tacrolimus and malonic-acid

FK506 (tacrolimus) is a 23-membered polyketide macrolide that possesses powerful immunosuppressant activity. In this study, feeding soybean oil into the fermentation culture of Streptomyces tsukubaensis improved FK506 production by 88.8%. To decipher the overproduction mechanism, comparative proteomic and metabolomic analysis was carried out. A total of 72 protein spots with differential expression in the two-dimensional gel electrophoresis (2-DE) were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS), and 66 intracellular metabolites were measured by gas chromatography-mass spectrometer (GC-MS). The analysis of proteome and metabolome indicated that feeding soybean oil as a supplementary carbon source could not only strengthen the FK506 precursor metabolism and energy metabolism but also tune the pathways related to transcriptional regulation, translation, and stress response, suggesting a better intracellular metabolic environment for the synthesis of FK506. Based on these analyses, 20 key metabolites and precursors of FK506 were supplemented into the soybean oil medium. Among them, lysine, citric acid, shikimic acid, and malonic acid performed excellently for promoting the FK506 production and biomass. Especially, the addition of malonic acid achieved the highest FK506 production, which was 1.56-fold of that in soybean oil medium and 3.05-fold of that in initial medium. This report represented the first comprehensive study on the comparative proteomics and metabolomics applied in S. tsukubaensis, and it would be a rational guidance to further strengthen the FK506 production.

Topics: Bacterial Proteins; Bioreactors; Citric Acid; Electrophoresis, Gel, Two-Dimensional; Fermentation; Gas Chromatography-Mass Spectrometry; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Gene Ontology; Immunosuppressive Agents; Lysine; Malonates; Metabolic Networks and Pathways; Metabolomics; Molecular Sequence Annotation; Proteomics; Shikimic Acid; Soybean Oil; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptomyces; Tacrolimus

Changes in mitochondrial integrity, reactive oxygen species release and Ca2+ handling are proposed to be involved in the pathogenesis of many neurological disorders including methylmalonic acidaemia and Huntington's disease, which exhibit partial mitochondrial respiratory inhibition. In this report, we studied the mechanisms by which the respiratory chain complex II inhibitors malonate, methylmalonate and 3-nitropropionate affect rat brain mitochondrial function and neuronal survival. All three compounds, at concentrations which inhibit respiration by 50%, induced mitochondrial inner membrane permeabilization when in the presence of micromolar Ca2+ concentrations. ADP, cyclosporin A and catalase prevented or delayed this effect, indicating it is mediated by reactive oxygen species and mitochondrial permeability transition (PT). PT induced by malonate was also present in mitochondria isolated from liver and kidney, but required more significant respiratory inhibition. In brain, PT promoted by complex II inhibition was stimulated by increasing Ca2+ cycling and absent when mitochondria were pre-loaded with Ca2+ or when Ca2+ uptake was prevented. In addition to isolated mitochondria, we determined the effect of methylmalonate on cultured PC12 cells and freshly prepared rat brain slices. Methylmalonate promoted cell death in striatal slices and PC12 cells, in a manner attenuated by cyclosporin A and bongkrekate, and unrelated to impairment of energy metabolism. We propose that under conditions in which mitochondrial complex II is partially inhibited in the CNS, neuronal cell death involves the induction of PT.

Topics: Animals; Antimycin A; Bongkrekic Acid; Brain; Calcimycin; Calcium; Catalase; Cell Survival; Cyclosporins; Dose-Response Relationship, Drug; Drug Interactions; Electron Transport Complex II; Enzyme Inhibitors; Female; In Vitro Techniques; Ionophores; Malonates; Membrane Potentials; Methylmalonic Acid; Mitochondria; NADP; Neurons; Nitro Compounds; Oxygen Consumption; PC12 Cells; Permeability; Propionates; Rats; Rotenone; Tacrolimus; Tetrazolium Salts; Thiazoles; Uncoupling Agents