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

耐铜木材腐烂真菌大多属于一类褐腐菌，它们通过生物合成草酸铜将铜沉淀成草酸铜，从而对含铜木材防腐剂进行解毒。因此，阐明这些耐铜真菌中草酸生物合成的生化机制是非常重要的。我们发现，大多数腐木担子菌都有乙醛酸循环酶，尽管它们是在富含葡萄糖的培养基上生长的，这与传统观点认为乙醛酸循环酶在生长在葡萄糖上的微生物中受到抑制形成了鲜明的对比。首次从耐铜担子菌Tyromyces(Fomitopsis)palustris中分离纯化并鉴定了乙醇酸循环中的关键酶异柠檬酸裂解酶(ICL)和苹果酸合成酶(MS)。重要的是，我们可以成功实现…ICL酶抑制剂对耐铜真菌Tyromyces palustris有较好的抑制作用，对草酸的生物氧合也有抑制作用。我们发现，三氯乙酸和乙醛酸环在草酸的生物合成中扮演着重要的角色，为真菌的生长产生能量。在褐腐菌的子实体形成过程中，草酸的生物合成停止，乙醛酸循环酶和草酸产生酶消失，而异位酸脱氢酶(ICDH)被激活并占据主导地位。结果进一步表明，在真菌生长的后期，ICDH在支持GABA途径的TCA循环中起着比ICL更重要的作用。重要的是，在这种真菌的整个生命周期中，它被发现缺乏a-酮戊二酸脱羧酶，而a-酮戊二酸脱羧酶是植物和动物正常TCA循环功能的关键酶。因此，乙醛酸循环和GABA途径在这些耐铜真菌中交替发挥重要作用，以补偿正常的TCA循环的功能丧失。较少

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