Abstract: Many clinically used drugs are derived from secondary metabolites that are biosynthesized in a modular fashion by the selection and condensation of small molecule building blocks. Chimeric biosynthetic pathways can be constructed in an attempt to produce analogues for drug discovery. Yet, the scope and utility of this combinatorial approach is limited by the inherent substrate specificity and poor functional modularity of most biosynthetic machinery. Here, we show that biosynthetic pathways are tolerant towards non-natural building blocks. Such promiscuity forms a platform for constructing new biosynthetic parts with substrate specificities orthogonal to those found in Nature. Accordingly, we describe a comprehensive program of enzyme engineering, directed evolution, and synthetic biology aimed at constructing artificial bacterial strains capable of producing complex natural products that are modified with unnatural chemical functionality. Our synthetic biology approach expands the synthetic capabilities of natural product diversification strategies and provides an improved understanding of the molecular basis for specificity in complex molecular assemblies.