The previously isolated non-phosphinothricin tripeptide producing Streptomyces viridochromogenes gene disruption mutant SP62/2 was used to identify and analyze genes encoding early steps of the phosphinothricin tripeptide biosynthesis. Cross-feeding and bioconversion experiments between SP62/2 and known non-phosphinothricin tripeptide producing mutants or presumptive phosphinothricin tripeptide precursors revealed that SP62/2 was blocked in step one or two of the phosphinothricin tripeptide biosynthesis. It was shown that the block in the biosynthesis is due to the integration of a temperature-sensitive plasmid by illegitimate recombination into the phosphinothricin tripeptide biosynthetic gene cluster. The corresponding region was isolated from the wild-type. A 2.7-kb DNA fragment was analyzed comprising three ORFs (ppm, ppd, orfX) which are probably translationally coupled. The ppm gene encodes a protein which is similar to PEP-phosphomutases and the deduced Ppd product shows similarity to the phosphonopyruvate decarboxylase from Streptomyces wedmorensis.