| Antibiotic biosynthesis and regulation mechanism is the basis of metabolic engineering to improve strain's productivities and obtain new hybrid antibiotics through the biosynthetic pathways' modification or reconstruction. To increase its production and obtain new pharmaceuticals of similar structure, the novel antibiotic AGPM biosynthesis and regulation mechanism was studied in this dissertation.First, feeding experiment and resting cell system were used to confirm precursors of AGPM. Different amino acids and branched fatty acids' effects on AGPM production were investigated, and the results showed those amino acids and branched fatty acids, such as valine, isoleucine, methionine, glutamine, acetate, propionate, butyrate could act as precursors and stimulate AGPM biosynthesis. Besides, these compounds had the same character –all could transform to the precursors of polyketide biosynthesis. So the polyketide pathway was presumed to be the main biosynthetic pathway of antibiotic AGPM. To testify above hypothesis, influences of different metabolic pathways' inhibitors on antibiotic AGPM biosynthesis were examined and the following results were obtained: Inhibitor of shikimic acid pathways -trimethylamine or inhibitors of mevalonic acid pathways-anthranilic acid and 3,4-dihydroxybenzoic acid had no effects on AGPM biosynthesis. Whereas, AGPM biosynthesis was severely inhibited by inhibitors of the key enzymes in the polyketide pathways, such as the specific inhibitors of β-ketoacyl-acyl carrier protein synthase –cerulenin, iodiacetamide or imidazole, the acyl-CoA carboxylase inhibitor-dioxane, and the thioesterase inhibitor- 2,4-dinitrofluorobenzene, methyl-malonyl CoA isomerase and valine dehydrogenase inhibitors- Cu2+,Zn2+ and Co2+ et al. Polyketide pathway was therefore confirmed as the main biosynthetic pathways of antibiotic AGPM by evidence both pro and con. The origin of carbon skeleton in antibiotic AGPM was further illustrated through stable isotope labeled experiment. 13C NMR chromatogram of AGPM indicated that the abundance of C-1, C-3, C-5, C-7, C-9, C-12, C-14, C-16, C-19, C-21, C-24, C-28 was enriched after adding CH3-13COONa. So it testified these carbons were incorporated from that carboxyl carbon in acetate. From polyketide synthesis mechanism, six acetates and the same number of propionates were analyzed being the main components of the carbon skeleton of AGPM. Thus, a biosynthetic model of AGPM carbon skeleton was advised to depict its biosynthesis course. Cosynthesis experiment of blocked mutants elucidated that antibiotic AGPM biosynthesis should also go through the following intermediates: A→B→C→AGPM, which were corresponding to the three kinds of blocked mutants. Characterization of them would ultimately illuminate the structure's modification course after the formation of AGPM carbon skeleton. To examine the regulation of cells on antibiotic biosynthesis, a kinetic model of AGPM fermentation and metabolic network model of Streptomyces leutogriseus were founded firstly. And the metabolic fluxes in different growth stage were analyzed by systemic linkup of them. The results showed: (1) Cells could regulate metabolic flux distribution in different growth stage, i.e. the flux of HMP pathway and TCA cycle increased while that of EMP pathway decreased in culture course; (2) Rigid nodes such as pyruvate, 5-P-ribulose, acetyl-CoA, 5-PRPP, 3-P-glycerate and oxaloacetic acid were identified to be the main targets of manipulation by metabolic engineering to improve strain's productivity. (3) Oxygen supply affected obviously on metabolic fluxes of cells in different growth stage and rational control of oxygen supply could increase antibiotic production. At last, the regulation mechanisms of glucose, ammonia and phosphate on AGPM biosynthesis were studies. The results showed glucose and phosphate had the same regulation mechanism, and both of them feedback inhibited catabolism of sugar through 6-P-glucose accumulation; while ammonium inhibited enzymes of EMP and HMP, but activa...
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