Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase

Background: A single modular polyketide synthase (PKS) gene cluster is resp onsible for production of both the 14-membered macrolide antibiotic picromy cin and the 12-membered macrolide antibiotic methymycin in Streptomyces ven ezuelae. Building on the success of the heterologous expression system engi neered using the erythromycin PKS, we have constructed an analogous system for the picromycin/methymycin PKS. Through heterologous expression and cons truction of a hybrid PKS, we have examined the contributions that the PKS, its internal thioesterase domain (pikTE) and the Pik TEII thioesterase doma in make in termination and cyclization of the two polyketide intermediates. Results: The picromycin/methymycin PKS genes were functionally expressed in the heterologous host Streptomyces lividans, resulting in production of bo th narbonolide and 10-deoxymethynolide (the precursors of picromycin and me thymycin, respectively). Go-expression with the Pik TEII thioesterase led t o increased production levels, but did not change the ratio of the two comp ounds produced, leaving the function of this protein largely unknown. Fusio n of the PKS thioesterase domain (pikTE) to 6-deoxyerythronolide B synthase (DEBS) resulted in formation of only 14-membered macrolactones. Conclusions: These experiments demonstrate that the PKS alone is capable of catalyzing the synthesis of both 14- and 12-membered macrolactones and fav or a model by which different macrolactone rings result from a combination of the arrangement between the module 5 and module 6 subunits in the picrom ycin PKS complex and the selectivity of the pikTE domain.