Stefan Leopold-Messer

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Leopold-Messer

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Stefan

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0000-0003-3490-3550

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Publications 1 - 10 of 10

A monodomain class II terpene cyclase assembles complex isoprenoid scaffolds
Moosmann, Philipp; Ecker, Felix; Leopold-Messer, Stefan; et al. (2020)
Nature Chemistry
Class II terpene cyclases, such as oxidosqualene and squalene-hopene cyclases, catalyse some of the most complex polycyclization reactions. They minimally exhibit a β,γ-didomain architecture that has been evolutionarily repurposed in a wide range of terpene-processing enzymes and likely resulted from a fusion of unidentified monodomain proteins. Although single domain class I terpene cyclases have already been identified, the corresponding class II counterparts have not been previously reported. Here we present high-resolution X-ray structures of a monodomain class II cyclase, merosterolic acid synthase (MstE). With a minimalistic β-domain architecture, this cyanobacterial enzyme is able to construct four rings in cytotoxic meroterpenoids with a sterol-like topology. The structures with bound substrate, product, and inhibitor provide detailed snapshots of a cyclization mechanism largely governed by residues located in a noncanonical enzyme region. Our results complement the few known class II cyclase crystal structures, while also indicating that archaic monodomain cyclases might have already catalyzed complex reaction cascades.
Animal-associated marine Acidobacteria with a rich natural-product repertoire
Leopold-Messer, Stefan; Chepkirui, Clara; Mabesoone, Mathijs F.J.; et al. (2023)
Chem
Sponges are well known as rich sources of bioactive natural products. Various studies suggest that many of these compounds are produced by symbiotic bacteria. However, substance supplies and functional insights about the producers remain limited because cultivation remains unsuccessful. To identify alternative, sustainable sources of sponge-derived polyketides, we computationally analyzed 5,289 characterized and orphan trans-acyltransferase polyketide synthases, enzymes with widespread roles in polyketide biosynthesis by bacterial symbionts. The workflow predicted animal-derived marine Acidobacteria of the family Acanthopleuribacteraceae with large sets of biosynthetic gene clusters to be enriched in sponge-type chemistry. Targeted compound isolation from a chiton-associated strain yielded congeners of the phorboxazoles and calyculins, potent and scarce cytotoxins exclusively known from sponges, and depsipeptides named acidobactamides. These first natural products of Acidobacteria and new fire coral metagenomic data on a third family member suggest animal-associated Acanthopleuribacteraceae as a rich source of sponge-type and other metabolites.
Evolution-guided engineering of trans-acyltransferase polyketide synthases
Mabesoone, Mathijs F. J.; Leopold-Messer, Stefan; Minas, Hannah A.; et al. (2024)
Science
Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis-acyltransferase (cis-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans-AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans-AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.
Evolution of combinatorial diversity in trans-acyltransferase polyketide synthase assembly lines across bacteria
Helfrich, Eric J.N.; Ueoka, Reiko; Chevrette, Marc G.; et al. (2021)
Nature Communications
Trans-acyltransferase polyketide synthases (trans-AT PKSs) are bacterial multimodular enzymes that biosynthesize diverse pharmaceutically and ecologically important polyketides. A notable feature of this natural product class is the existence of chemical hybrids that combine core moieties from different polyketide structures. To understand the prevalence, biosynthetic basis, and evolutionary patterns of this phenomenon, we developed transPACT, a phylogenomic algorithm to automate global classification of trans-AT PKS modules across bacteria and applied it to 1782 trans-AT PKS gene clusters. These analyses reveal widespread exchange patterns suggesting recombination of extended PKS module series as an important mechanism for metabolic diversification in this natural product class. For three plant-associated bacteria, i.e., the root colonizer Gynuella sunshinyii and the pathogens Xanthomonas cannabis and Pseudomonas syringae, we demonstrate the utility of this computational approach for uncovering cryptic relationships between polyketides, accelerating polyketide mining from fragmented genome sequences, and discovering polyketide variants with conserved moieties of interest. As natural combinatorial hybrids are rare among the more commonly studied cis-AT PKSs, this study paves the way towards evolutionarily informed, rational PKS engineering to produce chimeric trans-AT PKS-derived polyketides.
Host-associated marine bacteria – a rich source of bioactive polyketides
Leopold-Messer, Stefan (2022)
Natural products are specialized metabolites produced by living organisms. For millennia humans have used these compounds for ceremonial, recreational and medicinal purposes. Until today, natural products are an essential source for the development of novel drugs, which are necessary to combat emerging and hitherto incurable diseases. Modern natural product discovery is defined by the increasing number of available genome sequences and the development of powerful bioinformatic algorithms, which enable a method referred to as 'genome mining'. Thereby a bioinformatic analysis of genomes enables the prioritization of biosynthetic gene clusters (BGCs) and their respectively encoded enzymatic systems. The putative enzymatic activity can be used to predict potential biosynthetic products, which in the process of cultivation and isolation are later characterized. Since natural products mediate the communication and the defense between organisms, host-associated bacteria are particularly promising for the discovery of novel bioactive molecules. Here, genome mining is applied to two host-associated marine bacteria with an impressive biosynthetic potential, Acanthopleuribacter pedis isolated from a mollusk, and Gynuella sunshinyii from a plant. More specifically, this work focuses on the discovery of polyketides and peptides produced by the biosynthetic enzyme classes of transacyltransferase polyketide synthases (trans-AT PKSs) and non-ribosomal peptide synthetases (NRPSs). In chapter II an unbiased global analysis of all bacterial genomes published prior to June 2022 in the NCBI GenBank retrieved more than 5000 trans-AT PKSs and led to the identification the Acidobacterium A. pedis as a talented natural product producer. The large genome encodes 39 BGCs, three of which are trans-AT PKSs. De novo product prediction enabled isolation and characterization of two polyketides. One of them, 21- methoxy-calyculinamide is a congener of calyculin, a highly cytotoxic compound. It was previously isolated from a marine sponge but has been shown to be produced by uncultivated bacteria living inside the marine animal. The other polyketide, phorbactazole, is a novel compound. Its complex structure is reminiscent of the spongederived phorboxazole. These results show that alternative sustainable bacterial producers of sponge-like compounds can be identified. Furthermore, to the best of our knowledge,the two polyketides are the first natural products reported from Acidobacteria, a ubiquitous but poorly studied bacterial phylum. Chapter III describes the collaborative effort of identification, characterization, and total synthesis of the novel polyketides, janustatins, produced by G. sunshinyii. Genome mining enabled natural product prediction and subsequently the isolation of these scarce metabolites. The challenging structure elucidation was complemented by computational chemistry and bioinformatics to suggest an absolute configuration, which was then verified by total synthesis. Janustatins show a delayed cytotoxic effect against cancer cells in sub-nanomolar concentrations. In chapter IV the biosynthesis of janustatins by G. sunshinyii was studied. First, reliable genetic engineering tools that allow gene deletions, promoter replacement and complementation experiments were developed. With these methods, the role of two enzymes, JanA and JanB, in janustatin biosynthesis was investigated. Furthermore, promoter exchange in gene deletion strains enabled the isolation and characterization of biosynthetic intermediates. These results indicate that a hydroxylation and a desaturation catalyzed by JanA and JanB, respectively, render janustatins more cytotoxic. Additionally, we provide scientific evidence that the uncharacterized NRPS-para261 domain is essential for janustatin biosynthesis. Chapter V expands the use of the novel genetic engineering tools in G. sunshinyii. First, we used promoter replacement to enhance janustatin titers in G. sunshinyii. While significant increase in polyketide production has not been detected, these experiments show that BGCs can be rearranged in the genome of G. sunshinyii. Further, we applied the same strategy to an uncharacterized BGC. We were hoping that genetic engineering would facilitate identification of its product. So far, no target for isolation was retrieved. Lastly, we used the methods to engineer the biosynthesis of the polyketide lacunalide. Initial results indicate that deleting parts of the biosynthetic machinery yields strains which produce shortened polyketides. Overall, the experiments described in Chapter V pave the way to establishing G. sunshinyii as a bacterial polyketide factory. In this thesis, genome mining was employed to characterize novel polyketides from marine bacteria. The isolated natural products showed intriguing chemical structures and potent cytotoxicity. With the here-presented results, we shed light on the biosynthetic potential of bacteria isolated from host-organisms and emphasize their importance for natural product discovery in two examples. Development of genetic engineering methods has additionally enabled to study the biosynthesis of one of the polyketides presented. In the future these tools can be used to systematically study and modify polyketide synthases.
Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara
Schmidt, Konstantin; Santos-Matos, Gonçalo; Leopold-Messer, Stefan; et al. (2023)
eLife
Bacteria colonize specific niches in the animal gut. However, the genetic basis of these associations is often unclear. The proteobacterium Frischella perrara is a widely distributed gut symbiont of honey bees. It colonizes a specific niche in the hindgut and causes a characteristic melanization response. Genetic determinants required for the establishment of this association, or its relevance for the host, are unknown. Here, we independently isolated three point mutations in genes encoding the DNA-binding protein integration host factor (IHF) in F. perrara. These mutants abolished the production of an aryl polyene metabolite causing the yellow colony morphotype of F. perrara. Inoculation of microbiota-free bees with one of the mutants drastically decreased gut colonization of F. perrara. Using RNAseq, we found that IHF affects the expression of potential colonization factors, including genes for adhesion (type 4 pili), interbacterial competition (type 6 secretion systems), and secondary metabolite production (colibactin and aryl polyene biosynthesis). Gene deletions of these components revealed different colonization defects depending on the presence of other bee gut bacteria. Interestingly, one of the T6SS mutants did not induce the scab phenotype anymore despite colonizing at high levels, suggesting an unexpected role in bacteria-host interaction. IHF is conserved across many bacteria and may also regulate host colonization in other animal symbionts.
Genome-based discovery and total synthesis of janustatins, potent cytotoxins from a plant-associated bacterium
Ueoka, Reiko; Sondermann, Philipp; Leopold-Messer, Stefan; et al. (2022)
Nature Chemistry
Host-associated bacteria are increasingly being recognized as underexplored sources of bioactive natural products with unprecedented chemical scaffolds. A recently identified example is the plant-root-associated marine bacterium Gynuella sunshinyii of the chemically underexplored order Oceanospirillales. Its genome contains at least 22 biosynthetic gene clusters, suggesting a rich and mostly uncharacterized specialized metabolism. Here, in silico chemical prediction of a non-canonical polyketide synthase cluster has led to the discovery of janustatins, structurally unprecedented polyketide alkaloids with potent cytotoxicity that are produced in minute quantities. A combination of MS and two-dimensional NMR experiments, density functional theory calculations of C-13 chemical shifts and semiquantitative interpretation of transverse rotating-frame Overhauser effect spectroscopy data were conducted to determine the relative configuration, which enabled the total synthesis of both enantiomers and assignment of the absolute configuration. Janustatins feature a previously unknown pyridodihydropyranone heterocycle and an unusual biological activity consisting of delayed, synchronized cell death at subnanomolar concentrations.
Insights into Heterocycle Biosynthesis in the Cytotoxic Polyketide Alkaloid Janustatin A from a Plant-Associated Bacterium
Leopold-Messer, Stefan; Chawengrum, Pornsuda; Piel, Jörn (2025)
Biochemistry
Janustatin A is a potently cytotoxic polyketide alkaloid produced at trace amounts by the marine bacterial plant symbiont Gynuella sunshinyii. Its biosynthetic terminus features an unusual pyridine-containing bicyclic system of unclear origin, in which polyketide and amino acid extension units appear reversed compared to the order of enzymatic modules in the polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) assembly line. To elucidate unknown steps in heterocycle formation, we first established robust genome engineering tools in G. sunshinyii. A combination of gene deletion, complementation, production improvement, and NMR experiments then demonstrated that two desaturase homologues, JanA and JanB, are involved in hydroxylation and pyridine formation by desaturation, respectively. Structure-activity relationship studies showed that these modifications substantially increase the cytotoxicity and that the fully functionalized heterocyclic system is crucial for sub-nanomolar cytotoxicity. Isolation of the early post-PKS intermediate janustatin D with an already reversed heterocycle topology supports a noncanonical rearrangement process occurring on the PKS-NRPS assembly line.
Identification of trans-AT polyketide clusters in two marine bacteria reveals cryptic similarities between distinct symbiosis factors
Kačar, Dina; Cañedo, Librada M.; Rodríguez, Pilar; et al. (2021)
Environmental Microbiology
Glutarimide-containing polyketides are known as potent antitumoral and antimetastatic agents. The associated gene clusters have only been identified in a few Streptomyces producers and Burkholderia gladioli symbiont. The new glutarimide-family polyketides, denominated sesbanimides D, E and F along with the previously known sesbanimide A and C, were isolated from two marine alphaproteobacteria Stappia indica PHM037 and Labrenzia aggregata PHM038. Structures of the isolated compounds were elucidated based on 1D and 2D homo and heteronuclear NMR analyses and ESI-MS spectrometry. All compounds exhibited strong antitumor activity in lung, breast and colorectal cancer cell lines. Subsequent whole genome sequencing and genome mining revealed the presence of the trans-AT PKS gene cluster responsible for the sesbanimide biosynthesis, described as sbn cluster. Strikingly, the modular architecture of downstream mixed type PKS/NRPS, SbnQ, revealed high similarity to PedH in pederin and Lab13 in labrenzin gene clusters, although those clusters are responsible for the production of structurally completely different molecules. The unexpected presence of SbnQ homologues in unrelated polyketide gene clusters across phylogenetically distant bacteria, raises intriguing questions about the evolutionary relationship between glutarimide-like and pederin-like pathways, as well as the functionality of their synthetic products.
Artificial intelligence for natural product drug discovery
Mullowney, Michael W.; Duncan, Katherine R.; Elsayed, Somayah S.; et al. (2023)
Nature Reviews Drug Discovery
Developments in computational omics technologies have provided new means to access the hidden diversity of natural products, unearthing new potential for drug discovery. In parallel, artificial intelligence approaches such as machine learning have led to exciting developments in the computational drug design field, facilitating biological activity prediction and de novo drug design for molecular targets of interest. Here, we describe current and future synergies between these developments to effectively identify drug candidates from the plethora of molecules produced by nature. We also discuss how to address key challenges in realizing the potential of these synergies, such as the need for high-quality datasets to train deep learning algorithms and appropriate strategies for algorithm validation.

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Stefan Leopold-Messer

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