Biosynthetic Studies of C-P Compounds by the Use of a New Gene Modification Technique

本研究的目的是利用一种新的技术研究吸水链霉菌产生双丙氨磷的生物合成过程，更具体地说是研究非常独特的C-P键的形成机制双丙氨磷生物合成中的一种C-P键形成机制是由磷酸烯醇丙酮酸与膦甲酸反应生成膦基丙酮酸，从化学观点来看，它至少包含两个反应。然而，由于缺乏合适的阻断突变体，对这种机制的研究是不可行的。因此，我们尝试通过使用体外衍生突变引入突变来制备新型阻断突变体。结果，我们可以获得一个新的突变体。共合成实验，使用这种突变体和另一个已通过常规方法获得揭示了一个新的生物合成中间体在此反应中的参与。我们分离了这种代谢产物，并确定其结构为羧基膦酰基烯醇丙酮酸。此外，我们还纯化了将其转化为膦基丙酮酸的新酶，并克隆了相应的基因。我们利用同样的技术研究了另一个C-P键形成反应，即将磷酸烯醇丙酮酸转化为膦基丙酮酸。这种反应在产生C-P化合物的生物体中普遍存在。结果，我们成功地制备了不能催化该反应的阻断突变体，并克隆了对应于该反应的基因。此外，我们可以在另一种微生物中表达该基因，变铅青链霉菌。这一成功使我们能够更详细地研究C-P键的形成机制。因此，应该强调的是，我们所开发的技术是一个非常有用的方法来研究微生物代谢产物的生物合成机制。

The purpose of this research is to study the biosynthesis of bialaphos produced by Streptomyces hygroscopicus, more specifically the formation mechanisms of very unique C-P bonds, by the use of a new technique (in vitro derived mutation) which we have developed recently.One C-P bond forming mechanism in the biosynthesis of bialaphos, which consists of the reaction between phosphoenolpyruvate and phosphonoformic acid to give phosphinopyruvic acid, was considered to consist of at least two reactions from the chemical point of view. Investigation of this mechanism, however, was not feasible due to the lack of suitable blocked mutants. Therefore, we attempted to prepare new types of blocked mutants by introducing mutation through the use of in vitro derived mutation. As a result, we could obtain a new mutant. Cosynthesis experiments using this mutant and another one which had been obtained by a conventional method revealed the involvement of a new biosynthetic intermediate in this reaction. We isolated this metabolite and determined its structure to be carboxyphosphonoenolpyruvate. Furthermore, we purified the new enzyme which converted it to phosphinopyruvate, and cloned the corresponding gene.We utilized the same technique to investigate another C-P bond forming reaction, which transformed phosphoenolpyruvate to phosphonopyruvate. This reaction is ubiquitous among organisms which produce C-P compounds. As a result, we succeeded in preparation of blocked mutants unable to catalyze this reaction and we cloned the gene corresponding to this reaction. Furthermore, we could express this gene in another microorganism, Streptomyces lividans. This success enabled us to investigate the C-P bond formation mechanism in more detail. Therefore, it should be emphasized that the technique we have developed is a very useful method to study the biosynthetic mechanism of microbial metabolites.