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

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.

Exploring the Flexibility of the Glycopeptide Antibiotic Crosslinking Cascade for Extended Peptide Backbones.

The glycopeptide antibiotics (GPAs) are a clinically approved class of antimicrobial agents that classically function through the inhibition of bacterial cell-wall biosynthesis by sequestration of the precursor lipid II. The oxidative crosslinking of the core peptide by cytochrome P450 (Oxy) enzymes during GPA biosynthesis is both essential to their function and the source of their synthetic challenge. Thus, understanding the activity and selectivity of these Oxy enzymes is of key importance for the future engineering of this important compound class. Recent reports of GPAs that display an alternative mode of action and a wider range of core peptide structures compared to classic lipid II-binding GPAs raises the question of the tolerance of Oxy enzymes for larger changes in their peptide substrates. In this work, we explore the ability of Oxy enzymes from the biosynthesis pathways of lipid II-binding GPAs to accept altered peptide substrates based on a vancomycin template. Our results show that Oxy enzymes are more tolerant of changes at the N terminus of their substrates, whilst C-terminal extension of the peptide substrates is deleterious to the activity of all Oxy enzymes. Thus, future studies should prioritise the study of Oxy enzymes from atypical GPA biosynthesis pathways bearing C-terminal peptide extension to increase the substrate scope of these important cyclisation enzymes.

Kistamicin biosynthesis reveals the biosynthetic requirements for production of highly crosslinked glycopeptide antibiotics.

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.

Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer Streptomyces collinus Tü 365.

Streptomycetes are prolific sources of novel biologically active secondary metabolites with pharmaceutical potential. S. collinus Tü 365 is a Streptomyces strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein biosynthesis interacting with elongation factor EF-Tu. Genome Mining revealed 32 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of Streptomyces collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS-NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC-MS analyses, most of the uncharacterized gene clusters are not expressed under standard laboratory conditions. With this study we aimed to characterize the genome information of S. collinus Tü 365 to make use of gene clusters, which previously have not been described for this strain. We were able to connect the gene clusters of a lanthipeptide, a carotenoid, five terpenoid compounds, an ectoine, a siderophore and a spore pigment-associated gene cluster to their respective biosynthesis products.

Streptocollin, a Type†IV Lanthipeptide Produced by Streptomyces collinus Tü 365.

Lanthipeptides are ribosomally synthesized and post-translationally modified microbial secondary metabolites. Here, we report the identification and isolation of streptocollin from Streptomyces collinus Tü 365, a new member of class IV lanthipeptides. Insertion of the constitutive ermE* promoter upstream of the lanthipeptide synthetase gene stcL resulted in peptide production. The streptocollin gene cluster was heterologously expressed in S. coelicolor M1146 and M1152 with 3.5- and 5.5-fold increased yields, respectively. The structure and ring topology of streptocollin were determined by high resolution MS/MS analysis. Streptocollin contains four macrocyclic rings, with one lanthionine and three methyllanthionine residues. To the best of our knowledge, this is the first report on the isolation of a class IV lanthipeptide in preparative amounts, and on the successful heterologous expression of a class IV lanthipeptide gene cluster.

Complete genome sequence of the kirromycin producer Streptomyces collinus Tü 365 consisting of a linear chromosome and two linear plasmids.

Streptomyces collinus Tü 365 (DSMZ 40733), isolated from Kouroussa (Guinea), is the producer of the elfamycin family antibiotic kirromycin, which inhibits bacterial protein biosynthesis by interfering with elongation factor EF-Tu. Here, we report on the Streptomyces collinus Tü 365 complete genome sequence of the 8.27 MB chromosome and the two plasmids SCO1 and SCO2.

Stachyose in the cytosol does not influence freezing tolerance of transgenic Arabidopsis expressing stachyose synthase from adzuki bean.

We expressed the stachyose synthase from adzuki bean (Vigna angularis) in Arabidopsis thaliana, under the control of the constitutive CaMV 35S promoter. Transgenic lines had only trace amounts of stachyose under normal growth conditions but accumulated stachyose to similar levels as raffinose upon cold acclimation. Stachyose production did not alter the freezing tolerance of cold acclimated rosette leaves. Non-aqueous fractionation of sub-cellular compartments revealed that in cold acclimated plants, raffinose but not stachyose accumulated to a proportion higher than the compartment size fraction in the plastids. Since both oligosaccharides are synthesized in the cytosol, this provides evidence that the so far unknown raffinose transporter of the Arabidopsis chloroplast envelope does not efficiently transport stachyose. The failure of stachyose to influence freezing tolerance in Arabidopsis supports the hypothesis that raffinose family oligosaccharides might function in protecting the thylakoid but not the plasma membrane during freezing.