scholarly journals Untargeted metabolomics analysis of Ralstonia eutropha during plant oil cultivations reveals the presence of a fucose salvage pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Björn Gutschmann ◽  
Martina C. E. Bock ◽  
Stefan Jahns ◽  
Peter Neubauer ◽  
Christopher J. Brigham ◽  
...  
Keyword(s):  
De Novo ◽  
Ralstonia Eutropha ◽  
Wild Type ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Plant Oil ◽  
Microbial Physiology ◽  
Medium Chain Length ◽  
Metabolic States ◽  
Metabolomic Study

AbstractProcess engineering of biotechnological productions can benefit greatly from comprehensive analysis of microbial physiology and metabolism. Ralstonia eutropha (syn. Cupriavidus necator) is one of the best studied organisms for the synthesis of biodegradable polyhydroxyalkanoate (PHA). A comprehensive metabolomic study during bioreactor cultivations with the wild-type (H16) and an engineered (Re2058/pCB113) R. eutropha strain for short- and or medium-chain-length PHA synthesis has been carried out. PHA production from plant oil was triggered through nitrogen limitation. Sample quenching allowed to conserve the metabolic states of the cells for subsequent untargeted metabolomic analysis, which consisted of GC–MS and LC–MS analysis. Multivariate data analysis resulted in identification of significant changes in concentrations of oxidative stress-related metabolites and a subsequent accumulation of antioxidative compounds. Moreover, metabolites involved in the de novo synthesis of GDP-l-fucose as well as the fucose salvage pathway were identified. The related formation of fucose-containing exopolysaccharides potentially supports the emulsion-based growth of R. eutropha on plant oils.

scholarly journals Tung Oil-Based Production of High 3-Hydroxyhexanoate-Containing Terpolymer Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate-co-3-Hydroxyhexanoate) Using Engineered Ralstonia eutropha

Polymers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1084
Author(s):  
Hye Soo Lee ◽  
Sun Mi Lee ◽  
Sol Lee Park ◽  
Tae-Rim Choi ◽  
Hun-Suk Song ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Eleostearic Acid ◽  
Fossil Fuels ◽  
Ralstonia Eutropha ◽  
Wild Type ◽  
Medical Field ◽  
Tung Oil ◽  
Medium Chain Length ◽  
Partial Harvesting ◽  
Biodegradable Properties

Polyhydroxyalkanoates (PHAs) are attractive new bioplastics for the replacement of plastics derived from fossil fuels. With their biodegradable properties, they have also recently been applied to the medical field. As poly(3-hydroxybutyrate) produced by wild-type Ralstonia eutropha has limitations with regard to its physical properties, it is advantageous to synthesize co- or terpolymers with medium-chain-length monomers. In this study, tung oil, which has antioxidant activity due to its 80% α-eleostearic acid content, was used as a carbon source and terpolymer P(53 mol% 3-hydroxybytyrate-co-2 mol% 3-hydroxyvalerate-co-45 mol% 3-hydroxyhexanoate) with a high proportion of 3-hydroxyhexanoate was produced in R. eutropha Re2133/pCB81. To avail the benefits of α-eleostearic acid in the tung oil-based medium, we performed partial harvesting of PHA by using a mild water wash to recover PHA and residual tung oil on the PHA film. This resulted in a film coated with residual tung oil, showing antioxidant activity. Here, we report the first application of tung oil as a substrate for PHA production, introducing a high proportion of hydroxyhexanoate monomer into the terpolymer. Additionally, the residual tung oil was used as an antioxidant coating, resulting in the production of bioactive PHA, expanding the applicability to the medical field.

scholarly journals NAD+-Dependent Deacetylase Hst1p Controls Biosynthesis and Cellular NAD+ Levels in Saccharomyces cerevisiae

2003 ◽  
Vol 23 (19) ◽  
pp. 7044-7054 ◽  
Author(s):  
Antonio Bedalov ◽  
Maki Hirao ◽  
Jeffrey Posakony ◽  
Melisa Nelson ◽  
Julian A. Simon
Keyword(s):  
De Novo ◽  
Critical Role ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Array Analysis ◽  
Binding Partner ◽  
Dependent Protein ◽  
New Protein

ABSTRACT Nicotine adenine dinucleotide (NAD+) performs key roles in electron transport reactions, as a substrate for poly(ADP-ribose) polymerase and NAD+-dependent protein deacetylases. In the latter two processes, NAD+ is consumed and converted to ADP-ribose and nicotinamide. NAD+ levels can be maintained by regeneration of NAD+ from nicotinamide via a salvage pathway or by de novo synthesis of NAD+ from tryptophan. Both pathways are conserved from yeast to humans. We describe a critical role of the NAD+-dependent deacetylase Hst1p as a sensor of NAD+ levels and regulator of NAD+ biosynthesis. Using transcript arrays, we show that low NAD+ states specifically induce the de novo NAD+ biosynthesis genes while the genes in the salvage pathway remain unaffected. The NAD+-dependent deacetylase activity of Hst1p represses de novo NAD+ biosynthesis genes in the absence of new protein synthesis, suggesting a direct effect. The known Hst1p binding partner, Sum1p, is present at promoters of highly inducible NAD+ biosynthesis genes. The removal of HST1-mediated repression of the NAD+ de novo biosynthesis pathway leads to increased cellular NAD+ levels. Transcript array analysis shows that reduction in cellular NAD+ levels preferentially affects Hst1p-regulated genes in comparison to genes regulated with other NAD+-dependent deacetylases (Sir2p, Hst2p, Hst3p, and Hst4p). In vitro experiments demonstrate that Hst1p has relatively low affinity toward NAD+ in comparison to other NAD+-dependent enzymes. These findings suggest that Hst1p serves as a cellular NAD+ sensor that monitors and regulates cellular NAD+ levels.

scholarly journals Gambaran Asam Urat Serum pada vegetarian Lacto - ovo

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
Christy P. Hapendatu ◽  
Youla A. Assa ◽  
Michaela E. Paruntu
Keyword(s):  
Uric Acid ◽  
Nucleic Acid ◽  
Serum Uric Acid ◽  
Food Consumption ◽  
Sampling Method ◽  
De Novo ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Cross Sectional ◽  
High Uric Acid

Abstract: Purine is generated through three mechanisms: de novo synthesis, salvage pathway, and food consumption. Most purine derived from food is converted to uric acid directly, without the occurrence of nucleic acid network. Lacto-ovo vegetarian is a lifestyle that consist of consuming food like vegetables, fruits, nuts, tofu, tempe plus milk and eggs. The intake of food in vegetarian lacto-ovo, some of them are high in purines. The purpose of this research is to describe serum uric acid levels in lacto-ovo vegetarians. This study used a cross sectional with a total sampling as the sampling method. There were 25 respondents that participated in this study. From 25 respondents found as many as seven respondents (28%) who had high uric acid levels and 18 respondent (72%) had serum uric acid levels in the normal range.Keywords: Purin, Uric Acid, Vegetarian Lacto-ovoAbstrak : Purin dihasilkan melalui tiga mekanisme yaitu sintesis de novo, jalur penyelamatan, dan konsumsi makanan. Sebagian besar purin yang bersal dari makanan akan diubah menjadi asam urat secara langsung tanpa terjadinya asam nukleat jaringan. Vegetarian Lacto-ovo merupakan gaya hidup yang mengonsumsi golongan makanan seperti sayuran, buah-buahan, kacang-kacangan, tahu, tempe ditambah susu dan telur. Asupan makanan yang di konsumsi vegetarian lacto-ovo beberapa di antaranya merupakan bahan makanan yang tinggi purin. Tujuan dari penelitian ini untuk mengetahui gambaran kadar asam urat serum pada vegetarian lacto-ovo. Penelitian ini menggunakan desain penelitian deskriptif cross sectional dengan pengambilan sampel menggunakan metode total sampling. Penelitian ini diikuti oleh 25 responden. Dari 25 responden ditemukan sebanyak 7 responden (28%) yang memiliki kadar asam urat yang tinggi dan sebagian besar (72%) mempunyai kadar asam urat serum dalam batas normal.Kata kunci : Purin, Asam Urat, Vegetarian Lacto-ovo

scholarly journals Inhibition of De Novo Pyrimidine Nucleotide Synthesis By the Novel DHODH Inhibitor PTC299 Induces Differentiation and/or Death of AML Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5152-5152
Author(s):  
Marla Weetall ◽  
Kensuke Kojima ◽  
Sujan Piya ◽  
Christopher Trotta ◽  
John Baird ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
De Novo ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Dna Breaks ◽  
Advisory Committees ◽  
Pyrimidine Nucleotides ◽  
Nucleotide Synthesis ◽  
Pyrimidine Nucleotide

Background: Pyrimidine nucleotides are generated either by de novo synthesis or the salvage pathway in which pyrimidine nucleotides are obtained from the diet. Resting cells typically acquire adequate pyrimidine nucleotides from the salvage pathway. Rapidly proliferating cells, however, are dependent on the de novo synthesis of pyrimidine nucleotides. PTC299 is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for de novo pyrimidine nucleotide synthesis that had previously been in clinical trials for treatment of solid tumors. Results: Using 15N-labelled glutamine, we show that PTC299 reduces de novo pyrimidine nucleotide synthesis in PTC299-sensitive AML cell lines resulting in a depletion of total pyrimidine nucleotides. In parallel to reduction in pyrimidine nucleotides, PTC 299 leads to accumulation of DHO, the substrate of DHODH and unexpectedly, an accumulation of N-carbamoyl aspartate the metabolite above DHO in the de novo pyrimidine nucleotide synthesis pathway. PTC299 was broadly active against leukemia and lymphoma lines, with 80% of the AML lines tested showing sensitivity. Treatment of AML cell lines with PTC299 induced differentiation as shown by increased CD14 and/or reduced proliferation. Using isogenic AML lines, we show that PTC299 reduces the proliferation of both p53 wildtype and p53 deficient leukemia calls with similar potency as measured by the concentration of PTC299 required to reduce cell number by 50% (CC50). In cells expressing wildtype p53, PTC299 increases p53 activation. However, p53- wildtype cells undergo increased apoptosis whereas p53-deficience cells undergo necrosis. PTC299 induced a G1/S cell cycle arrest, also independent of p53 status. PTC299 increased H2A.X (a marker of double stranded DNA breaks) in both p53 wildtype and p53 deficient cells. These data suggest that the depletion of nucleotides results in stalling at the replication fork, and subsequent DNA-breaks. Conclusion: De novo pyrimidine nucleotide synthesis is critical for AML survival and proliferation. Depletion of nucleotides results in reduced proliferation, triggering either differentiation and/or cell death. Disclosures Weetall: PTC Therapeutics: Employment. Trotta:PTC Therapeutics: Employment. Baird:PTC Therapeutics: Employment. O'Keefe:PTC Therapeutics: Employment. Furia:PTC Therapeutics: Employment. Borthakur:PTC Therapeutics: Consultancy; Janssen: Research Funding; AbbVie: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; NKarta: Consultancy; AstraZeneca: Research Funding; Xbiotech USA: Research Funding; Incyte: Research Funding; GSK: Research Funding; Oncoceutics, Inc.: Research Funding; Novartis: Research Funding; Agensys: Research Funding; BMS: Research Funding; Oncoceutics: Research Funding; Cantargia AB: Research Funding; Bayer Healthcare AG: Research Funding; Eisai: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Polaris: Research Funding; Merck: Research Funding; Cyclacel: Research Funding; Eli Lilly and Co.: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Arvinas: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Strategia Therapeutics: Research Funding. Spiegel:PTC Therapeutics: Consultancy.

Mechanism of pyridoxine 5'-phosphate accumulation in PLPBP protein-deficiency

2022 ◽  
Author(s):  
Tomokazu Ito ◽  
Honoka Ogawa ◽  
Hisashi Hemmi ◽  
Diana M. Downs ◽  
Tohru Yoshimura
Keyword(s):  
De Novo ◽  
Binding Protein ◽  
Vitamin B ◽  
Wild Type ◽  
Salvage Pathway ◽  
Intracellular Accumulation ◽  
Pyridoxal Kinase ◽  
Genetic Studies ◽  
E Coli ◽  
Metabolic Systems

The pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) plays an important role in vitamin B 6 homeostasis. Loss of this protein in organisms such as Escherichia coli and humans disrupts the vitamin B 6 pool and induces intracellular accumulation of pyridoxine 5'-phosphate (PNP), which is normally undetectable in wild-type cells. The accumulated PNP could affect diverse metabolic systems through inhibition of some PLP-dependent enzymes. In this study, we investigated the as yet unclear mechanism of intracellular accumulation of PNP by the loss of PLPBP protein encoded by yggS in E. coli . Genetic studies using several PLPBP-deficient strains of E. coli lacking known enzyme(s) in the de novo or salvage pathway of vitamin B 6 , which includes pyridoxine (amine) 5'-phosphate oxidase (PNPO), PNP synthase, pyridoxal kinase, and pyridoxal reductase, demonstrated that neither the flux from the de novo pathway nor the salvage pathway solely contributed to the PNP accumulation caused by the PLPBP mutation. Studies with the strains lacking both PLPBP and PNPO suggested that PNP shares the same pool with PMP, and showed that PNP levels are impacted by PMP levels and vice versa . We show that disruption of PLPBP lead to perturb PMP homeostasis, which may result in PNP accumulation in the PLPBP-deficient strains. Importance A PLP-binding protein PLPBP from the conserved COG0325 family has recently been recognized as a key player in vitamin B 6 homeostasis in various organisms. Loss of PLPBP disrupts vitamin B 6 homeostasis and perturbs diverse metabolisms, including amino acid and α-keto acid metabolism. Accumulation of PNP is a characteristic phenotype of the PLPBP deficiency and is suggested to be a potential cause of the pleiotropic effects, but the mechanism of the PNP accumulation was poorly understood. In this study, we show that fluxes for PNP synthesis/metabolism are not responsible for the accumulation of PNP. Our results indicate that PLPBP is involved in the homeostasis of pyridoxamine 5'-phosphate, and its disruption may lead to the accumulation of PNP in PLPBP-deficiency.

scholarly journals DRES-09. REGULATORY EFFECTS OF THE CILIARY GTPASE ARL13B ON PURINE METABOLISM IN GBM

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi73-vi73
Author(s):  
Miranda Saathoff ◽  
Jack Shireman ◽  
Eunus Ali ◽  
Cheol Park ◽  
Issam Ben-Sahra ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Specific Activity ◽  
Purine Metabolism ◽  
P Value ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Dna Double Strand Breaks ◽  
Purine Nucleotides ◽  
Regulatory Effects

Abstract Glioblastoma (GBM) is the most common form of adult primary brain cancer. Despite an aggressive treatment regimen – surgical resection, irradiation, and temozolomide (TMZ) chemotherapy – patients’ prognosis is still grim. TMZ acts by methylating purines, specifically at the O6 and N7 positions of guanine, to induce cytotoxic DNA double-strand breaks. We thus wanted to explore how purine metabolism may contribute to TMZ-resistance. In mammalian cells, purine nucleotides can be recycled by the salvage pathway or generated via de novo synthesis. The salvage pathway is energetically inexpensive relative to de novo thus, highly proliferative GBM cells preferentially utilize the salvage pathway. We have shown that salvage synthesis is reduced in response to TMZ (p-value=0.0021), hinting that the cells may utilize de novo to evade therapy induced alkylation of purines. Using immunoprecipitation-mass spectroscopy analysis, we found a novel interaction between the ciliary GTPase ARL13B and IMPDH2, the rate-limiting enzyme in de novo synthesis. We have shown that this interaction, occurring at the C-terminal domain of ARL13B, plays a significant role in the regulation of purine biosynthesis as abolishing it through ARL13B knockdown reduced flux through de novo (p-value< 0.0001) synthesis as measured by the specific activity of IMPDH2. Further, the lentiviral-mediated rescue of ARL13B brings IMPDH2 activity back to basal levels (p< 0.0001). Given its canonical function as a GTPase, we hypothesize that ARL13B acts as a novel regulator of de novo synthesis by sequestering GDP, allowing IMPDH2 to sense and respond to the cytosolic levels of guanine nucleotides. Without ARL13B the de novo pathway is halted, forcing the cells to rely on salvage to replenish nucleotide pools. Reliance on this pathway in the presence of TMZ causes cells to incorporate damaged nucleotides as a result of the drug’s alkylating action leading to the increased therapeutic efficacy of TMZ.

scholarly journals Thiamine Is Synthesized by a Salvage Pathway in Rhizobium leguminosarum bv. viciae Strain 3841

2006 ◽  
Vol 188 (18) ◽  
pp. 6661-6668 ◽  
Author(s):  
R. Karunakaran ◽  
K. Ebert ◽  
S. Harvey ◽  
M. E. Leonard ◽  
V. Ramachandran ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
De Novo ◽  
Fluorescent Microscopy ◽  
Good Growth ◽  
Wild Type ◽  
Salvage Pathway ◽  
Mesorhizobium Loti ◽  
Reverse Transcription Pcr

ABSTRACT In the absence of added thiamine, Rhizobium leguminosarum bv. viciae strain 3841 does not grow in liquid medium and forms only “pin” colonies on agar plates, which contrasts with the good growth of Sinorhizobium meliloti 1021, Mesorhizobium loti 303099, and Rhizobium etli CFN42. These last three organisms have thiCOGE genes, which are essential for de novo thiamine synthesis. While R. leguminosarum bv. viciae 3841 lacks thiCOGE, it does have thiMED. Mutation of thiM prevented formation of pin colonies on agar plates lacking added thiamine, suggesting thiamine intermediates are normally present. The putative functions of ThiM, ThiE, and ThiD are 4-methyl-5-(β-hydroxyethyl) thiazole (THZ) kinase, thiamine phosphate pyrophosphorylase, and 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) kinase, respectively. This suggests that a salvage pathway operates in R. leguminosarum, and addition of HMP and THZ enabled growth at the same rate as that enabled by thiamine in strain 3841 but elicited no growth in the thiM mutant (RU2459). There is a putative thi box sequence immediately upstream of the thiM, and a gfp-mut3.1 fusion to it revealed the presence of a promoter that is strongly repressed by thiamine. Using fluorescent microscopy and quantitative reverse transcription-PCR, it was shown that thiM is expressed in the rhizosphere of vetch and pea plants, indicating limitation for thiamine. Pea plants infected by RU2459 were not impaired in nodulation or nitrogen fixation. However, colonization of the pea rhizosphere by the thiM mutant was impaired relative to that of the wild type. Overall, the results show that a thiamine salvage pathway operates to enable growth of Rhizobium leguminosarum in the rhizosphere, allowing its survival when thiamine is limiting.

scholarly journals De novo synthesis and salvage pathway coordinately regulates polyamine homeostasis and determines T cell proliferation and function

2020 ◽  
Author(s):  
Ruohan Wu ◽  
Xuyong Chen ◽  
Siwen Kang ◽  
Tingting Wang ◽  
JN Rashida Gnanaprakasam ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
De Novo ◽  
T Cell Proliferation ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Polyamine Biosynthesis ◽  
Living Organisms ◽  
A Minor

AbstractRobust and effective T cell-mediated immune responses require proper allocation of metabolic resources through metabolic pathways to sustain the energetically costly immune response. As an essential class of polycationic metabolites ubiquitously present in all living organisms, the polyamine pool is tightly regulated by biosynthesis and salvage pathway. We demonstrated that arginine is a major carbon donor and glutamine is a minor carbon donor for polyamine biosynthesis in T cells. Accordingly, the dependence of T cells can be partially relieved by replenishing the polyamine pool. In response to the blockage of de novo synthesis, T cells can rapidly restore the polyamine pool through a compensatory increase in polyamine uptake from the environment, indicating a layer of metabolic plasticity. Simultaneously blocking synthesis and uptake depletes the intracellular PA pool, inhibits T cell proliferation, suppresses T cell inflammation, indicating the potential therapeutic value of targeting the polyamine for managing inflammatory and autoimmune diseases.

scholarly journals Differential role of nicotinamide adenine dinucleotide deficiency in acute and chronic kidney disease

2020 ◽  
Vol 36 (1) ◽  
pp. 60-68
Author(s):  
Anna Faivre ◽  
Elena Katsyuba ◽  
Thomas Verissimo ◽  
Maja Lindenmeyer ◽  
Renuga Devi Rajaram ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Nicotinamide Adenine Dinucleotide ◽  
De Novo ◽  
Nicotinamide Adenine ◽  
Sequencing Analysis ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Nad Synthesis ◽  
Ckd Stage

Abstract Background Nicotinamide adenine dinucleotide (NAD+) is a ubiquitous coenzyme involved in electron transport and a co-substrate for sirtuin function. NAD+ deficiency has been demonstrated in the context of acute kidney injury (AKI). Methods We studied the expression of key NAD+ biosynthesis enzymes in kidney biopsies from human allograft patients and patients with chronic kidney disease (CKD) at different stages. We used ischaemia–reperfusion injury (IRI) and cisplatin injection to model AKI, urinary tract obstruction [unilateral ureteral obstruction (UUO)] and tubulointerstitial fibrosis induced by proteinuria to investigate CKD in mice. We assessed the effect of nicotinamide riboside (NR) supplementation on AKI and CKD in animal models. Results RNA sequencing analysis of human kidney allograft biopsies during the reperfusion phase showed that the NAD+de novo synthesis is impaired in the immediate post-transplantation period, whereas the salvage pathway is stimulated. This decrease in de novo NAD+ synthesis was confirmed in two mouse models of IRI where NR supplementation prevented plasma urea and creatinine elevation and tubular injury. In human biopsies from CKD patients, the NAD+de novo synthesis pathway was impaired according to CKD stage, with better preservation of the salvage pathway. Similar alterations in gene expression were observed in mice with UUO or chronic proteinuric glomerular disease. NR supplementation did not prevent CKD progression, in contrast to its efficacy in AKI. Conclusion Impairment of NAD+ synthesis is a hallmark of AKI and CKD. NR supplementation is beneficial in ischaemic AKI but not in CKD models.

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