How to engineer giant enzymes: A methodology for mutagenesis of polyketide synthases in native hosts

Natural products are a fascinating source of chemical diversity and their biosynthetic pathways of biological complexity. The investigation and engineering of biosynthetic pathways towards polyketides in Actinomycetes provides challenges across all steps of the mutagenesis procedure. The typically GC-rich and long genes require robust PCR protocols. The resulting amplicons, often exceeding 10 kbp in length, require equally robust cloning procedures. Finally, the genetic manipulation of Actinomycetes, especially Streptomyces spp., calls for specialized procedures, in particular when the construction of several hundred variants is needed. This chapter will detail methods for all three steps of the process and have been previously used to generate numerous polyketide synthase variants in several Actinomycete species.