Open Access

Andreas Tietze

• The growing number of infections with multi-resistant bacteria or the current COVID-19 pandemic put compounds with therapeutic properties into the public focus. Non-ribosomal peptides (NRPs) are natural products that are already marketed as antibiotics, cytotoxic agents or immunosuppressants. Their biological activities rely on the structural diversity including non-proteinogenic amino acids (AAs), heterocycles or modifications like methylation or acylation. The biosynthesis of NRPs is carried out by non-ribosomal peptide synthetases (NRPSs). These multifunctional megaenzymes show a modular architecture like in an assembly-line. Each module is thereby responsible for the incorporation and modification of one AA and therefore contains different catalytic domains. The adenylation (A) domain recognizes and activates its specific substrate in an ATP-dependent manner which is transferred to a 4’-phosphopantetheine cofactor post-translationally attached to the thiolation (T) domain. Peptide bond formation between two T domain bound substrates catalysed by the condensation (C) domain transfers the growing peptide chain to the following module. Such a C-A-T module can be extended with optional domains to integrate structural diversity and a terminal thioesterase (TE) domain usually releases the peptide via hydrolysis or intramolecular attack of nucleophiles. Inspired by the modular architecture, NRPS engineering deals with the modification of NRPs in order to increase biological activities, circumvent bacterial resistances or create de novo peptides. This can be achieved by mutasynthesis or modification of the substrate binding pocket as well as single and multiple domain substitution. However, the few successful approaches led to impaired enzymes and did not establish a general applicable guideline. In the first publication as part of this work, the development of such a guideline comprising three rules is addressed. First, the A-T-C tridomain named exchange unit (XU) is seen as a catalytic unit instead of a module. When using them as building blocks, the C domain’s specificity for the AA of the following XU has to be considered as second rule. Third, a conserved WNATE motif within the C-A linker depicts the fusion point of the XUs. Upon heterologous expression of the cloned plasmids in E. coli and high performance liquid chromatography coupled mass spectrometry-based analysis of the extracts, the ambactin-producing NRPS from Xenorhabdus was reprogrammed with one and two XUs. This only leads to a moderate loss of production titre or an even higher one when the AA configuration was changed by introducing a dual condensation/epimerization (C/E) domain. The pentamodular GameXPeptide-producing NRPS was reconstructed using up to five XUs of four different NRPSs and even completely de novo synthetases were created. The second publication describes the exchange unit condensation domain (XUC) concept and relies on a fusion point between the two subdomains (N-terminal CDsub and C-terminal CAsub) of the C domain’s V-shaped pseudodimeric structure which generates A-T didomains with flanking CAsub and CDsub. These hybrid C domain-forming building blocks depict an improvement to the XU concept by avoiding the drawback of C domain specificity. This allows a more flexible NRPS engineering that can e.g. enable peptide library design. Furthermore, beside a combination of both concepts within one NRPS and a transfer to Bacillus NRPSs, the use of XUC with relaxed A domain specificity allowed further peptide modifications by introducing non-natural AAs. The third publication deals with aldehyde and alcohol-generating reductase (R) domains which depict an alternative for peptide release in NRPSs. A promoter exchange in X. indica identified a pyrazine-producing NRPS with a minimal architecture of an A, T and R domain and was therefore termed ATRed. R domains were additionally used in engineered NRPSs to produce pyrazinones and derivatives thereof by XU substitution although most constructs failed to show production. Beyond that, an R domain has been shown to replace a TE domain in wild type synthetases leading to slightly modified NRPs and the postulated biosynthesis was incidentally revised. Furthermore, an NRPS with terminal R domain was engineered to produce a free peptide aldehyde, which are known to be potent proteasome inhibitors. For the above mentioned ATReds, the presence of up to three coding regions was further identified in 20 different Xenorhabdus strains but only six of them were verified to produce pyrazines. All ATReds share variable sequence similarities among each other and were subsequently divided into three subtypes. One subtype is supposed to perform the pyrazine biosynthesis via a non-canonical catalytic triad.