including quantitative analysis of its vitamers in the yeast Scheffersomyces stipitis

BackgroundThe demand for naturally derived products is continuously growing. Nutraceuticals such as pre- and post-biotics, antioxidants and vitamins are prominent examples in this scenario, but many of them are mainly produced by chemical synthesis. The global folate market is expected to register a CAGR of 5.3% from 2019 to 2024 and reach USD 1.02 billion by the end of 2024. Vitamin B9, commonly known as folate, is an essential micronutrient for humans. Acting as a cofactor in one-carbon transfer reactions, it is involved in many biochemical pathways, among which the synthesis of nucleotides and amino acids. In addition to plants, many microorganisms can naturally produce it, and this can pave the way for establishing production processes. In this work, we explored the use of Scheffersomyces stipitis for the production of natural vitamin B9 by microbial fermentation as a sustainable alternative to chemical synthesis.ResultsGlucose and xylose are the main sugars released during the pretreatment and hydrolysis processes of several residual lignocellulosic biomasses (such as corn stover, wheat straw or bagasse). We optimized the growth conditions in minimal medium formulated with these sugars and investigated the key role of oxygenation and nitrogen source on folate production. Vitamin B9 production was first assessed in shake flasks and then in bioreactor, obtaining a folate production up to 3.7 ± 0.07 mg/L, which to date is the highest found in literature when considering wild type microorganisms. Moreover, the production of folate was almost entirely shifted toward reduced vitamers, which are those metabolically active for humans.ConclusionsFor the first time, the non-Saccharomyces yeast S. stipitis was used to produce folate. The results confirm its potential as a microbial cell factory for folate production, which can be also improved both by genetic engineering strategies and by fine-tuning the fermentation conditions and nutrient requirements.

Methotrexate recognition by the human reduced folate carrier SLC19A1.

Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during cell division1,2. Mammals lack de novo folate synthesis pathways and thus rely on folate uptake from the extracellular milieu3. The human reduced folate carrier (hRFC, also known as SLC19A1) is the major importer of folates into the cell1,3, as well as chemotherapeutic agents such as methotrexate4-6. As an anion exchanger, RFC couples the import of folates and antifolates to anion export across the cell membrane and it is a major determinant in methotrexate (antifolate) sensitivity, as genetic variants and its depletion result in drug resistance4-8. Despite its importance, the molecular basis of substrate specificity by hRFC remains unclear. Here we present cryo-electron microscopy structures of hRFC in the apo state and captured in complex with methotrexate. Combined with molecular dynamics simulations and functional experiments, our study uncovers key determinants of hRFC transport selectivity among folates and antifolate drugs while shedding light on important features of anion recognition by hRFC.

The iron–sulfur cluster assembly (ISC) protein Iba57 executes a tetrahydrofolate-independent function in mitochondrial [4Fe–4S] protein maturation

Mitochondria harbor the bacteria-inherited iron–sulfur cluster assembly (ISC) machinery to generate [2Fe–2S; iron–sulfur (Fe–S)] and [4Fe–4S] proteins. In yeast, assembly of [4Fe–4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe–4S] proteins. Both YgfZ and Iba57 are structurally related to an enzyme family catalyzing THF-dependent one-carbon transfer reactions including GcvT of the glycine cleavage system. On this basis, a universally conserved folate requirement in ISC-dependent [4Fe–4S] protein biogenesis was proposed. To test this idea for mitochondrial Iba57, we performed genetic and biochemical studies in Saccharomyces cerevisiae, and we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum. We provide three lines of evidence for the THF independence of the Iba57-catalyzed [4Fe–4S] protein assembly pathway. First, yeast mutants lacking folate show no defect in mitochondrial [4Fe–4S] protein maturation. Second, the 3D structure of Iba57 lacks many of the side-chain contacts to THF as defined in GcvT, and the THF-binding pocket is constricted. Third, mutations in conserved Iba57 residues that are essential for THF-dependent catalysis in GcvT do not impair Iba57 function in vivo, in contrast to an exchange of the invariant, surface-exposed cysteine residue. We conclude that mitochondrial Iba57, despite structural similarities to both YgfZ and THF-binding proteins, does not utilize folate for its function.

Targeting HOTAIRM1 ameliorates glioblastoma by disrupting mitochondrial oxidative phosphorylation and serine metabolism

SummarySerine hydroxymethyltransferase 2 (SHMT2), which catalyzes the conversion of serine to glycine and one-carbon transfer reactions in mitochondria, is significantly upregulated in glioblastoma (GBM). However, the mechanism by which the stability of SHMT2 gene expression is maintained to drive GBM tumorigenesis has not been clarified. Herein, through microarray screening, we identified that HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) modulates the SHMT2 level in various GBM cell lines. Serine catabolism and mitochondrial oxidative phosphorylation activities were decreased by HOTAIRM1 inhibition. Mechanistically, according to our mass spectrometry and eCLIP-seq results, HOTAIRM1 can bind to PTBP1 and IGF2BP2. Furthermore, HOTAIRM1 maintains the stability of SHMT2 by promoting the recognition of an m6A site and the interaction of PTBP1/IGF2BP2 with SHMT2 mRNA. The stability of HOTAIRM1 can also be enhanced and results in positive feedback regulation to support the progression of GBM. Thus, targeting HOTAIRM1 could be a promising metabolic therapy for GBM.

Folic acid supplementation during pregnancy alters behavior in male rat offspring: nitrative stress and neuroinflammatory implications.

Pregnancy diet can impact offspring's neurodevelopment, metabolism, redox homeostasis, and inflammatory status. In pregnancy, folate demand is increased due to the requirement for one-carbon transfer reactions. The present study was proposed to investigate the effect of folic acid supplementation throughout pregnancy on a battery of behavior tests (olfactory preference, motor activity, exploratory capacity, habituation, memory, anxiety- and depression-like behavior). Redox homeostasis and neuroinflammatory status in cerebral cortex were also investigated. After pregnancy confirmation, the pregnant rats were randomly divided into two groups, according to the diet: group 1, (control) standard diet (2mg/kg diet of folic acid) and group 2, supplemented diet with 4mg/kg diet of folic acid. Throughout the gestational period, the pregnant rats received experimental diets. Results show that the supplemented diet with 4mg/kg diet of folic acid throughout pregnancy impaired memory and motricity of the offspring when compared with control (standard diet). It was also observed an increase in anxiety- and depression-like behavior in this group. Nitrite levels increased in cerebral cortex of the offspring, when compared to control group. In contrast, iNOS expression and immunocontent were not altered. Moreover, we identify an increase in TNF-α, IL-1β, IL-6, IL-10, and MCP-1 gene expression in the cerebral cortex. In conclusion, our study showed that the supplemented diet with 4mg/kg diet of folic acid throughout pregnancy may cause behavioral and biochemical changes in the male offspringGraphical abstract After pregnancy confirmation, the pregnant rats were randomly divided into two groups, according to the diet: group 1, (control) standard diet (2mg/kg diet of folic acid) and group 2, supplemented diet with 4mg/kg diet of folic acid. Throughout the gestational period, the pregnant rats received experimental diets. Results show that folic acid supplementation did not impair the mother-pup relationship. We showed that supplemented diet with 4mg/kg diet of folic acid during pregnancy impairs memory and motricity of the offspring when compared with standard diet. It was also observed an increase in anxiety- and depression-like behavior in this group. Nitrative stress and neuroinflammation parameters were increased in the cerebral cortex of the offspring. ROS, reactive oxygen species.

Natural variation of folate content in cowpea (Vigna unguiculata) germplasm and its correlation with the expression of the GTP cyclohydrolase I coding gene

Cowpea (Vigna unguiculata) is one of the most important sources of vitamins, protein, and minerals for Africa, Latin America and Asia. In addition, grains are a rich source of folates. Folates are cofactors for the one-carbon transfer reactions needed for many biochemical processes. Their deficiency is associated with several diseases. In this study, we investigated variation in total folate levels in cowpea genotypes from Northern Brazil, Western Africa and Southwestern Asia and the correlation with VugchI gene expression. Results revealed that VugchI transcripts were observed in all studied developmental stages [10-, 15-, 20- and 25-days post anthesis (dpa)], reaching a maximum at 20 dpa. Folate content ranged from 780.7–177.2 μg/100 g of dried seeds, and the highest amounts were observed in traditional varieties cultivated in Nigeria and Northern Brazil. A statistically significant correlation was observed between the amounts of total folate and the expression of the VugchI gene at 20 dpa. Data showed a large variation in the total folate content that could be exploited by breeding programs to generate higher-folate varieties. It would be important to combine both high folate content and agriculturally relevant traits, which would lead farmers to adopt modern varieties enriched with folates.

Reduced Methionine Synthase (Mtr) Expression, a Model of Vitamin B12 Deficiency, Leads to Uracil Accumulation in Mitochondrial and Nuclear DNA

ObjectivesIntegrity of both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) is affected by nutritional and environmental factors. Folate and B12 are water-soluble B-vitamins that act as cofactors in folate-mediated one-carbon metabolism (FOCM), a series of one-carbon transfer reactions that support several essential cell processes including nucleotide biosynthesis. Impairments in FOCM, such as folate or vitamin B12 deficiency, can disturb thymidylate (dTMP, the “T” base in DNA) synthesis and lead to uracil misincorporation to DNA. Both folate and B12 deficiency as well as genetic polymorphisms affecting FOCM function are associated with increased uracil accumulation and DNA damage in nDNA. However, dTMP synthesis occurs in multiple cellular compartments (cytosol, nucleus, and mitochondria), and it is not well defined how FOCM impairments affects mtDNA integrity and nucleotide pool balance between compartments. MethodsCurrently, we are using in vivo and in vitro experiments to assess nucleotide synthesis and uracil accumulation in a functional model of vitamin B-12 deficiency with decreased expression of methionine synthase (Mtr), one of two B12-requiring enzymes. Tissues from Mtr+/+,+/– mice and Mtr+/+,+/- mouse-derived cells were exposed to either folate-replete or folate-deplete conditions to explore the combined effects of folate and B12 deficiency on nucleotide synthesis capacity and integrity of both nDNA and mtDNA. ResultsWe have developed and validated a novel real-time PCR-based assay to quantify uracil misincorporation into mtDNA. In mouse liver, we observed a significant increase of uracil in mtDNA with decreased Mtr expression as well as an interaction between Mtr genotype and folate status. Mouse embryonic fibroblasts (MEFs) demonstrated perturbed dTMP synthesis and increased uracil accumulation in nDNA with decreased Mtr expression. ConclusionsThese data suggest that functional B12 deficiency, as modeled by decreased Mtr expression, disrupts nucleotide synthesis in more than one cellular compartment and increases uracil accumulation not only in nDNA, but also in mtDNA. Reduced Mtr expression may cause a redistribution of folates in the cytoplasm which impacts mitochondrial folate levels, and subsequently mitochondrial dTMP synthesis. Funding SourcesN/A.

Folate and Inflammation – links between folate and features of inflammatory conditions

Folate serves as a cofactor for one-carbon (1C) transfer reactions. These reactions are involved in the synthesis of DNA nucleotides, the amino acid methionine, and in the regulation of homocysteine (Hcy) levels. Emerging evidence suggests that these reactions have roles in the development and maintenance of inflammatory responses, with optimal folate availability having key importance in preventing endothelial dysfunction and DNA instability. Low folate levels are commonly observed in chronic inflammatory diseases, indicating that inadequate folate may be involved in the pathogenesis of inflammatory conditions or that chronic inflammation increases folate requirements. These findings highlight folate interventions as a potential treatment in inflammatory disorders. However, current understanding of folate and its influence on inflammatory phenotypes is limited. Evidence indicates that the relationship between folate and inflammation is dependent on several factors, including the timing of intervention, dosage, and interaction with environment and genes. These factors require further investigation before recommendations for folate intake can be made for the prevention and treatment of inflammation. This review outlines the emerging role of folate in inflammation and key factors that may influence this relationship.

Abstract 4791: Impact of folate transport redundancies on the therapy with tumor-targeted and untargeted antifolates

Abstract Folates participate in one-carbon (C1) transfer reactions in normal and cancer cells. Antifolate therapeutics disrupt cytosolic C1 metabolism, and have long been a mainstay for the therapy of a number of cancers. There are three major folate transporter systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). RFC is broadly expressed in tissues and tumors and is characterized by a neutral pH optimum. PCFT has more limited tissue distribution but is widely expressed in human solid tumors and exhibits an acidic pH optimum. FRα is expressed in a subset of solid tumors including epithelial ovarian cancer (EOC) but shows limited expression in most normal tissues. We previously discovered novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl compounds typified by AGF94 which show selective cellular uptake by FRα and/or PCFT over RFC, in contrast to the classical antifolates methotrexate (MTX) and pemetrexed (PMX) which are transported by both PCFT and RFC, and to a lesser extent by FRα. We showed that AGF94 had enhanced antitumor activities by targeting cytosolic C1 metabolism to PCFT-expressing tumors but lacking RFC, reflecting contraction of cellular tetrahydrofolate pools. In EOC cells, AGF94 showed substantial in vitro inhibition of cell proliferation independent of FRα expression, as long as PCFT was present. We systematically explored the impact of folate transporter redundancies among PCFT, FRα and RFC in determining anti-tumor efficacy of novel tumor-targeted and classical antifolates. Towards this goal, we engineered cell line models from PCFT-, FR-, and RFC-null HeLa cells to express doxycycline (DOX)-inducible FRα or RFC. We used these models to constitutively express PCFT together with DOX-inducible FRα or RFC. These were characterized for transporter expression and function, as well as intracellular folate levels, with/without DOX induction. The relative contributions of RFC, PCFT and FRα to transport function were evaluated from pH 5.5 to 7.4, with radiolabeled MTX and AGF347 which targets both cytosolic and mitochondrial C1 metabolism. We assessed antiproliferative activities of classical antifolates (PMX, MTX, PT523) versus tumor-targeted compounds (e.g., AGF94, AGF102, AGF347) in these models with/without DOX. Our results establish that co-expression of the major folate transporters can have variable and surprisingly disparate and substantial impacts on anti-tumor efficacies of both classical and tumor-targeted antifolates. Our study identified critical determinants of anti-tumor activity with classical and tumor-targeted antifolates, including relative levels of folate transporter expression and transporter specificity, and the impact of intracellular folate levels and extracellular pH. Citation Format: Zhanjun Hou, Carrie O’Connor, Changwen Ning, Adrianne Wallace-Povirk, Josephine Frühauf, Md. Junayed Nayeen, Aleem Gangjee, Larry H. Matherly. Impact of folate transport redundancies on the therapy with tumor-targeted and untargeted antifolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4791.

Metabolic control of BRISC-SHMT2 assembly regulates immune signalling.

SummarySHMT2 regulates one-carbon transfer reactions essential for amino acid and nucleotide metabolism, using PLP as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active, tetrameric state. SHMT2 also promotes inflammatory cytokine signaling by interacting with the BRISC deubiquitylase (DUB) complex, although it is unclear if this function relates to metabolism. We reveal the cryo-EM structure of human BRISC-SHMT2 complex at 3.8 Å resolution. The BRISC complex is a U-shaped dimer of four subunits and SHMT2 sterically blocks the BRCC36 active site and inhibits DUB activity. Only the inactive SHMT2 dimer, but not the active, PLP-bound tetramer binds and inhibits BRISC. BRISC mutations that disrupt SHMT2 binding impaired type I interferon signaling in response to inflammatory stimuli. Intracellular PLP levels regulated BRISC-SHMT2 interaction and inflammatory cytokine responses. These data reveal a new mechanism of metabolite regulation of DUB activity and inflammatory signaling.

Medicines associated with folate-homocysteine-methionine pathway disruption.

Folate is vital for cell development and growth. It is involved in one-carbon transfer reactions essential for the synthesis of purines and pyrimidines. It also acts in conjunction with cobalamin (vitamin B12) as a fundamental cofactor in the remethylation cycle that converts homocysteine to methionine. A deficiency in folate or vitamin B12 can lead to elevated homocysteine level, which has been identified as an independent risk factor in several health-related conditions. Adequate folate levels are essential in women of childbearing age and in pregnant women, and folate deficiency is associated with several congenital malformations. Low folate levels can be caused by dietary deficiencies, a genetic predisposition or treatment with medicines that affect folate concentration. Women who are pregnant or of child-bearing age commonly use medicines, so it is important to identify the basic biochemical mechanisms by which medicines interfere with the folate-homocysteine-methionine pathway. This review focuses on prescription medicines associated with folate disruption. It also summarizes their undesirable/toxic effects. Recommendations regarding folate supplementation during medical therapy are also reviewed.

Recent Developments in Folate Nutrition.

The term folate (vitamin B9) refers to a group of water-soluble compounds that are nutritionally essential for the support of optimal human health and development. Folates participate in numerous one-carbon transfer reactions, including the methylation of important biomolecules (lipids, amino acids, DNA). A deficiency of folate leads to pathological outcomes including anemia and impairments in reproductive health and fetal development. Due to the linkage of impaired folate status with an increased prevalence of neural tube defects (NTDs) in babies, several jurisdictions required the fortification of the food supply with folic acid, a synthetic and stable form of folate. Data from the postfortification era have provided strong evidence for the reduction of NTDs due to folic acid fortification. However, concern is now growing with respect to the amount of synthetic folic acid within the human food supply. Excess folic acid intake has been linked to a masking of vitamin B12 deficiency, and concerns regarding the promotion of folate-sensitive cancers, including colorectal cancer. New strategies to ensure the supply of optimal folate to at-risk populations may be needed, including the use of biofortification approaches, in order to address recent concerns.

Neural tube defects and folic acid in Japan: Prologue introduction ‐ Understanding of the current status of Japan and the proposal from Japanese Teratology Society

Neural tube defects and folic acid in Japan: Prologue introduction ‐ Understanding of the current status of Japan and the proposal from Japanese Teratology Society

Role of folate in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of chronic liver conditions that are characterized by steatosis, inflammation, fibrosis, and liver injury. The global prevalence of NAFLD is rapidly increasing in proportion to the rising incidence of obesity and type 2 diabetes. Because NAFLD is a multifaceted disorder with many underlying metabolic abnormalities, currently, there is no pharmacological agent that is therapeutically approved for the treatment of this disease. Folate is a water-soluble B vitamin that plays an essential role in one-carbon transfer reactions involved in nucleic acid biosynthesis, methylation reactions, and sulfur-containing amino acid metabolism. The liver is the primary organ responsible for storage and metabolism of folates. Low serum folate levels have been observed in patients with obesity and diabetes. It has been reported that a low level of endogenous folates in rodents perturbs folate-dependent one-carbon metabolism, and may be associated with development of metabolic diseases such as NAFLD. This review highlights the biological role of folate in the progression of NAFLD and its associated metabolic complications including obesity and type 2 diabetes. Understanding the role of folate in metabolic disease may position this vitamin as a potential therapeutic for NAFLD.

Tetrahydrofolate Modulates Floral Transition through Epigenetic Silencing.

Folates, termed from tetrahydrofolate (THF) and its derivatives, function as coenzymes in one-carbon transfer reactions and play a central role in synthesis of nucleotides and amino acids. Dysfunction of cellular folate metabolism leads to serious defects in plant development; however, the molecular mechanisms of folate-mediated cellular modifications and physiological responses in plants are still largely unclear. Here, we reported that THF controls flowering time by adjusting DNA methylation-regulated gene expression in Arabidopsis (Arabidopsis thaliana). Wild-type seedlings supplied with THF as well as the high endogenous THF content mutant dihydrofolate synthetase folypoly-Glu synthetase homolog B exhibited significant up-regulation of the flowering repressor of Flowering Wageningen and thereby delaying floral transition in a dose-dependent manner. Genome-wide transcripts and DNA methylation profiling revealed that THF reduces DNA methylation so as to manipulate gene expression activity. Moreover, in accompaniment with elevated cellular ratios between monoglutamylated and polyglutamylated folates under increased THF levels, the content of S-adenosylhomo-Cys, a competitive inhibitor of methyltransferases, was obviously higher, indicating that enhanced THF accumulation may disturb cellular homeostasis of the concerted reactions between folate polyglutamylation and folate-dependent DNA methylation. In addition, we found that the loss-of-function mutant of CG DNA methyltransferase MET1 displayed much less responsiveness to THF-associated flowering time alteration. Taken together, our studies revealed a novel regulatory role of THF on epigenetic silencing, which will shed lights on the understanding of interrelations in folate homeostasis, epigenetic variation, and flowering control in plants.

Effects of Folic Acid and 5‐Methyltetrahydrofolate Supplementation on Folate Metabolism and One‐Carbon Transfer Reactions in a Mouse Model

Because of potential adverse health effects of folic acid (FA) supplementation, 5‐methyltetrahydrofolate (5MTHF) has been proposed as a safe alternative to provide supplemental levels of folate. However, the effects of 5MTHF versus FA supplementation on folate metabolism and one‐carbon transfer reactions have not been well studied in an in vivo model. We compared the supplemental effects of FA and 5‐MTHF on the expression of genes involved in intracellular folate metabolism and one‐carbon transfer reactions in the liver using a mouse model. Postweaning C57BL/6 mice were randomized to receive diets containing 2, 10, 20 mg FA/kg of diet or their molar equivalent 5MTHF diets for 12 weeks. Gene expression in the liver was assessed using real‐time RT‐qPCR. Plasma and hepatic folate concentrations accurately reflected supplemental levels of both FA and 5MTHF (p‐trend<0.0001). Expression of thymidylate synthase (Tyms) in the liver was not significantly different between FA and 5MTHFR at each molar equivalent supplemental levels. However, 20 mg 5MTHF supplementation was associated with a significant downregulation of Tyms compared with 10 mg 5MTHF (p<0.05). Expression of dihydrofolate reductase (Dhfr) was significantly higher with 5MTHF supplementation compared with FA supplementation at each molar equivalent concentration (p<0.0001). Expression of methylenetetrahydrofolate reductase (Mthfr) was significantly higher in the 10 mg 5MTHF group relative to its molar equivalent FA group (p<0.001), and relative to the 20 mg 5MTHF group (p<0.05). Expression of methionine synthase (Mtr) was significantly greater in all 5MTHF groups relative to their molar equivalent FA groups (p<0.001). Expression of DNA methyltransferase 1 (Dnmt1) was not significantly different between the 5MTHF and FA groups at each molar equivalent concentration. However, Dnmt1 expression was greater in the 10 mg FA and 5‐MTHF group relative to the 2 mg FA group and 20 mg 5‐MTHF group, respectively (p<0.05). Expression of Dnmt3a was greater in all 5MTHF groups relative to their molar equivalent FA groups (p<0.05), and was significantly greater in the 20 mg 5MTHF group relative to the 2 mg 5MTHF group (p<0.05). Expression of Dnmt3b was greater in the 10 and 20 mg 5MTHF groups relative to their equimolar FA groups. Our results suggest that 5MTHF supplementation elicits differential effects on the expression of genes involved in folate metabolism and one‐carbon transfer reactions compared with FA supplementation. Studies are underway to determine functional ramifications associated the observed differential gene expression between 5MTHF and FA supplementation.

Folates in Plants: Research Advances and Progress in Crop Biofortification.

Folates, also known as B9 vitamins, serve as donors and acceptors in one-carbon (C1) transfer reactions. The latter are involved in synthesis of many important biomolecules, such as amino acids, nucleic acids and vitamin B5. Folates also play a central role in the methyl cycle that provides one-carbon groups for methylation reactions. The important functions fulfilled by folates make them essential in all living organisms. Plants, being able to synthesize folates de novo, serve as an excellent dietary source of folates for animals that lack the respective biosynthetic pathway. Unfortunately, the most important staple crops such as rice, potato and maize are rather poor sources of folates. Insufficient folate consumption is known to cause severe developmental disorders in humans. Two approaches are employed to fight folate deficiency: pharmacological supplementation in the form of folate pills and biofortification of staple crops. As the former approach is considered rather costly for the major part of the world population, biofortification of staple crops is viewed as a decent alternative in the struggle against folate deficiency. Therefore, strategies, challenges and recent progress of folate enhancement in plants will be addressed in this review. Apart from the ever-growing need for the enhancement of nutritional quality of crops, the world population faces climate change catastrophes or environmental stresses, such as elevated temperatures, drought, salinity that severely affect growth and productivity of crops. Due to immense diversity of their biochemical functions, folates take part in virtually every aspect of plant physiology. Any disturbance to the plant folate metabolism leads to severe growth inhibition and, as a consequence, to a lower productivity. Whereas today's knowledge of folate biochemistry can be considered very profound, evidence on the physiological roles of folates in plants only starts to emerge. In the current review we will discuss the implication of folates in various aspects of plant physiology and development.

Causes, Consequences and Public Health Implications of Low B-Vitamin Status in Ageing.

The potential protective roles of folate and the metabolically related B-vitamins (vitamins B12, B6 and riboflavin) in diseases of ageing are of increasing research interest. The most common cause of folate and riboflavin deficiencies in older people is low dietary intake, whereas low B12 status is primarily associated with food-bound malabsorption, while sub-optimal vitamin B6 status is attributed to increased requirements in ageing. Observational evidence links low status of folate and the related B-vitamins (and/or elevated concentrations of homocysteine) with a higher risk of degenerative diseases including cardiovascular disease (CVD), cognitive dysfunction and osteoporosis. Deficient or low status of these B-vitamins alone or in combination with genetic polymorphisms, including the common MTHFR 677 C → T polymorphism, could contribute to greater disease risk in ageing by causing perturbations in one carbon metabolism. Moreover, interventions with the relevant B-vitamins to optimise status may have beneficial effects in preventing degenerative diseases. The precise mechanisms are unknown but many have been proposed involving the role of folate and the related B-vitamins as co-factors for one-carbon transfer reactions, which are fundamental for DNA and RNA biosynthesis and the maintenance of methylation reactions. This review will examine the evidence linking folate and related B-vitamins with health and disease in ageing, associated mechanisms and public health implications.

Folic Acid Production by Engineered Ashbya gossypii

Folic acid (vitamin B9) is the common name of a number of chemically related compounds (folates), which play a central role as cofactors in one-carbon transfer reactions. Folates are involved in the biosynthesis and metabolism of nucleotides and amino acids, as well as supplying methyl groups to a broad range of substrates, such as hormones, DNA, proteins, and lipids, as part of the methyl cycle. Humans and animals cannot synthesize folic acid and, therefore, need them in the diet. Folic acid deficiency is an important and underestimated problem of micronutrient malnutrition affecting billions of people worldwide. Therefore, the addition of folic acid as food additive has become mandatory in many countries thus contributing to a growing demand of the vitamin. At present, folic acid is exclusively produced by chemical synthesis despite its associated environmental burdens. In this work, we have metabolically engineered the industrial fungus Ashbya gossypii in order to explore its potential as a natural producer of folic acid. Overexpression of FOL genes greatly enhanced the synthesis of folates and identified GTP cyclohydrolase I as the limiting step. Metabolic flux redirection from competing pathways also stimulated folic acid production. Finally, combinatorial engineering synergistically increased the production of different bioactive forms of the folic vitamin. Overall, strains were constructed which produce 146-fold (6595µg/L) more vitamin than the wild-type and by far represents the highest yield reported.