Guidelines for Optimizing Type S Nonribosomal Peptide Synthetases

[Image: see text] Bacterial biosynthetic assembly lines, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), play a crucial role in the synthesis of natural products that have significant therapeutic potential. The ability to engineer these biosynthetic assembly lines offers opportunities to produce artificial nonribosomal peptides, polyketides, and their hybrids with improved properties. In this study, we introduced a synthetic NRPS variant, termed type S NRPS, which simplifies the engineering process and enables biocombinatorial approaches for generating nonribosomal peptide libraries in a parallelized high-throughput manner. However, initial generations of type S NRPSs exhibited a bottleneck that led to significantly reduced production yields. To address this challenge, we employed two optimization strategies. First, we truncated SYNZIPs from the N- and/or C-terminus of the NRPS. SYNZIPs comprise a large set of well-characterized synthetic protein interaction reagents. Second, we incorporated a structurally flexible glycine–serine linker between the NRPS protein and the attached SYNZIP, aiming to improve dynamic domain–domain interactions. Through an iterative optimization process, we achieved remarkable improvements in production yields, with titer increases of up to 55-fold compared to the nonoptimized counterparts. These optimizations successfully restored production levels of type S NRPSs to those observed in wild-type NRPSs and even surpassed them. Overall, our findings demonstrate the potential of engineering bacterial biosynthetic assembly lines for the production of artificial nonribosomal peptides. In addition, optimizing the SYNZIP toolbox can have valuable implications for diverse applications in synthetic biology, such as metabolic engineering, cell signaling studies, or engineering of other multienzyme complexes, such as PKSs.