Cosynthesis in mixed culture and protoplast fusion of non-producing mutants of Streptomyces hygroscopicus which may produce biosynthetic intermediates of bialaphos (AMPBA) were studied. Non-producing mutants were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment, and six stable non-producing mutants were used for cosynthesis and protoplast fusion studies. The biosynthetic block sites of the non-producers were consistent with studies of the biosynthetic pathway of AMPBA.Bialaphos (AMPBA) is a metabolite of Streptomyces hygroscopicus ATCC 21705 having strong herbicide activity). The structure of this substance was determined to be 2-amino-4-(hydroxy)(methyl)-phosphinoylbutyrylalanylalanine2), and is the first natural product to have the unique C-P-C bond of the phosphinothricyl moiety). In previous papers4,5), we have described non-producing mutants of AMPBA producer obtained by mutagenesis with NTG treatment; these mutants produced derivatives or intermediates of AMPBA. We have further investigated the biosynthetic pathway of AMPBA by the technique of mixed culture cosynthesis and protoplast fusion using six additional AMPBA nonproducing mutants of S. hygroscopicus. The present paper deals with the determination of blocked sites in non-producers and the results of a complementation study by cosynthesis and protoplast fusion. Materials and MethodsOrganisms Streptomyces hygroscopicus ATCC 21705 was used parental strain. Proteus sp. MB-838 was used for the assay of AMPBA. Media Seed culture medium (S-1) was prepared with 2.0 % soluble starch, 1.0 % peptone, 0.3 % meat extract, 0.05 % KZHPO4, pH 7.0. Agar medium (A-4: 0.4% glucose, 0.35% wheat germ, 0.23% soluble vegetative protein, 0.03 % KH2PO4 0.0001 % CoCl2.6H2O, 2.0 % agar, pH 7.0) was used for cosynthesis experiments and selection of non-producing mutants by the use of agar piece method.') Cosynthesis on an Agar Plate Cosynthesis on agar plates was carried out by modifying the procedure derived by DELIC et al.') and FURUMAI et a1.8•0). The mycelial suspension of two non-producing mutants was harvested from S-1 medium (0.1 ml each) and spread about 1 -2 mm apart on opposite halves of an A-4 medium plate. The plate was incubated for 7 days at 28°C. An agar strip, 5 x 60 mm was cut from the plate at right angles to the line of separation between the two mutants. This strip was plated on the surface of an agar plate seeded with Proteus sp. MB-838, and incubated at 32°C overnight. The appearance of an inhibition zone along the agar strip was considered a sign of the production of AMPBA.
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