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

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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.