【作者】 陈贵斌；

【导师】 胡宗定； 元英进；

【作者基本信息】 天津大学 ， 生物化工， 2003， 博士

【摘要】 抗生素生物合成和调控机制是代谢工程改造生物合成途径用来提高菌种生产能力以及获得新型杂合抗生素的基础。为了提高新型抗生素AGPM的生产以及获得类似结构新药物，本论文首次开展其生物合成和调控机制的研究。首先，采用刺激实验和静息细胞培养实验法来确定AGPM的合成前体。考察了不同氨基酸和脂肪酸等对抗生素合成的影响，结果表明缬氨酸、异亮氨酸、蛋氨酸、谷氨酸以及乙酸、丙酸、丁酸等具有前体的作用，能够刺激抗生素的生产。而且它们都具有转化为聚酮合成前体的共同特征。所以聚酮途径被认为是抗生素AGPM合成的主要途径。为了证明抗生素AGPM由聚酮合成，进而考察不同代谢途径的抑制剂对抗生素AGPM合成的影响，结果表明莽草酸途径的抑制剂三甲胺和甲羟戊酸途径的抑制剂邻氨基苯甲酸及3,4-二羟苯甲酸，对抗生素AGPM 的生物合成没有影响；而许多聚酮合成关键酶的抑制剂对抗生素AGPM的生物合成具有强烈的抑制作用，如β-酮酯酰-ACP合成酶专一性抑制剂浅蓝菌素和碘乙酰胺、咪唑、乙酰CoA羧化酶抑制剂二噁烷、硫酯酶抑制剂2,4-二硝基氟苯、甲基丙二酰异构酶和缬氨酸脱氢酶抑制剂Cu2+、Zn2+和Co2+等。从而从正反两面都证明了抗生素AGPM由聚酮途径合成。同位素标记法进一步被用来阐明AGPM分子中碳骨架的来源。结果显示CH3-13COONa标记后C-1、C-3、C-5、C-7、C-9、C-12、C-14、C-16、C-19、C-21、C-24、C-28自然丰度增加，说明它们来自乙酸钠的羧基碳，从而根据聚酮合成的机制判断AGPM的碳骨架主要由6分子乙酸以及相同数目的丙酸活性单位构成，另外两个碳原子可能来自蛋氨酸。按聚酮合成机理，提出抗生素AGPM碳骨架生物合成模型。应用阻断突变株进一步进行互补共合成实验，阐明了在碳骨架形成后，抗生素AGPM的生物合成还需经历A→B→C→AGPM等多个中间产物合成的步骤，它们分别与三类阻断突变株相对应。分离这些中间产物最终将阐明抗生素AGPM骨架合成后的一些结构修饰过程。为了阐明菌体自身对抗生素AGPM生物合成的调控，建立了适合抗生素AGPM间歇式发酵的动力学模型和藤黄灰链霉菌的代谢网络模型，将两者有机结合对菌体不同生长时期的代谢流分布进行分析，结果表明：（1）在不同生长时期，菌体自身对胞内各主要代谢途径的代谢流分布具有调控作用，即随发酵时间增加，EMP途径代谢通量明显的下降，而HMP途径和TCA循环的代谢通量增加；（2）丙酮酸、5-磷酸核酮糖、乙酰CoA、5-磷酸核糖焦磷酸以及磷酸<WP=4>甘油酸和草酰乙酸等刚性节点是代谢工程提高菌种产素能力的主要操作对象；（3）供氧状况对不同时期的胞内代谢流分布有明显的影响，合理的供氧有利于AGPM的高产。另外，对培养基中的葡萄糖、铵离子或者磷酸盐等组分对抗生素AGPM生物合成的调控机理进行研究，实验表明了葡萄糖和磷酸盐主要是通过糖的磷酸化以及6-磷酸葡萄糖的积累反馈抑制糖分解代谢对抗生素AGPM的生物合成进行调控；而铵离子则通过抑制糖代谢EMP途径和HMP途径的酶（6－磷酸脱氢酶），激活TCA循环的酶，如琥珀酸脱氢酶等机制对抗生素AGPM生物合成进行调控。根据以上实验结论，最终提出AGPM高产菌种推理选育的一些策略。更多还原

【Abstract】 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 <WP=6>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更多还原