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An enzymatic activity catalyzing P-methylation of Af-acetyldemethylphosphinothricin, a biosynthetic intermediate of the herbicide bialaphos, was detected in a cell extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism. The gene coding for this P-methylation enzyme in the bialaphos biosynthetic gene cluster was also expressed in Streptomyces lividans. The methyl donor of the reaction was determined to be methylcobalamin. The P-methylation enzyme utilized both TV-acetyldemethylbialaphos and A^-acetyldemethylphosphinothricin as substrates. 781Bialaphos (BA) is a tripeptide produced by Streptomyces hygroscopicus SF-1293, and is now in use as a herbicide2). This compoundpossesses a direct carbon-phosphorus-carbon bond system that is quite unique amongnatural products. Wehave previously characterized most of the biosynthetic pathway of BAconsisting of at least 13 steps including three C-P bond-formation steps. One is the intramolecular rearrangement of phosphoenolpyruvic acid (PEP) in step 1 catalyzed by PEP phosphomutase3). Another is the intramolecular rearrangement of carboxyphosphonoenolpyruvate (CPEP) in step 5 catalyzed by CPEP phosphonomutase4), and the last is P-methylation of TV-acetyldemethylphosphinothricin (TV-Ac DMPT)in step 125). We have recently isolated PEP phosphomutase3) and CPEP phosphonomutase1'4'6* and determined their corresponding nucleotide sequences7). The biosynthetic mechanism of the C-P-C bond formation which presumably proceeds by P-methylation of a phosphinic acid derivative(s), however, has remained unclear.In previous reports5'8'9*, we determined the pathway from demethylphosphinothricin (DMPT)to BA as shown in Fig. 1. Mutant NP45 lacks the ability to catalyze methylation ofphosphinic acid derivatives such as TV-Ac DMPT or 7V-acetyldemethylbialaphos (TV-Ac DMBA). The detailed reaction sequence, however, remained to be clarified. Wealso reported that DMPTand demethylbialaphos (DMBA) were accumulated in the fermentation broth of the parent strain cultivated in the absence of cobalt ion5). Thus we assumed that cobalt ion was essential for this P-methylation. Since this seemed to be one of the key steps in the BAbiosynthesis, we have attempted to purify from S. hygroscopicus the enzyme catalyzing the formation of P-methyl derivative(s) from phosphinic acid(s).Here we report the detection of the jP-methylation activity in cell extracts of the BAproducing organism and its expression in Streptomyces lividans.For part 111).

In the course of a screening program, a novel quinolone antibiotic, YM-30059 (Fig. 1) was isolated from the culture broth of Arthrobacter sp. YL-02729S. YM-30059 exhibited relatively high antibacterial activities against Gram-positive bacteria including multiple-drug resistant Staphylococcus aureus and S. epidermidis. The present paper deals with the taxonomy of the producing organism, fermentation, isolation, structure elucidation and biological activity of the new antibiotic.

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