Modular polyketide synthases catalyze the biosynthesis of medicinally impor
tant natural products through an assembly-line mechanism. Although these me
gasynthases display very precise overall selectivity, we show that their co
nstituent modules are remarkably tolerant toward diverse incoming acyl chai
ns. By appropriate engineering of linkers, which exist within and between p
olypeptides, it is possible to exploit this tolerance to facilitate the tra
nsfer of biosynthetic intermediates between unnaturally linked modules. Thi
s protein engineering strategy also provides insights into the evolution of
modular polyketide synthases.