<jats:title>ABSTRACT</jats:title> <jats:p> Polyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT <jats:sub>0</jats:sub> in the nystatin PKS loading module NysA with the propionate-specific AT <jats:sub>1</jats:sub> from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT <jats:sub>0</jats:sub> domain by site-directed mutagenesis were accomplished. However, none of the NysA mutants constructed was able to initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids were functional, providing for different levels of nystatin biosynthesis. An interplay between certain residues in AT <jats:sub>0</jats:sub> and an active site residue in the ketosynthase (KS)-like domain of NysA in initiation of nystatin biosynthesis was revealed. Some hybrids between the NysA and RimA loading modules carrying the NysA AT <jats:sub>0</jats:sub> domain were able to prime rimocidin PKS with both acetate and butyrate units upon complementation of a <jats:italic>rimA</jats:italic> -deficient mutant of the rimocidin/CE-108 producer <jats:named-content content-type="genus-species">Streptomyces diastaticus</jats:named-content> . Expression of the PimS0 loading module from the pimaricin producer in the same host, however, resulted in production of CE-108 only. Taken together, these data indicate relaxed substrate specificity of NysA AT <jats:sub>0</jats:sub> domain, which is counteracted by a strict specificity of the first extender module KS domain in the nystatin PKS of <jats:named-content content-type="genus-species">Streptomyces noursei</jats:named-content> .