<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Exploring the use of a blue pigment-producing NRPS as a tagging method to easily detect engineered NRPs"^^ . "Nonribosomal peptide synthetases (NRPSs) are modular mega-enzymes found in bacteria and fungi that produce nonribosomal peptides (NRPs) in an assembly line fashion. Each module is in charge of adding a specific amino acid (AA) to the growing peptide chain. Three basic domains constitute one NRPS module: the adenylation (A), peptidyl carrier protein (PCP) and condensation (C) domains. The A domain recognizes and activates the AA. An external enzyme, the PPtase, attaches a phosphopantetheine (PPant) arm to the PCP domain which then picks up the activated AA and delivers it to the C domain. The C domain recognizes the growing peptide chain (donor) as well as the new AA (acceptor) and fuses the two together. A special feature of NRPSs is their ability to recognize and incorporate not only proteinogenic AAs, but also other building blocks like fatty acids (FAs) or non-proteinogenic AAs. All building blocks can be further modified through the action of additional domains: epimerization (E), methylation (M) and oxidation (Ox) domains, among others. In this manner a great variety of different NRPs can be synthesized, many of which are bioactive and exhibit anti-microbial or anti-cancer properties. Thus, it is highly desirable to understand how NRPS domains and modules function and find ways to genetically re-engineer them for custom NRP production.\r\nSince the discovery of NRPSs, many efforts have already been made to engineer these enzymes in order to create custom NRPs, but general design rules yet remain elusive. The successful attempts to re-create functional NRPS for the production of novel NRPs include: (i) mutations of the A domain, (ii) subdomain modifications and (iii) rearrangements on the module level. Yet, many engineered NRPSs exhibit only slow reaction rates and low product yields. In some cases, the desired NRP products cannot be detected at all, possibly due to additional control mechanisms that have not been taken into account during the engineering process, such as substrate specificity of C domains. Hence there is still a great need to identify the general rules for successful NRPS engineering in order to exploit the ever-growing molecular toolbox of newly discovered NRPSs for recombinant production of novel bioactive compounds.\r\nIn this work I present my attempts to develop an approach to easily monitor the outcome of NRPS manipulation using a pigment-producing synthetase as a genetic tag. To this end, I first investigated two homologous synthetases, IndC and BpsA, which produce the blue pigment indigoidine, and mutants thereof and revised the proposed biosynthesis mechanism. I then created a series of fusion constructs between modules coming from different NRPSs and IndC/BpsA to test if indigoidine-tagged peptides could be produced. I identified a promising construct for which point mutations in the upstream module resulted in weaker or null pigment production. However, the expected indigoidine-tagged AA was not detectable, which could be due to the fact that indigoidine production inevitably leads to the separation of the donor AA. These results raised further questions as to whether in a native NRPS, the same modifications lead to congruent effects in neighboring modules. I addressed this question using a fragment of a non-engineered NRPS to monitor the activity of the native and modified versions in an in vitro assay, which I present in the last part of the results. Surprisingly, the effects of the same set of modifications on neighboring modules did not only differ between the engineered NRP-pigment synthetase and the native NRPS, but also between different modules within the native NRPS. These results hint at highly individual behavior of NRPS modules, depending on the context they are in."^^ . "2019" . . . . . . . "Anna"^^ . "Degen"^^ . "Anna Degen"^^ . . . . . . "Exploring the use of a blue pigment-producing NRPS as a tagging method to easily detect engineered NRPs (PDF)"^^ . . . "190123_Thesis_AnnaDegen_archive.pdf"^^ . . . "Exploring the use of a blue pigment-producing NRPS as a tagging method to easily detect engineered NRPs (Other)"^^ . . . . . . "indexcodes.txt"^^ . . "HTML Summary of #26403 \n\nExploring the use of a blue pigment-producing NRPS as a tagging method to easily detect engineered NRPs\n\n" . "text/html" . . . "500 Naturwissenschaften und Mathematik"@de . "500 Natural sciences and mathematics"@en . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .