The biologically active compound mensacarcin is produced by Streptomyces bottropensis. The cosmid cos2 contains a large part of the mensacarcin biosynthesis gene cluster. Heterologous expression of this cosmid in Streptomyces albus J1074 led to the production of the intermediate didesmethylmensacarcin (DDMM). In order to gain more insights into the biosynthesis, gene inactivation experiments were carried out by λ-Red/ET-mediated recombination, and the deletion mutants were introduced into the host S. albus. In total, 23 genes were inactivated. Analysis of the metabolic profiles of the mutant strains showed the complete collapse of DDMM biosynthesis, but upon overexpression of the SARP regulatory gene msnR1 in each mutant new intermediates were detected. The compounds were isolated, and their structures were elucidated. Based on the results the specific functions of several enzymes were determined, and a pathway for mensacarcin biosynthesis is proposed.

Mensacarcin, a potential antitumour drug, is produced by Streptomyces bottropensis. The structure consists of a three-membered ring system with many oxygen atoms. Of vital importance in this context is an epoxy moiety in the side chain of mensacarcin. Our studies with different mensacarcin derivatives have demonstrated that this epoxy group is primarily responsible for the cytotoxic effect of mensacarcin. In order to obtain further information about this epoxy moiety, inactivation experiments in the gene cluster were carried out to identify the epoxy-forming enzyme. Therefore the cosmid cos2, which covers almost the complete type II polyketide synthase (PKS) gene cluster, was heterologously expressed in Streptomyces albus. This led to production of didesmethylmensacarcin, due to the fact that methyltransferase genes are missing in the cosmid. Further gene inactivation experiments on this cosmid showed that MsnO8, a luciferase-like monooxygenase, introduces the epoxy group at the end of the biosynthesis of mensacarcin. In addition, the protein MsnO8 was purified, and its crystal structure was determined to a resolution of 1.80 Å.