<jats:title>Abstract</jats:title><jats:p>Mensacarcin is a potent cytotoxic agent isolated from <jats:italic>Streptomyces bottropensis.</jats:italic> It possesses a high content of oxygen atoms and two epoxide groups, and shows cytostatic and cytotoxic activity comparable to that of doxorubicin, a widely used drug for antitumor therapy. Another natural compound, rishirilide A, was also isolated from the fermentation broth of <jats:italic>S. bottropensis</jats:italic>. Screening a cosmid library of <jats:italic>S. bottropensis</jats:italic> with minimal PKS‐gene‐specific primers revealed the presence of three different type II polyketide synthase (PKS) gene clusters in this strain: the <jats:italic>msn</jats:italic> cluster (mensacarcin biosynthesis), the <jats:italic>rsl</jats:italic> cluster (rishirilide biosynthesis), and the <jats:italic>mec</jats:italic> cluster (putative spore pigment biosynthesis). Interestingly, luciferase‐like oxygenases, which are very rare in <jats:italic>Streptomyces</jats:italic> species, are enriched in both the <jats:italic>msn</jats:italic> cluster and the <jats:italic>rsl</jats:italic> cluster. Three cosmids, cos2 (containing the major part of the <jats:italic>msn</jats:italic> cluster), cos3 (harboring the <jats:italic>mec</jats:italic> cluster), and cos4 (spanning probably the whole <jats:italic>rsl</jats:italic> cluster) were introduced into the general heterologous host <jats:italic>Streptomyces albus</jats:italic> by intergeneric conjugation. Expression of cos2 and cos4 in <jats:italic>S. albus</jats:italic> led to the production of didesmethylmensacarcin (DDMM, a precursor of mensacarcin) and the production of rishirilide A and B (a precursor of rishirilide A), respectively. However, no product was detected from the expression of cos3. In addition, based on the results of isotope‐feeding experiments in <jats:italic>S. bottropensis</jats:italic>, a putative biosynthesis pathway for mensacarcin is proposed.