Expanding The Chemical Space of RiPPs in Rare Actinobacetria Employing a Tunable Metabologenomic Approach

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URI: http://hdl.handle.net/10900/119130
Dokumentart: PhDThesis
Date: 2022-08-17
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Pharmazie
Advisor: Groß, Harald (Prof. Dr.)
Day of Oral Examination: 2021-08-17
DDC Classifikation: 500 - Natural sciences and mathematics
Other Keywords:
Nocardia terpenica
License: http://tobias-lib.uni-tuebingen.de/doku/lic_ohne_pod.php?la=de http://tobias-lib.uni-tuebingen.de/doku/lic_ohne_pod.php?la=en
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The intrinsic driving force of finding new RiPP scaffolds is largely attributed to their appreciable biological/physiological functions, however the constantly growing interest in disclosing novel architectures currently extends beyond the classical needs. For example, RiPP-BGCs regularly provide a rich pool of enzymatic machineries that can install specific, in some cases rare, tailorings expanding the biobrick toolbox in synthetic biology and chemistry. Although the analyses of different genome sequences of Nocardia spp. disclosed a huge cryptic biosynthetic potential waiting to be unearthed, the discovery of such genetically encoded entities is insurmountable. Thus, to partially overcome the typical limitations offered by the common silence of biosynthetic gene clusters (BGCs) and the inherited challenge to connect the genotype(s) with the chemotype(s), a tunable metabologenomic approach was developed within the frame of this work. The application of the OSMAC concept in tandem with the configured analytical strategy in Nocardia terpenica IFM 0406, 0706T and Longimycelium tulufanense CGMCC 4.5737 enabled to chart unprecedented chemical space belonging to RiPPs with three novel molecular families. The first study case comprises a genome-oriented prioritization of a novel BGC associated with a new-to-nature combination of multiple post-translational enzymes in N. terpenica IFM 0406. It was hypothesized that the gene cluster will in turn assemble a novel chemical entity blending three different class-defining post-translational modifications (PTMs). Recruiting a synergism of bioinformatics, stable isotope labeling experiments and NMR spectroscopy facilitated the successful identification, isolation and structural characterization of three new RiPPs, named nocathioamides A-C. Aside from structurally featuring unprecedented PTM in the form of a macrocyclic imide bridge, they inaugurate the first-in-nature combinatorial tribrid RiPPs hovering over three different biosynthetic machineries of lanthipeptides, LAPs and thioamitides. The second investigation deals with the deorphanization of an additional unique RiPP-BGC found in N. terpenica genomes. The bioinformatic annotation and prediction pinpoint to an unknown scaffold belonging to class II lasso peptide which is additionally decorated with a possible oxidative event(s). A quick bioinformatic survey revealed the presence of homologous BGCs coding for similar entities in other bacteria exemplified by Longimycelium tulufanense. Relying on the predicted core peptides in tandem with the tuned workflow, the encoded products were deciphered from both isolates, termed nocapeptins and longipeptins. Nocapeptins, from N. terpenica, and longipeptins, from L. tulufanense, symbolize the founders of a novel molecular family of lasso peptides in which a conserved regional PTM as a unique oxidative crosslink is structurally appended. Aside from the characteristic skeletal inter-linkage embedded in longipeptin A, the structure could leverage the modification scope with further tailoring events like hydroxylation and methylation, delivering thereby the most tailored lasso architecture till now. The discovery of nocathioamides and nocapeptins stretched with longipeptins portrays a proof of concept to the effectiveness of employing the technique of genome mining prioritization to unearth a new chemical space with uncharted biosynthetic enzymes from underexplored species.

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