Biology subjects

Kegler, C.

Publications and source records attributed to Kegler, C..

4 recordsLinked to original sources

bioRxiv2026

Heterologous Reconstitution of 4-Amidopentadienoate Biosynthesis Identifies the PKS Machinery Responsible for Warhead Formation

The 4-amidopentadienoate (APD) motif is a rare and bioactive pharmacophore found in the cyclic lipodepsipeptide natural products BE-43547, rakicidins, microtermolides, and related compounds. Although several biosynthetic gene clusters associated with this family have been identified, the enzymatic origin of the APD warhead has remained unknown. Here, we investigated APD formation using NRPS engineering to install the unusual APD-associated polyketide synthase (PKS) module from the BE-43547 biosynthetic pathway into heterologous nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) assembly lines expressed in Escherichia coli. The engineered hybrids produced the designed lipopeptide containing the APD moiety, demonstrating that the core biosynthetic machinery is sufficient for APD installation and that no trans-acting enzymes are required. Total synthesis of the target compound enabled structural validation by co-elution, MS/MS analysis, and NMR spectroscopy. Production titers of 59 ug/L were achieved in the heterologous host. Systematic mutagenesis further revealed that the conserved dehydratase His-Asp catalytic dyad is essential for APD formation, whereas an additional highly conserved histidine is dispensable. These findings establish the biosynthetic origin of the APD warhead and provide a foundation for incorporating this privileged pharmacophore into engineered peptide scaffolds.

biochemistry↗

bioRxiv2026

Site-Specific Introduction of Non-Canonical Amino Acids into natural and engineered Non-Ribosomal Peptides

The incorporation of non-canonical amino acids (ncAAs) into proteins, developed in the past 20 years, has opened new avenues with respect to protein structure, protein modification, protein-protein interaction or enzyme catalysis beyond what is possible with the 20 proteinogenic AAs. Although >300 unusual building blocks including several ncAAs have been described in nonribosomal peptides (NRPs) naturally, we aimed to further expand the scope of the underlying nonribosomal peptide synthetases (NRPS) to incorporate ncAAs beyond the naturally available ones. We have therefore systematically screened for ncAA accepting NRPS systems, applied NRPS engineering to transfer the respective ncAA-accepting parts into other NRPSs and thereby created novel peptides that were further derivatized in post-enzymatic chemical synthesis reactions directly in bacterial culture extracts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/738027v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@8968ddorg.highwire.dtl.DTLVardef@143a852org.highwire.dtl.DTLVardef@dfac73org.highwire.dtl.DTLVardef@b2c6fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Evolutionary Inspired Engineering of Megasynthetases

Many clinically used drugs are derived from or inspired by bacterial natural products that often are biosynthesised via non-ribosomal peptide synthetases (NRPS), giant megasynthases that activate and join individual amino acids in an assembly line fashion. Since NRPS are not limited to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would allow the biotechnological generation of complex peptides including linear, cyclic and further modified natural product analogues, e.g. to optimise natural product leads. Here we describe a detailed phylogenetic analysis of several bacterial NRPS that led to the identification of a new recombination breakpoint within the thiolation (T) domain that is important for natural NRPS evolution. From this, an evolution-inspired eXchange Unit between T domains (XUT) approach was developed which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

synthetic biology↗

bioRxiv2018

Structure-based redesign of docking domain interactions modulates the product spectrum of a rhabdopeptide-synthesizing NRPS

Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by docking domains (DDs) to synthesize well-defined peptide products. In contrast to these classical NRPSs, the subunits of rhabdopeptide/xenortide producing NRPSs can act iteratively and in different order resulting in libraries of peptide products. In order to define the structural and thermodynamic basis for their unusual interaction patterns, we determined the structures of all N-terminal DDs (NDDs) as well as of an NDD-CDD complex and characterized all putative DD interactions thermodynamically for one such system. Key amino acid residues for DD interactions were identified that upon their exchange not only changed the DD affinity but also resulted in rationally predictable changes in peptide production. A simple set of recognition rules for DD interactions was identified that also operates in other megasynthase complexes.

biochemistry↗