Resurrecting ancestral antibiotics: unveiling the origins of modern lipid II targeting glycopeptides

Article https://doi.org/10.1038/s41467-023-43451-4 Resurrecting ancestral antibiotics: unveiling the origins of modern lipid II targeting glycopeptides Received: 10 August 2023 Check for updates 1234567890():,; 1234567890():,; Accepted: 9 November 2023 Mathias H. Hansen 1,2,3,8, Martina Adamek 4,5,6,8, Dumitrita Iftime4,8, Daniel Petras4, Frauke Schuseil 4,5,6, Stephanie Grond 7, Evi Stegmann Max J. Cryle 1,2,3 & Nadine Ziemert 4,5,6 4,6 , Antibiotics are central to modern medicine, and yet they are mainly the products of intra and inter-kingdom evolutionary warfare. To understand how nature evolves antibiotics around a common mechanism of action, we investigated the origins of an extremely valuable class of compounds, lipid II targeting glycopeptide antibiotics (GPAs, exemplified by teicoplanin and vancomycin), which are used as last resort for the treatment of antibiotic resistant bacterial infections. Using a molecule-centred approach and computational techniques, we first predicted the nonribosomal peptide synthetase assembly line of paleomycin, the ancestral parent of lipid II targeting GPAs. Subsequently, we employed synthetic biology techniques to produce the predicted peptide and validated its antibiotic activity. We revealed the structure of paleomycin, which enabled us to address how nature morphs a peptide antibiotic scaffold through evolution. In doing so, we obtained temporal snapshots of key selection domains in nonribosomal peptide synthesis during the biosynthetic journey from ancestral, teicoplanin-like GPAs to modern GPAs such as vancomycin. Our study demonstrates the synergy of computational techniques and synthetic biology approaches enabling us to journey back in time, trace the temporal evolution of antibiotics, and revive these ancestral molecules. It also reveals the optimisation strategies nature has applied to evolve modern GPAs, laying the foundation for future efforts to engineer this important class of antimicrobial agents. Natural products form one of the most important sources of medicinal compounds, with modern medicine reliant on antibiotics that often originate from biosynthesis in various microorganisms1. Indeed, thousands of compounds have been isolated from natural sources— with many more predicted—that display enormous structural diversity. These compounds are mainly produced as so called secondary or specialised metabolites by organisms and represent important adaptive characteristics that have been subjected to natural selection 1 Department of Biochemistry and Molecular Biology, The Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia. 2EMBL Australia, Monash University, Clayton, VIC 3800, Australia. 3ARC Centre of Excellence for Innovations in Peptide and Protein Science, Monash University, Clayton, VIC 3800, Australia. 4Interfaculty Institute of Microbiology and Infection Medicine Tübingen, Cluster of Excellence ‘Controlling Microbes to Fight Infections’, University of Tübingen, Tübingen, Germany. 5German Centre for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany. 6Institute for Bioinformatics and Medical Informatics (IBMI), University of Tübingen, Tübingen, Germany. 7Institute of Organic Chemistry, University of Tübingen, e-mail: ; Tübingen, Germany. 8These authors contributed equally: Mathias H. Hansen, Martina Adamek, Dumitrita Iftime. ; Nature Communications | (2023)14:7842 1 Article https://doi.org/10.1038/s41467-023-43451-4 during evolution2,3. Given the importance of biosynthetic processes to access complex natural products at scale, understanding how such pathways evolve is crucial information if we are to successfully reengineer such assemblies to allow the formation of new, designer compounds with improved properties. The biosynthesis of natural products is typically encoded by biosynthetic gene clusters (BGCs), which usually include genes for precursor and core biosynthesis, post-core biosynthesis, regulation, resistance, and transport. Genome analysis has shown that BGCs—and hence natural products—evolve through a range of processes including the recombination of specific subclusters, gene conversion, gene duplication and horizontal gene transfer4. However, although some evolutionary models have been proposed2,5, little is understood about the molecular mechanisms of how natural products, arguably the largest and most economically important source of chemical diversity on the planet, have evolved. Recent exciting work to address this has made use of ancient DNA to investigate natural products from the Pleistocene era6, although longer evolutionary timescales are doubtless challenging for such an approach. In this work, we have sought to understand the evolutionary history of lipid II targeting glycopeptide antibiotics (GPAs), a vital class of nonribosomal peptides used in the clinic for the treatment of resistant bacterial infections and exemplified by vancomycin (Van) and teicoplanin (Tei) (Fig. 1)7,8. Various types of GPAs are known9, Van-type (I) which extend beyond the lipid II targeting GPAs under investigation here to type V GPAs such as corbomycin10 and kistamicin11 that possess altered structures and modes of action (Fig. 1). All GPAs contain a multicyclic peptide core structure that is assembled through the combined activity of a nonribosomal peptide synthetase (NRPS) machinery12 and cytochrome P450 monooxygenases, which catalyse a cascade of oxidative crosslinking reactions13. The peptide core of GPAs is largely composed of aromatic amino acids including nonproteinogenic amino acids such as 4-hydroxyphenylglycine (Hpg), 3,5-dihydroxyphenylglycine (Dpg), and β-hydroxytyrosine (Bht). Curiously, one key difference across the biosynthetic pathways for lipid II targeting GPAs is the formation of Bht, which is either obtained by tyrosine (Tyr) oxidation on the NRPS as in the teicoplanin (Tei) pathway or generated offline and directly incorporated as in the vancomycin (Van) pathway. Beyond variations in Bht formation and the core peptide, diversity within the GPA family is expanded yet further through modifications to the post-peptide assembly process7,8. With the exception of type V GPAs, GPAs function by interrupting bacterial cell wall biosynthesis through the sequestration of the peptidoglycan precursor lipid II7. Whilst lipid II targeting GPAs—such as Tei and Van—share a conserved mechanism of action, they differ in the structures of their peptide cores and the BGCs encoding these antibiotics. Earlier phylogenetic reconciliation indicated that the origins of Pek-type (I) α-van-β-glc Me-β-glc Dpg- Bht-Hpg--Hpg--Asn- Bht -Leu Cl Cl Ris-type (III) Dpg- Bht-Hpg--Hpg -Glu -Tyr -Ala Cl Tei-type (IV) α-rha β-NAc-gls FA-β-gls α-ara-α-man-β-glc Dpg-Bht -Hpg--Hpg--Dpg- Tyr -Hpg Cl Dpg-Bht -Hpg--Hpg--Dpg- Bht -Hpg Cl α-man α-man Type V Avo-type (II) α-ria α-ria-β-glc O-α-man Dpg- Bht-Hpg--Hpg--Hpg-Bht -Hpg Cl α-rha Fig. 1 | S (...truncated)