Harnessing the promiscuity of natural product biosynthesis: a platform for engineering new pathways with orthogonal specificities

Many natural products are biosynthesized in a modular fashion by the selection and condensation of small molecule building blocks. Chimeric biosynthetic apparatus can be constructed in an attempt to produce analogues for drug discovery. Yet, the scope and utility of such approaches is limited by the inherent substrate specificity and poor functional modularity of most biosynthetic components. Here, we show that several types of biosynthetic machinery is more tolerant towards non-natural building blocks than has been previously recognized. 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 regioselectively modified with non-natural 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.