An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases

Bacterial type I polyketide synthases (PKSs) generate a structurally diverse group of natural products with a wide range of biological activities. Hybrid type I PKSs in which domains of one multifunctional polypeptide are replaced with components from heterologous systems have generated significant interest over the past decade. Almost invariably only one or several specific hybrids are made at a time and tested for functionality. This approach is slow, dependent upon a fortuitous choice of specific fusions points, and often leads to inactive or minimally active hybrid systems. We describe herein a method for generating and screening a library of hybrid pikAI complementation plasmids (encoding the loading domain and the first two extension domains of pikromycin PKS) able to restore pikromycin in a BB138 Streptomyces venezuelae pikAI-deletion mutant. In the first step the plasmid sequence encoding the loading domain AT₀-ACP₀ was replaced by a counter selectable marker, sacB. DNA family shuffling was then used to generate a diverse library of chimeric AT₀-ACP₀ fragments, which were used to replace sacB by λ-Red-mediated in vivo recombination in an Escherichia coli host. This method resulted in the rapid and efficient generation of a large number of hybrid pikAI complementation plasmids, which were used to transform S.venezuelae BB138. A bioassay of over 4000 of these transformants successfully revealed three different PikAI hybrids which were able to lead to pikromycin production. The study suggests that most of the hybrids are not detectably functional, and underscores the need to generate and screen large and diverse libraries in which different fusion points are tried. The methodologies applied in this study address this need and can be used for directed evolution of any component of the PikPKS, and potentially other type I PKS systems.