PROJECT SUMMARY/ABSTRACT Bacteria from the order Actinomycetales, especially those from the genus Streptomyces, are some of the most prolific producers of bioactive natural products, including over 75% of commercially available antibiotics. Although other orders of bacteria harbor biosynthetic pathways, the metabolic giftedness of actinomycetes is the gold standard for pharmaceutical discovery. In particular, they harbor many pathways that are modular such as type I polyketide synthases (PKSs) and type I non-ribosomal peptide synthetases (NRPSs), which have been noted for their considerable potential for engineered biosynthesis for well over 30 years. However, actinomycetes present a number of challenges with their growth profiles and genetic systems that serve as less than ideal as heterologous hosts, especially for rapid profiling of engineered systems or for protein production for enzymology studies. Furthermore, their high GC genomes (~75% GC) complicate routine molecular biology, genetic manipulations, and transcriptional and translational efficiency. This is particularly the case with megasynthase enzymes such as PKSs and NRPSs, which are large in size and frequently express poorly, as truncated products, or do not fold correctly. While such issues are discussed in the natural products enzymology community, few systematic studies exist in the literature to understand the functional consequences of heterologous host choice and refactoring, especially at the level at which it affects protein function. Furthermore, many of the systems that would be ideal to profile megasynthases (such as in vitro transcription-translation; TXTL) create irregularities with regard to truncation and folding, which are also poorly characterized. We will examine the functional consequences of host and refactoring-dependent expression on such actinomycetal megasynthases at the protein level. The characterization described in this proposal will lead to a more systematic understanding of the benefits and drawbacks of different systems of heterologous expression for different applications which include characterizing and profiling engineered megasynthases and ultimately producing small molecule metabolites. This work will afford the realization of the full potential of proteins from actinomycetes as a rich source for synthetic biology parts for engineered metabolism.