<jats:title>ABSTRACT</jats:title><jats:sec><jats:label /><jats:p>Cloning and engineering of natural product biosynthetic pathways followed by heterologous expression in a tractable host is a widely used approach for expression and genetic modification of microbial secondary metabolites. Herein, we employed <jats:italic>ccdB</jats:italic> counterselection combined with oligonucleotide‐mediated recombineering to efficiently create point mutations in a complex nonribosomal peptide synthetase (NRPS) from <jats:italic>Photorabdus luminescens</jats:italic> directing the biosynthesis of luminmides. After in depth analysis of the luminmide production profile from the native NRPS, single and double point mutations were rationally constructed within the adenylation (A) domain from NRPS module 3 which turned out to have a broad substrate tolerance. Expression of mutated versions of the 15.6 kb NRPS gene <jats:italic>plu3263</jats:italic> in <jats:italic>E. coli</jats:italic> led to alterations in luminmide production profiles and allowed to direct the biosynthesis towards certain derivatives. These results demonstrate the suitability of counterselection recombineering for site‐directed mutagenesis of complex expression constructs, e.g., genes encoding NRPS biosynthetic pathways in multi‐copy plasmids. Biotechnol. Bioeng. 2015;112: 1343–1353. © 2015 Wiley Periodicals, Inc.</jats:sec>