Responsible for the Vitamin B12 dependent methylation of the beta-lactam ring in the thienamycin (thn) gene cluster. It is currently not known what the methyl donor is, but in keeping with the other B12-dependent methyltransferases, it has been shown as methylcobalamin here. Mutagenesis performed on the thnP gene product demonstrated that thnP is essential for thienamycin biosynthesis. However, the mutant retained the ability to produce cephamycin, thus showing that ThnP is not involved in cephamycin C biosynthesis.
Rodríguez M, Núñez LE, Braña AF, Méndez C, Salas JA, Blanco G
Mutational analysis of the thienamycin biosynthetic gene cluster from Streptomyces cattleya
▸ Abstract
The generation of non-thienamycin-producing mutants with mutations in the thnL, thnN, thnO, and thnI genes within the thn gene cluster from Streptomyces cattleya and their involvement in thienamycin biosynthesis and regulation were previously reported. Four additional mutations were independently generated in the thnP, thnG, thnR, and thnT genes by insertional inactivation. Only the first two genes were found to play a role in thienamycin biosynthesis, since these mutations negatively or positively affect antibiotic production. A mutation of thnP results in the absence of thienamycin production, whereas a 2- to 3-fold increase in thienamycin production was observed for the thnG mutant. On the other hand, mutations in thnR and thnT showed that although these genes were previously reported to participate in this pathway, they seem to be nonessential for thienamycin biosynthesis, as thienamycin production was not affected in these mutants. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of all available mutants revealed some putative intermediates in the thienamycin biosynthetic pathway. A compound with a mass corresponding to carbapenam-3-carboxylic acid was detected in some of the mutants, suggesting that the assembly of the bicyclic nucleus of thienamycin might proceed in a way analogous to that of the simplest natural carbapenem, 1-carbapen-2-em-3-carboxylic acid biosynthesis. The accumulation of a compound with a mass corresponding to 2,3-dihydrothienamycin in the thnG mutant suggests that it might be the last intermediate in the biosynthetic pathway. These data, together with the establishment of cross-feeding relationships by the cosynthesis analysis of the non-thienamycin-producing mutants, lead to a proposal for some enzymatic steps during thienamycin assembly.
