Supplementary data are available at Bioinformatics online.

We thank the patients and their families for their trust in taking part in this study. The study was academically funded and supported by the Medical University Vienna, the General Hospital Vienna, and the Research Center for Molecular Medicine (CeMM) of the Austrian Academy of Sciences. We gratefully acknowledge funding from the Vienna Science and Technology Fund (LS16-034 to GSF and UJ), the Austrian Science Fund (F4704-B20 to PV, F4711-B20 to GSF, and P27132-B20 to PBS), and the European Molecular Biology Organization Long Term Fellowship (1543-2012 to GIV, 733-2016 to TP). BS acknowledges

Non-ribosomal peptide synthetases (NRPSs) are enzymes that catalyze ribosome-independent production of small peptides, most of which are bioactive. NRPSs act as peptide assembly lines where individual, often interconnected modules each incorporate a specific amino acid into the nascent chain. The modules themselves consist of several domains that function in the activation, modification and condensation of the substrate. NRPSs are evidently modular, yet experimental proof of the ability to engineer desired permutations of domains and modules is still sought. Here, we use a synthetic-biology approach to create a small library of engineered NRPSs, in which the domain responsible for carrying the activated amino acid (T domain) is exchanged with natural or synthetic T domains. As a model system, we employ the single-module NRPS IndC from Photorhabdus luminescens that produces the blue pigment indigoidine. As chassis we use Escherichia coli. We demonstrate that heterologous T domain exchange is possible, even for T domains derived from different organisms. Interestingly, substitution of the native T domain with a synthetic one enhanced indigoidine production. Moreover, we show that selection of appropriate inter-domain linker regions is critical for functionality. Taken together, our results extend the engineering avenues for NRPSs, as they point out the possibility of combining domain sequences coming from different pathways, organisms or from conservation criteria. Moreover, our data suggest that NRPSs can be rationally engineered to control the level of production of the corresponding peptides. This could have important implications for industrial and medical applications.