銅耐性腐朽菌に特有なシュウ酸合成複合酵素系の機能解析とその制御法の開発

耐铜木材腐烂真菌是一种棕腐担子菌属真菌，通过草酸盐的生物合成将铜沉淀成草酸铜，从而解毒含铜木材防腐剂。因此，阐明这些耐铜真菌中草酸盐生物合成的生化机制具有重要意义。本次调查的新发现总结如下。我们发现，尽管在富葡萄糖培养基中生长，但大多数木材腐烂的担子菌都具有乙醛酸循环酶，这与在葡萄糖培养基中生长的微生物中乙醛酸循环酶被抑制的经典观点形成鲜明对比。我们首次从一种耐铜真菌担子菌Tyromyces (Fomitopsis) palustris中纯化并鉴定了glyxolyate循环的关键酶，如异柠檬酸裂解酶（ICL）和苹果酸合成酶（MS）。重要的是，ICL酶抑制剂可以有效地抑制草酸盐的生物合成，更有效地控制耐铜真菌palustris Tyromyces。我们发现TCA和乙醛酸循环在草酸的生物合成中发挥重要作用，为真菌的生长提供能量4。褐腐菌在子实体形成过程中，草酸生物合成停止，乙醛酸循环酶和草酸生成酶消失，而异位脱氢酶（ICDH）被激活并占主导地位。结果进一步表明，在真菌生长后期，ICDH比ICL更重要地支持GABA途径运行TCA循环。重要的是，在这种真菌的整个生命周期中，发现它缺乏a-酮戊二酸脱羧酶，这是植物和动物正常TCA循环功能的关键酶。因此，在这些耐铜真菌中，乙醛酸循环和GABA途径交替发挥重要作用，以补偿正常TCA循环的功能障碍。少

Copper-tolerant wood rotting fungi most of which belong to a group of brown-rot basidiomycestes have been recognized to detoxicate copper-contining wood preservertives by precipitating copper into copper oxalate by biosynthesis of oxlate. Thus, it is very important to elucidate biochemical mechanisms for oxalate biosyntheis in these copper-tolerant fungi.New findings obtained by this ivestigation are summized as follows.1. We have discovered that most of the wood-rotting basidiomyceses have the glyoxylate cycle enzymes although thy were grown on gluose-rich medium, which is sharply contrasted with the clasical view that the glyolxylate cycle enzymes are repressed in microorganisms grown on glucose.2. We sucessfuly purified and characterized the key enzymes of the glyxolylate cycle such as isocitrate lyase (ICL) and malate synthase (MS) for the first time from a basidiomycetous fungus Tyromyces (Fomitopsis) palustris, which is a copper-tolerant fungus.3. Importanlty, we could successful … More ly control the copper-tolerant fungus Tyromyces palustris with ICL enzyme inhibior, which also found potently inhibit the oxalate bioxynthesis. We have found that both TCA and glyoxylate cycles play an important role in biosynthesis of oxalic acid to produce energy for the fungal growth4. During the fruitbody formation of the brown-rot fungus, the oxalate biosynthesis was ceased and the glyoxylate cycle enzymes and oxalate producing enzynes disappeared, whereas isocitate dehydrogenase (ICDH) was activated and predominate over the ICL.The results further indicate that at the later stage of thefungal growth ICDH played more important role than ICL to support the GABA route for operation of the TCA cycle.5. Importantly, throughout the life cycle of thisfungus, it was found to lack a-ketoglutarate decarbxylase which is a key enzyme of normal TCA cycle functioning in both plants and animals. Thus, the glyoxylate cycle and GABA route play alternately importantl role to compensate the desfunction of the normal TCA cycle in these copper-tolerant fungi. Less