| Fredericamycin (FDM) A and leinamycin (LNM) are antitumor antibiotics isolated from Streptomyces griseus and Streptomyces atroolivaceus, respectively. Because of their unprecedented molecular structures, unique modes of action, and potent biological activities, investigation of their biosynthesis would provide further insights for polyketide synthases (PKSs) and nonribosomal synthetases (NRPSs) and pave the way for generating novel FDM and LNM analogues by combinatorial biosynthesis. The following four specific topics have thus been addressed:; FdmW was characterized to be a phosphopantetheinyl transferase dedicated for FDM biosynthesis. It prefers its cognate substrate, the FdmH acyl carrier protein (ACP) to heterologous ACPs in vitro. Inactivation of fdmW reduced FDM yield to 7% of the wild-type strain, and complementation of the DeltafdmW mutant by expressing fdmW in trans restored FDM production.; The Lnm PKS-NRPS interface is characterized by a mutated ketoacyl synthase (KS) domain and an unusual ACP domain. In vivo site-directed mutagenesis of the KS, ACP and the KS of the following module support that they are all essential for LNM biosynthesis. The unusual ACP could be phosphospantetheinylated in vitro. These results suggest a novel mechanism for transition from peptide to polyketide biosynthesis.; Isotope feeding experiments with 13C labeled precursors suggest that the C-1', C-2' and C-3' carbons in LNM are from propionate. A three gene cassette was identified to be involved in the biosynthesis of the three carbons. The three genes were individually inactivated and the resultant mutants abolished LNM production, while accumulated six novel hybrid peptide-polyketides without the dithiolane ring. These results support the attachment of the three carbons via a 3-hydroxy-3-methylglutyryl CoA synthase catalyzed Claisen condensation, which is likely the first step for the formation of the dithiolane moiety.; LnmO was determined as a CRP/FNR type transcriptional regulator by in silico analysis. Gene inactivation and complementation experiments support that it is a pathway-specific transcriptional activator for LNM biosynthesis. Introduction of an additional copy of lnmO into the anticancer compound TG-25 producing strain led to an approximate 3-fold yield increase. |
