Tautomycetin is a specific protein phosphatase I inhibitor. In an effort to elucidate the biosynthetic mechanism of tautomycetin, we inactivated genes of the tautomycetin biosynthetic gene cluster, tauI, tauO, and tauK, which encode for putative P450 oxidase, citryl-CoA lyase, and esterase enzymes, respectively. The mutant STQ0606 (${delta}tauO$) did not produce any detectable amount of tautomycetin intermediates but could convert dialkylmaleic anhydride to tautomycetin, strongly indicating that TauO was involved in dialkylmaleic anhydride biosynthesis. STQ1211 (${delta}tauK$) accumulated dialkylmaleic anhydride, whereas the cofermentation of STQ1211 (${delta}tauK$) and STQ0606 (${delta}tauO$) restored the production of tautomycetin. Together, these results suggest that TauK was responsible for the conjugation of dialkylmaleic anhydride and the polyketide moiety in tautomycetin biosynthesis. The disruption of tauI resulted in the accumulation of 5-des-keto-tautomycetin, revealing that TauI was responsible for the oxidation at C5 as the last step. Although the shunt pathways were involved in the biosynthesis of tautomycetin, the main post-polyketide synthase tailoring steps were dehydration, decarboxylation and oxidation, taking place consecutively. This study allowed us to predict the biosynthesis of tautomycetin more accurately and provided novel insights into the mechanism of the biosynthesis of tautomycetin.