Engineering polyketide synthases and nonribosomal peptide synthetases

•We review engineering of polyketide synthases and nonribosomal peptide synthetases.•Rational and evolutionary strategies for altering building block specificity are highlighted.•Reengineering protein:protein interactions have recently become viable.•These advances will provide new parts for natural product diversification.

Naturally occurring polyketides and nonribosomal peptides with broad and potent biological activities continue to inspire the discovery of new and improved analogs. The biosynthetic apparatus responsible for the construction of these natural products has been the target of intensive protein engineering efforts. Traditionally, engineering has focused on substituting individual enzymatic domains or entire modules with those of different building block specificity, or by deleting various enzymatic functions, in an attempt to generate analogs. This review highlights strategies based on site-directed mutagenesis of substrate binding pockets, semi-rational mutagenesis, and whole-gene random mutagenesis to engineer the substrate specificity, activity, and protein interactions of polyketide and nonribosomal peptide biosynthetic machinery.