Cosynthesis and protoplast fusion by mutants of bialaphos (AMPBA) producing Streptomyces hygroscopicus.

Abstract

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.

The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus

“…S . hygroscopicus strains containing cloned bap genes were inoculated onto agar plugs containing A4 medium (Ogawa et al, 19836) and grown for 5 d at 28 "C. The plugs were transferred to the surface of bialaphos assay medium seeded with lawns of the bialaphos-sensitive indicator bacterium, Bacillus subtilis ATCC 6633. Bialaphos production was detected as a zone of growth inhibition.…”

Enzyme Chemistry

“…Incorporation experiments with [13C]-labelled glucose and acetate have established the basic building blocks for the phosphinothricin skeleton while extensive investigations with a series of mutants blocked in antibiotic production have led to the identification of the major intermediates of the bialaphos biosynthetic pathway (Seto et al, 1983a(Seto et al, , 1983bOgawa et al, 1983;Imai etal., 1984Imai etal., , 1985Seto etal., 1984), illustrated in Fig. Incorporation experiments with [13C]-labelled glucose and acetate have established the basic building blocks for the phosphinothricin skeleton while extensive investigations with a series of mutants blocked in antibiotic production have led to the identification of the major intermediates of the bialaphos biosynthetic pathway (Seto et al, 1983a(Seto et al, , 1983bOgawa et al, 1983;Imai etal., 1984Imai etal., , 1985Seto etal., 1984), illustrated in Fig.…”

Exploration of the Use of the “Bialaphos Genes” for Improving Bioherbicide Efficacy

“…In plants, bialaphos is metabolized into phosphinothricin which inhibits glutalnine synthetase (GS)4 activity, leading to ammonia buildup and phytotoxicity (50,51). Bialaphos is produced as a product of conventional microbial fermentation (32,33).…”