The bialaphos biosynthetic genes of Streptomyces hygroscopicus: cloning and analysis of the genes involved in the alanylation step.

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Abstract
We have isolated and studied the genes involved in the alanylation step in the biosynthesis of a herbicide, bialaphos which is produced by Streptomyces hygroscopicus. Three bialaphos-nonproducing mutants, NP60, NP61 and NP62, isolated from S. hygroscopicus by treatment with N-methyl-N''-nitro-N-nitrosoguanidine were defective for the alanylation step and were not restored to productivity by any locus of the gene cluster previously cloned. Three plasmids were isolated using NP60, NP61 and NP62 as recipients. The genes which restored productivity to NP61 and NP62 hybridized to the contiguous region of the bialaphos biosynthetic gene cluster. The gene cluster involved in the bialaphos production was about 35 kb long. The gene which restored productivity to NP60 did not hybridize to the bialaphos biosynthetic gene cluster MV3 and MV4, putative alanylation blocked mutants, were derived from a bialaphos producer by gene replacement of an unidentified region of the biosynthetic gene cluster with an in vitro altered DNA sequence. The genes which restored productivity to VM3 and VM4 were located between the genes which code for phosphinomethylmalic acid synthase and demethylphosphinothricin acetyltransferase in the cluster. These results suggest that multiple genes are involved in the alanylation step.