Precursor Vitamin Research Articles

Co-delivery of vitamin and amino acid within MOFs for oxidative stress-based tumor gas therapy

As an alternative to chemotherapy, emerging gas therapy is considered a “green” treatment due to its minimal side effects. However, even typical gas molecules like nitric oxide (NO) face challenges such as a very short half-life (1.5–6 min), poor targeting, and limited therapeutic effects. This study employs a one-pot method to simultaneously encapsulate the NO donor L-arginine (L-Arg) and the H2O2 precursor Vitamin K3 (VK3) into the pores of zeolitic imidazolate framework-8 (ZIF-8), achieving their co-delivery to tumor sites to address these issues. Furthermore, ZIF-8 is functionalized with hyaluronic acid (HA) to impart active targeting properties to tumor tissues. In the acidic tumor microenvironment, pH-sensitive ZIF-8 degrades, releasing VK3 and L-Arg. Under the action of the NAD(P)H quinone oxidoreductase-1 (NQO1) enzyme, VK3 generates H2O2, increasing oxidative stress levels in the tumor microenvironment, and reacts with L-Arg to produce NO, thereby achieving tumor oxidative stress-based gas therapy. Both in vitro and in vivo experiments showed good tumor treatment effects, with a tumor inhibition rate of up to 90.5 % and minimal impact on normal tissues and organs. This approach demonstrates efficient loading, controlled release, and significant anti-tumor performance, offering new insights into gas and reactive oxygen species (ROS) synergistic therapy.

Acute oxalate nephropathy: exploring the role of excess dietary oxalate intake.

Acute oxalate nephropathy is a rare but important cause of severe acute kidney injury. We report here two cases presenting as unexplained AKI which were confirmed histologically to be due to acute oxalate nephropathy. Dietary oxalate or its precursor vitamin C was the cause of oxalate exposure in both of these cases. While one patient recovered, another continued to need dialysis and succumbed to underlying metastatic cancer. This cause should be suspected in all patients presenting with unexplained AKI, and detailed history about dietary intake of oxalate or vitamin C should be inquired.

The vitamin B5/coenzyme A axis: A target for immunomodulation?

Coenzyme A (CoA) serves as a vital cofactor in numerous enzymatic reactions involved in energy production, lipid metabolism, and synthesis of essential molecules. Dysregulation of CoA-dependent metabolic pathways can contribute to chronic diseases, such as inflammatory diseases, obesity, diabetes, cancer, and cardiovascular disorders. Additionally, CoA influences immune cell activation by modulating the metabolism of these cells, thereby affecting their proliferation, differentiation, and effector functions. Targeting CoA metabolism presents a promising avenue for therapeutic intervention, as it can potentially restore metabolic balance, mitigate chronic inflammation, and enhance immune cell function. This might ultimately improve the management and outcomes for these diseases. This review will more specifically focus on the contribution of pathways regulating the availability of the CoA precursor Vitamin B5/pantothenate in vivo and modulating the development of Th17-mediated inflammation, CD8-dependent anti-tumor immunity but also tissue repair processes in chronic inflammatory or degenerative diseases.

Use of 25-hydroxyvitamin D3 in diets for sows: A review.

Dietary supplementation with 25-hydroxyvitamin D3 (25OHD3), as an alternative source of vitamin D, is becoming increasingly popular due to its commercialization and more efficient absorbability. The addition of 25OHD3 rather than its precursor vitamin D3 can circumvent the 25-hydroxylation reaction in the liver, indicating that supplementation of 25OHD3 can rapidly improve the circulating vitamin D status of animals. Emerging experiments have reported that maternal 25OHD3 supplementation could increase sow performances and birth outcomes and promote circulating vitamin D status of sows and their offspring. Increased milk fat content was observed in many experiments; however, others demonstrated that adding 25OHD3 to lactating sow diets increased the contents of milk protein and lactose. Although an inconsistency between the results of different experiments exists, these studies suggested that maternal 25OHD3 supplementation could alter milk composition via its effects on the mammary gland. Previous studies have demonstrated that adding 25OHD3 to sow diets could improve the mRNA expressions of insulin-induced gene 1 (INSIG1) and sterol regulatory element-binding protein 1 (SREBP1) in the mammary gland cells from milk and increase the mRNA expressions of acetyl-CoA carboxylase α (ACCα) and fatty acid synthase (FAS) in the mammary gland tissue. Maternal 25OHD3 supplementation promotes skeletal muscle development of piglets before and after parturition, and improves bone properties including bone density and bone breaking force in lactating sows and their piglets. Interestingly, 25OHD3 supplementation in sow diets could improve neonatal bone development via regulation of milk fatty acid composition related to bone metabolism and mineralization. In this review, we also discuss the effects of adding 25OHD3 to sow diets on the gut bacterial metabolites of suckling piglets, and propose that butyrate production may be associated with bone health. Therefore, to better understand the nutritional functions of maternal 25OHD3 supplementation, this paper reviews advances in the studies of 25OHD3 for sow nutrition and provides references for practical application.

In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii.

Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (β-carotene) and drug (violacein) titers. We found that S.boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled β-carotene synthesis (194 μg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional β-carotene recovered in feces was 56-fold higher than the β-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S.boulardii living in the mammalian gut and provides a set of tools for modulating these titers.

The vitamin E long-chain metabolite α-13′-COOH affects macrophage foam cell formation via modulation of the lipoprotein lipase system

The α-tocopherol-derived long-chain metabolite (α-LCM) α-13′-carboxychromanol (α-13′-COOH) is formed via enzymatic degradation of α-tocopherol (α-TOH) in the liver. In the last decade, α-13′-COOH has emerged as a new regulatory metabolite revealing more potent or even different effects compared with its vitamin precursor α-TOH. The detection of α-13′-COOH in human serum has further strengthened the concept of its physiological relevance as a potential regulatory molecule. Here, we present a new facet on the interaction of α-13′-COOH with macrophage foam cell formation. We found that α-13′-COOH (5 μM) increases angiopoietin-like 4 (ANGPTL4) mRNA expression in human THP-1 macrophages in a time- and dose-dependent manner, while α-TOH (100 μM) showed no effects. Interestingly, the mRNA level of lipoprotein lipase (LPL) was not influenced by α-13′-COOH, but α-TOH treatment led to a reduction of LPL mRNA expression. Both compounds also revealed different effects on protein level: while α-13′-COOH reduced the secreted amount of LPL protein via induction of ANGPTL4 cleavage, i.e. activation, the secreted amount of LPL in the α-TOH-treated samples was diminished due to the inhibition of mRNA expression. In line with this, both compounds reduced the catalytic activity of LPL. However, α-13′-COOH but not α-TOH attenuated VLDL-induced lipid accumulation by 35%. In conclusion, only α-13′-COOH revealed possible antiatherogenic effects due to the reduction of VLDL-induced foam cell formation in THP-1 macrophages. Our results provide further evidence for the role of α-13′-COOH as a functional metabolite of its vitamin E precursor.

Pathway Engineering for Beta-Carotene and Carotenoid Biosynthesis in Y. lipolytica.

β-carotene is an increasingly sought-after organic pigment with antioxidant properties and a vitamin precursor. Yarrowia lipolytica, though unable to naturally synthesize carotenoids, can produce high amounts of the precursor acetyl-CoA making it a promising host for metabolic engineering towards novel biotechnological production of carotenoids. Here, we describe a synthetic biology methodology for Y. Lipolytica metabolic engineering based on Golden Gate DNA assembly for the generation of a multigene cassette, subsequent transformation enabling β-carotene biosynthesis, and quantification of the compound.

Clinical Evidence for Targeting NAD Therapeutically.

Nicotinamide adenine dinucleotide (NAD) pharmacology is a promising class of treatments for age-related conditions that are likely to have a favorable side effect profile for human use, given the widespread use of the NAD precursor vitamin B3 supplements. However, despite several decades of active investigation and numerous possible biochemical mechanisms of action suggested, only a small number of randomized and adequately powered clinical trials of NAD upregulation as a therapeutic strategy have taken place. We conducted a systematic review of the literature, following the PRISMA guidelines, in an attempt to determine whether or not the human clinical trials performed to date support the potential benefits of NAD supplementation in a range of skin, metabolic and age-related conditions. In addition, we sought medical indications that have yielded the most promising results in the limited studies to date. We conclude that promising, yet still speculative, results have been reported for the treatment of psoriasis and enhancement of skeletal muscle activity. However, further trials are required to determine the optimal method of raising NAD levels, identifying the target conditions, and comparisons to the present standard of care for these conditions. Lastly, pharmacological methods that increase NAD levels should also be directly compared to physiological means of raising NAD levels, such as exercise programs and dietary interventions that are tailored to older individuals, and which may be more effective.

Nicotinamide riboside with pterostilbene (NRPT) increases NAD+ in patients with acute kidney injury (AKI): a randomized, double-blind, placebo-controlled, stepwise safety study of escalating doses of NRPT in patients with AKI

BackgroundPreclinical studies have identified both NAD+ and sirtuin augmentation as potential strategies for the prevention and treatment of AKI. Nicotinamide riboside (NR) is a NAD+ precursor vitamin and pterostilbene (PT) is potent sirtuin activator found in blueberries. Here, we tested the effect of combined NR and PT (NRPT) on whole blood NAD+ levels and safety parameters in patients with AKI.MethodsWe conducted a randomized, double-blind, placebo-controlled study of escalating doses of NRPT in 24 hospitalized patients with AKI. The study was comprised of four Steps during which NRPT (5 subjects) or placebo (1 subject) was given twice a day for 2 days. NRPT dosing was increased in each Step: Step 1250/50 mg, Step 2500/100 mg, Step 3750/150 mg and Step 41,000/200 mg. Blood NAD+ levels were measured by liquid chromatography-mass spectrometry and safety was assessed by history, physical exam, and clinical laboratory testing.ResultsAKI resulted in a 50% reduction in whole blood NAD+ levels at 48 h compared to 0 h in patients receiving placebo (p = 0.05). There was a trend for increase in NAD+ levels in all NRPT Steps individually at 48 h compared to 0 h, but only the change in Step 2 reached statistical significance (47%, p = 0.04), and there was considerable interindividual variability in the NAD+ response to treatment. Considering all Steps together, NRPT treatment increased NAD+ levels by 37% at 48 h compared to 0 h (p = 0.002). All safety laboratory tests were unchanged by NRPT treatment, including creatinine, estimated glomerular filtration rate (eGFR), electrolytes, liver function tests, and blood counts. Three of 20 patients receiving NRPT reported minor gastrointestinal side effects.ConclusionNRPT increases whole blood NAD+ levels in hospitalized patients with AKI. In addition, NRPT up to a dose of 1000 mg/200 mg twice a day for 2 days is safe and well tolerated in these patients. Further studies to assess the potential therapeutic benefit of NRPT in AKI are warranted.Trial registrationNCT03176628, date of registration June 5th, 2017.

Nicotinamide riboside supplementation corrects deficits in oxytocin, sociability and anxiety of CD157 mutants in a mouse model of autism spectrum disorder

Oxytocin (OT) is a critical molecule for social recognition and memory that mediates social and emotional behaviours. In addition, OT acts as an anxiolytic factor and is released during stress. Based on the activity of CD38 as an enzyme that produces the calcium-mobilizing second messenger cyclic ADP-ribose (cADPR), CD157, a sister protein of CD38, has been considered a candidate mediator for the production and release of OT and its social engagement and anti-anxiety functions. However, the limited expression of CD157 in the adult mouse brain undermined confidence that CD157 is an authentic and/or actionable molecular participant in OT-dependent social behaviour. Here, we show that CD157 knockout mice have low levels of circulating OT in cerebrospinal fluid, which can be corrected by the oral administration of nicotinamide riboside, a recently discovered vitamin precursor of nicotinamide adenine dinucleotide (NAD). NAD is the substrate for the CD157- and CD38-dependent production of cADPR. Nicotinamide riboside corrects social deficits and fearful and anxiety-like behaviours in CD157 knockout males. These results suggest that elevating NAD levels with nicotinamide riboside may allow animals with cADPR- and OT-forming deficits to overcome these deficits and function more normally.

Toward a Stable and Potent Coenzyme A-Targeting Antiplasmodial Agent: Structure-Activity Relationship Studies of N-Phenethyl-α-methyl-pantothenamide.

Pantothenamides (PanAms) are potent antiplasmodials with low human toxicity currently being investigated as antimalarials with a novel mode of action. These structural analogues of pantothenate, the vitamin precursor of the essential cofactor coenzyme A, are susceptible to degradation by pantetheinase enzymes present in serum. We previously discovered that α-methylation of the β-alanine moiety of PanAms increases their stability in serum and identified N-phenethyl-α-methyl-pantothenamide as a pantetheinase-resistant PanAm with potent, on-target, and selective antiplasmodial activity. In this study, we performed structure-activity relationship investigations to establish whether stability and potency can be improved further through alternative modification of the scissile amide bond and through substitution/modification of the phenyl ring. Additionally, for the first time, the importance of the stereochemistry of the α-methyl group was evaluated in terms of stability versus potency. Our results demonstrate that α-methylation remains the superior choice for amide modification, and that while monofluoro-substitution of the phenyl ring (that often improves ADME properties) was tolerated, N-phenethyl-α-methyl-pantothenamide remains the most potent analogue. We show that the 2S,2'R-diastereomer is far more potent than the 2R,2'R-diastereomer and that this cannot be attributed to preferential metabolic activation by pantothenate kinase, the first enzyme of the coenzyme A biosynthesis pathway. Unexpectedly, the more potent 2S,2'R-diastereomer is also more prone to pantetheinase-mediated degradation. Finally, the results of in vitro studies to assess permeability and metabolic stability of the 2S,2'R-diastereomer suggested species-dependent degradation via amide hydrolysis. Our study provides important information for the continued development of PanAm-based antimalarials.

A novel way to synthesize pantothenate in bacteria involves β-alanine synthase present in uracil degradation pathway.

Pantothenate is an indispensable vitamin precursor of the synthesis of coenzyme A (CoA), a key metabolite required in over 100 metabolic reactions. β‐Alanine (β‐ala) is an indispensable component of pantothenate. Due to the metabolic relevance of this pathway, we assumed that orthologous genes for ß‐alanine synthesis would be present in the genomes of bacteria, archaea, and eukaryotes. However, comparative genomic studies revealed that orthologous gene replacement and loss of synteny occur at high frequency in panD genes. We have previously reported the atypical plasmid‐encoded location of the pantothenate pathway genes panC and panB (two copies) in R. etli CFN42. This study also revealed the unexpected absence of a panD gene encoding the aspartate decarboxylase enzyme (ADC), required for the synthesis of β‐ala. The aim of this study was to identify the source of β‐alanine in Rhizobium etli CFN42. In this study, we present a bioinformatic analysis and an experimental validation demonstrating that the source of β‐ala in this R. etli comes from β‐alanine synthase, the last enzyme of the uracil degradation pathway.

High Dose of Dietary Nicotinamide Riboside Induces Glucose Intolerance and White Adipose Tissue Dysfunction in Mice Fed a Mildly Obesogenic Diet.

Nicotinamide riboside (NR) is a nicotinamide adenine dinucleotide (NAD+) precursor vitamin. The scarce reports on the adverse effects on metabolic health of supplementation with high-dose NR warrant substantiation. Here, we aimed to examine the physiological responses to high-dose NR supplementation in the context of a mildly obesogenic diet and to substantiate this with molecular data. An 18-week dietary intervention was conducted in male C57BL/6JRccHsd mice, in which a diet with 9000 mg NR per kg diet (high NR) was compared to a diet with NR at the recommended vitamin B3 level (control NR). Both diets were mildly obesogenic (40 en% fat). Metabolic flexibility and glucose tolerance were analyzed and immunoblotting, qRT-PCR and histology of epididymal white adipose tissue (eWAT) were performed. Mice fed with high NR showed a reduced metabolic flexibility, a lower glucose clearance rate and aggravated systemic insulin resistance. This was consistent with molecular and morphological changes in eWAT, including sirtuin 1 (SIRT1)-mediated PPARγ (proliferator-activated receptor γ) repression, downregulated AKT/glucose transporter type 4 (GLUT4) signaling, an increased number of crown-like structures and macrophages, and an upregulation of pro-inflammatory gene markers. In conclusion, high-dose NR induces the onset of WAT dysfunction, which may in part explain the deterioration of metabolic health.

Safety and Metabolism of Long-term Administration of NIAGEN (Nicotinamide Riboside Chloride) in a Randomized, Double-Blind, Placebo-controlled Clinical Trial of Healthy Overweight Adults

Nicotinamide riboside (NR) is a newly discovered nicotinamide adenine dinucleotide (NAD+) precursor vitamin. A crystal form of NR chloride termed NIAGEN is generally recognized as safe (GRAS) for use in foods and the subject of two New Dietary Ingredient Notifications for use in dietary supplements. To evaluate the kinetics and dose-dependency of NR oral availability and safety in overweight, but otherwise healthy men and women, an 8-week randomized, double-blind, placebo-controlled clinical trial was conducted. Consumption of 100, 300 and 1000 mg NR dose-dependently and significantly increased whole blood NAD+ (i.e., 22%, 51% and 142%) and other NAD+ metabolites within 2 weeks. The increases were maintained throughout the remainder of the study. There were no reports of flushing and no significant differences in adverse events between the NR and placebo-treated groups or between groups at different NR doses. NR also did not elevate low density lipoprotein cholesterol or dysregulate 1-carbon metabolism. Together these data support the development of a tolerable upper intake limit for NR based on human data.

S136 REPROGRAMMING HEMATOPOIETIC STEM CELL FUNCTION VIA MODULATION OF MITOCHONDRIAL ACTIVITY

Background:Hematopoietic stem cells (HSCs) are a small group of cells that ensure a steady production of all mature blood cells throughout an organism's lifetime. HSCs mediate this process by giving rise to progenitors that in turn produce terminally differentiated mature blood cell lineages via several rounds of cell division and well‐orchestrated differentiation steps. Cellular metabolism has emerged as a crucial regulator guiding this complex process. Especially, HSCs differ from their committed progeny by relying primarily on anaerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy production. This distinct metabolic state protects the HSCs from cellular damage inflicted by reactive oxygen species (ROS) in active mitochondria. However, whether this change in the metabolic program is the cause or a consequence of the unique function of HSCs remains unknown.Aims:Having previously shown that mitochondrial activity can be used as a reliable readout for HSC fate, here we asked if modulation of mitochondrial activity results in enhancement of HSC function.Methods:We used in vitro mitochondrial activity and in vivo long‐term blood reconstitution assays as readouts of murine and human HSC functionality.Results:We previously demonstrated that modulation of mitochondrial metabolism in murine and human HSCs, by chemical uncouplers of the electron transport chain or via NAD+ boosting agent Nicotinamide Riboside (NR), results in better long‐term blood production in serially transplanted mice (Vannini N∗, Girotra M∗. et al., Nature Communication 2016 and, Vannini et al., Cell Stem Cell, in press). Here we proceeded to carry out a screen, using mitochondrial activity as readout, to identify metabolic modulators that enhance HSC function. We found two novel candidates, a natural compound and a vitamin precursor, that modulate mitochondrial activity in both mouse and human HSCs, and resulted in enhanced HSC function post bone‐marrow transplantation. Interestingly, we found that these candidates mediate their effects partially by inducing mitophagy, supporting recent studies highlighting the role of mitophagy as a key driver of HSC function. Moreover, our preliminary analysis reveals that they mediate similar effects in aged human HSCs, making them ideal candidates to revert age‐associated myeloid bias in human patients.Summary/Conclusion:Our data establishes a causal relationship between mitochondrial metabolism and HSC function via mitophagy. It also provides a valuable tool to identify optimal ex vivo conditions for HSC expansion, to improve the outcome for patients suffering from bone marrow insufficiency and to restore the functionality of the immune system in aged individuals.

Synthesis, structural characterization, DNA/protein binding and antioxidant activities of binuclear Ni(II) complexes containing ONS chelating ligands bridged by 1,3-bis(diphenylphosphino)propane

Binuclear Ni(II) complexes of 4-diethylaminosalicylaldehyde-4(N)-substituted thiosemicarbazones bridged by 1,3-bis(diphenylphosphino)propane have been synthesized and characterized. A spectroscopic evaluation of their binding ability with DNA and protein was carried out, the results of which implied a better affinity for the complexes. The free radical scavenging activity of the complexes was found to be greater than those observed for the ligands, the metal precursor and standard Vitamin C.

Chronic\xa0nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy middle-aged and older adults

Nicotinamide adenine dinucleotide (NAD+) has emerged as a critical co-substrate for enzymes involved in the beneficial effects of regular calorie restriction on healthspan. As such, the use of NAD+ precursors to augment NAD+ bioavailability has been proposed as a strategy for improving cardiovascular and other physiological functions with aging in humans. Here we provide the evidence in a 2 × 6-week randomized, double-blind, placebo-controlled, crossover clinical trial that chronic supplementation with the NAD+ precursor vitamin, nicotinamide riboside (NR), is well tolerated and effectively stimulates NAD+ metabolism in healthy middle-aged and older adults. Our results also provide initial insight into the effects of chronic NR supplementation on physiological function in humans, and suggest that, in particular, future clinical trials should further assess the potential benefits of NR for reducing blood pressure and arterial stiffness in this group.

Vitamin K2 in multiple sclerosis patients.

SummaryBackgroundVitamin K2 (VK2) belongs to the vitamin K family and comprises a number of subtypes differing in length of side chains consisting of isoprenoid groups (menaquinone-n, MK-n). It is essential for a number of physiological functions although the full spectrum of activity has not yet been elucidated. Due to its role in protection of mitochondrial damage, VK2 could be relevant in preventing disease progress in multiple sclerosis (MS).MethodsWe measured VK2 serum levels by the double antibody sandwich Enzyme-linked Immunosorbent Assay (ELISA) technique in MS patients and age and sex matched controls, both under vitamin D supplementation, and related it to disease characteristics and treatment.ResultsOverall, 45 MS patients (31 females and 39 of the relapsing-remitting type) and 29 healthy controls (19 females) were included in the analysis. The MS patients had vastly lower VK2 blood levels than controls (235 ± 100 ng/ml vs. 812 ± 154 ng/ml, respectively). Female patients had significantly lower VK2 levels than males and a decrease with age by approximately 10% per decade was found. The VK2 levels were lower with increasing numbers of attacks per year and were higher in patients with optic nerve lesions. No consistent relationship with medications was detected.ConclusionThe substantially lower levels of VK2 in MS patients could be due to depletion, lower production in the gut, diminished absorption or, less likely, reduced intake of precursor vitamin K1. The role of VK2 in MS development and progress deserves further study.Electronic supplementary materialThe online version of this article (10.1007/s00508-018-1328-x) contains supplementary material, which is available to authorized users.

Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD+ levels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study

NRPT is a combination of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor vitamin found in milk, and pterostilbene (PT), a polyphenol found in blueberries. Here, we report this first-in-humans clinical trial designed to assess the safety and efficacy of a repeat dose of NRPT (commercially known as Basis). NRPT was evaluated in a randomized, double-blind, and placebo-controlled study in a population of 120 healthy adults between the ages of 60 and 80 years. The study consisted of three treatment arms: placebo, recommended dose of NRPT (NRPT 1X), and double dose of NRPT (NRPT 2X). All subjects took their blinded supplement daily for eight weeks. Analysis of NAD+ in whole blood demonstrated that NRPT significantly increases the concentration of NAD+ in a dose-dependent manner. NAD+ levels increased by approximately 40% in the NRPT 1X group and approximately 90% in the NRPT 2X group after 4 weeks as compared to placebo and baseline. Furthermore, this significant increase in NAD+ levels was sustained throughout the entire 8-week trial. NAD+ levels did not increase for the placebo group during the trial. No serious adverse events were reported in this study. This study shows that a repeat dose of NRPT is a safe and effective way to increase NAD+ levels sustainably.

A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of β‐carotene

The increasing market demands of β-carotene as colorant, antioxidant and vitamin precursor, requires novel biotechnological production platforms. Yarrowia lipolytica, is an industrial organism unable to naturally synthesize carotenoids but with the ability to produce high amounts of the precursor Acetyl-CoA. We first found that a lipid overproducer strain was capable of producing more β-carotene than a wild type after expressing the heterologous pathway. Thereafter, we developed a combinatorial synthetic biology approach base on Golden Gate DNA assembly to screen the optimum promoter-gene pairs for each transcriptional unit expressed. The best strain reached a production titer of 1.5 g/L and a maximum yield of 0.048 g/g of glucose in flask. β-carotene production was further increased in controlled conditions using a fed-batch fermentation. A total production of β-carotene of 6.5 g/L and 90 mg/g DCW with a concomitant production of 42.6 g/L of lipids was achieved. Such high titers suggest that engineered Y. lipolytica is a competitive producer organism of β-carotene.