The evolution of glycopeptide antibiotics Martina Adamek 1,2,3 , Frauke Adam 1,2,3 , Max Cryle 4,5,* , Athina Gavrilidou 1,2,3 , Mathias Hansen 4,5 , Dumitrita Iftime 1 , Evi Stegmann 1* , Nadine Ziemert 1,2,3,* 1 University of Tuebingen, Germany, 2 German Centre for Infection Research (DZIF), Germany, 3 Institute for Bioinformatics and Medical Informatics (IBMI), University of Tübingen, Germany, 4 Department of Biochemistry and Molecular Biology, Monash University, Australia, 5 EMBL Australia, Monash University, Australia Glycopeptide antibiotics (GPAs) are NRPS (nonribosomal peptide-synthetase) derived heptapeptides that are produced by Actinomycetota. They are important last resort antibiotics active against Gram-positive bacteria. What is striking, is the huge versatility of GPAs. They differ in the AA composition of the heptapeptide backbone as well as in numerous modifications by tailoring enzymes, such as glycosylation, methylation, acylation or halogenation. In our study we investigated how the GPAs evolved to such a high diversity by means of studying the phylogenetic history of the main glycopeptide biosynthesis genes. We were able to show that GPA biosynthetic gene clusters were undergoing extensive horizontal gene transfer (HGT). This concerns the entire biosynthesis gene cluster as such, but also various tailoring enzymes and functional domains. Using ancestral state reconstruction and ancestral sequence reconstruction we were able to predict how the ancestor of the GPAs known today might have looked like, and at which point in evolution certain traits were acquired, while others were lost. The GPA scaffold evolved from a more complex to a simpler structure, meaning the GPA ancestor had seven aromatic amino acids with four crosslinks encoded on four NRPS genes, while the type I GPAs as seen today have five aromatic and two aliphatic amino acids with only three crosslinks on three NRPS genes. This happened through recombination of functional domains and complete modules around the same evolutionary timescale as the loss of the crosslinking monooxygenase. Furthermore, the GPA ancestor molecule was glycosylated and halogenated. Reconstruction of the evolutionary history of the tailoring enzymes showed that they evolved through duplication and were acquired via HGT mostly from other Actinobacteria. Gene gain or loss happened much more often than alterations of the NRPS backbone. Recombination analysis further allowed us to reconstruct the extent of evolution through recombination vs. point mutations and thereby to predict which parts of the GPA backbone are flexible and which are highly conserved. The knowledge how evolution has shaped the glycopeptides leading to successful structure alterations will be of help to guide engineering and synthetic biology approaches for NRPS assembly lines.
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© The Author(s), 2022
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