The timing of introduction of the unusually placed Delta(11,13) diene system in ansamitocin (AP) biosynthesis was probed by synthesizing optically active potential tri- and tetraketide intermediates as their SNAC thioesters. An AP-nonproducing mutant Actinosynnema pretiosum was complemented by the R enantiomer of the triketide and by the tetraketide with rearranged double bonds, but not by the tetraketide carrying the double bonds in conjugation to the thioester function. The results show that the double bonds are installed in their final positions during processing of the nascent polyketide on module 3 of the asm PKS and that KS4 of the PKS acts as a gatekeeper which accepts only a tetraketide with shifted double bonds as substrate for further processing.