Biology subjects

Bode, H. B.

Publications and source records attributed to Bode, H. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Heterologous Reconstitution of 4-Amidopentadienoate Biosynthesis Identifies the PKS Machinery Responsible for Warhead Formation

The 4-amidopentadienoate (APD) motif is a rare and bioactive pharmacophore found in the cyclic lipodepsipeptide natural products BE-43547, rakicidins, microtermolides, and related compounds. Although several biosynthetic gene clusters associated with this family have been identified, the enzymatic origin of the APD warhead has remained unknown. Here, we investigated APD formation using NRPS engineering to install the unusual APD-associated polyketide synthase (PKS) module from the BE-43547 biosynthetic pathway into heterologous nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) assembly lines expressed in Escherichia coli. The engineered hybrids produced the designed lipopeptide containing the APD moiety, demonstrating that the core biosynthetic machinery is sufficient for APD installation and that no trans-acting enzymes are required. Total synthesis of the target compound enabled structural validation by co-elution, MS/MS analysis, and NMR spectroscopy. Production titers of 59 ug/L were achieved in the heterologous host. Systematic mutagenesis further revealed that the conserved dehydratase His-Asp catalytic dyad is essential for APD formation, whereas an additional highly conserved histidine is dispensable. These findings establish the biosynthetic origin of the APD warhead and provide a foundation for incorporating this privileged pharmacophore into engineered peptide scaffolds.

biochemistry↗

bioRxiv2026

Development of a High-throughput in vivo Assay for the Determination of Adenylation Domain Specificities

Natural product synthesis by non-ribosomal peptide synthetases (NRPS) is greatly defined by the substrate selectivity of the adenylation (A) domains. Previous assays for specificity determination were mainly performed in vitro and were requiring protein purification. In this work, we developed - based on NRPS engineering - a novel in vivo assay suitable for high-throughput application named ASCR (A domain screening). Using the recently described XUT fusion sites, A domains and their upstream condensation domains were assembled as di-domains to characterized NRPS model system, which allowed detection of defined tripeptide products via mass spectrometry directly after cell culture extraction. We evaluated the assay by screening in total 54 A domains from five known and seven uncharacterized NRPS, covering a broad range organism taxonomy and GC content of the investigated NRPS-encoding genes. Additionally, we applied the assay to elucidate and confirm the structures of novel cyclic pentapeptides derived from three novel NRPS from Photorhabdus temperata K122.

biochemistry↗

bioRxiv2026

Site-Specific Introduction of Non-Canonical Amino Acids into natural and engineered Non-Ribosomal Peptides

The incorporation of non-canonical amino acids (ncAAs) into proteins, developed in the past 20 years, has opened new avenues with respect to protein structure, protein modification, protein-protein interaction or enzyme catalysis beyond what is possible with the 20 proteinogenic AAs. Although >300 unusual building blocks including several ncAAs have been described in nonribosomal peptides (NRPs) naturally, we aimed to further expand the scope of the underlying nonribosomal peptide synthetases (NRPS) to incorporate ncAAs beyond the naturally available ones. We have therefore systematically screened for ncAA accepting NRPS systems, applied NRPS engineering to transfer the respective ncAA-accepting parts into other NRPSs and thereby created novel peptides that were further derivatized in post-enzymatic chemical synthesis reactions directly in bacterial culture extracts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/738027v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@8968ddorg.highwire.dtl.DTLVardef@143a852org.highwire.dtl.DTLVardef@dfac73org.highwire.dtl.DTLVardef@b2c6fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Derivatization of the non-ribosomal peptide pyrrolizixenamide using NRPS engineering

Pyrrolizidine alkaloids (PA) are well-known and widespread natural products from plants, which have also been identified in several different bacteria. In the latter case, the core structure is constructed by a non-ribosomal peptide synthetase (NRPS), which then undergoes oxidative ring contraction catalyzed by a Baeyer-Villiger monooxygenase. By deploying various NRPS engineering strategies, we have successfully generated five novel peptides carrying the unusual PA moiety at their C-terminus. Nonetheless, efforts to obtain a larger library of PAs were unsuccessful. Combined computational modelling and docking experiments suggest that this failure stems from the strict specificity of the thioesterase (TE) domain at the end of the NRPS, which discriminates against peptides carrying more than two amino acids. Our work thus suggests protein design strategies by which this intrinsic limitation to NRPS engineering may be overcome in future.

biochemistry↗

bioRxiv2026

Acyl amides derived from a bacterial symbiont control development of its nematode host

Symbiotic bacteria frequently regulate host development through small-molecule signals. In the entomopathogenic Steinernema nematode - Xenorhabdus bacterium symbiosis, which is widely used in biological pest control, the signals that trigger nematode development have remained unknown. Using a phenotypic screen of Xenorhabdus mutants, we identified bacterial acyl amides, which we named stripteamides, as signals required for efficient recovery of Steinernema infective juveniles. Stripteamides are synthesized from phenylethylamine and activated fatty acid substrates by StaS, a previously uncharacterized amide synthase. Mechanistic and structural analyses indicate that StaS evolved from the initiation module of a nonribosomal peptide synthetase (NRPS) and was repurposed to generate signaling metabolites for host development. Stripteamide signaling is conserved across Xenorhabdus species and operates in different Steinernema hosts. These findings reveal a new function for widespread bacterial acyl amides that previously have been shown as cytotoxic and affecting bacterial quorum sensing, highlighting the context dependence of microbial natural product language.

microbiology↗

bioRxiv2026

Split-Indigoidine synthetase as optical reporter for benchmarking protein-protein interactions

Indigoidine is a blue pigment biosynthesized by a single-module Non-Ribosomal Peptide Synthetase (NRPS) using L-glutamine as substrate. Despite its potential as a colorimetric reporter, no such system has been established from it to date. We used a recently characterized interdomain fusion site located between its adenylation (A) and thiolation (T) domains to develop the Indi2GO system, which provides a naked-eye detectable and quantitative optical readout of transient and covalent protein-protein-interaction (PPI) in living cells. Indi2GO enables high-throughput benchmarking and optimization of PPI tools in a standard 96-well plate reader format, without requiring exogenous substrates, specialized equipment or complex analytical workflows. We demonstrate its broad applicability with three widely used protein-protein interaction tools: SYNZIPS, inteins, and the SpyTag:SpyCatcher system. We used Indi2GO to validate novel SYNZIP pairs, which we used in NRPS engineering, highlighting its applicability for the development of novel PPI-mediating tools in the context of NRPS engineering and synthetic biology.

synthetic biology↗

bioRxiv2025

Drift, selection and convergence in the evolution of a nonribosomal peptide

Nonribosomal peptides play important roles in microbial ecology and medicine, for example as toxins and antibiotics such as penicillin1. Like ordinary proteins, nonribosomal peptides are subject to evolutionary change, which is driven by rampant recombinations of their synthetases2. This makes it difficult to study the evolutionary histories of nonribosomal peptides, because recombination is a serious challenge for phylogenetic inference3. Here we use recombination-aware phylogenetics and ancestral sequence reconstruction to retrace the evolution of GameXPeptides, nonribosomal peptides entomopathogenic bacteria use to kill their insect prey4. By untangling the complex histories of their synthetases, we discover dozens of new GameXPeptides and show striking patterns of structural convergence. In analogy to classic statistical test for natural selection acting on nucleotide mutations, we develop a similar test for whether selection acts to fix recombinations. We provide statistical and experimental evidence of both neutral genetic drift and natural selection in the diversification of GameXPeptides. GameXPeptides variants rapidly evolved highly insect specific toxicities, suggesting that their diversification may be driven by an evolutionary arms race. Our work suggests that small, fast evolving alteration to nonribosomal peptides can overcome host resistance on short timescales, with important implications for the evolution of resistance evading antibiotics.

evolutionary biology↗

bioRxiv2025

Split inteins for generating combinatorial non-ribosomal peptide libraries

Engineering Non-Ribosomal Peptide Synthetases (NRPS) is a promising strategy for discovering new bioactive compounds, which can serve as valuable leads for drug development, such as new antibiotics. However, their engineering is hampered by the limited availability of molecular tools for the efficient heterologous expression of their large biosynthetic gene clusters. In fact, a single NRPS gene can already exceed the size limits of standard cloning vectors. In this study, we establish split inteins as a novel tool for NRPS engineering to enable the expression of single, covalently linked NRPS proteins from multiple plasmids and to perform cloning-free module swapping. Using the xenotetrapeptide synthetase as model system, we show that an NRPS can be split into three parts and reconstituted via trans-splicing using two orthogonal inteins at four different engineering sites. Based on this tripartite platform we build a library comprising 21 plasmids and generated 324 hybrid NRPS by combinatorial transformation. More than half were catalytically active, producing over 200 novel peptides. This intein-based technology provides a modular platform for generating natural product-like peptide libraries, expanding biocatalytically accessible chemical space.

synthetic biology↗

bioRxiv2025

Identification of pseudotetraivprolide from Pseudomonas entomophila give novel insights into the biosynthesis of detoxin/rimosamide-like anti-antibiotics

Novel variants of known natural product (NP) classes might guide our understanding of biosynthesis, mode of action and potential application as drugs for all members of such NP classes. Here we describe a novel member of the widespread detoxine/rimosamide-like (DRL) natural products named pseudotetraivprolide from Pseudomonas strains, which has the characteristic DRL-activity of protecting Bacillus cereus against the antibiotic blasticidin S. The generation of several deletion and complemented mutants, heterologous expression experiments, identification and structure elucidation of several derivatives, chemical synthesis of main derivatives, enzymatic characterization of individual biochemical steps and detailed homology modelling of enzyme complexes were performed. This allowed us to show the primary metabolism-derived malonyl CoA-ACP transacylase (AT) FabD acting as trans-AT in the biosynthesis, suggest an order for all late-stage modifications and provide a function for the three conserved hypothetical proteins PipDFG acting as last-step acetylation complex thereby stabilizing the final product.

microbiology↗

bioRxiv2025

Identification and biosynthesis of xildivaline, a novel and widespread peptide deformylase inhibitor from Gammaproteobacteria

Xenorhabdus strains, Gram-negative bacteria pathogenic to insects and symbionts to nematodes of the genus Steinernema are prolific producers of various natural products. Here we describe the xisABCDE biosynthesis gene cluster from Xenorhabdus hominickii responsible for the production of xildivalines. These non-ribosomal peptide and polyketide hybrids act as peptide deformylase inhibitor (PDI) and occur also in other Gammaproteobacteria, especially Vibrio. Their structure and biosynthesis were fully elucidated despite their instability, highlighting a rare trans-methylation of their N-terminus. Subsequently, the structure of the responsible methyltransferase XisE and the peptide deformylase XisD, serving as resistance mechanism, were elucidated by X-ray crystallography, allowing insights into the function and the mode of action of this novel class of PDIs.

microbiology↗

bioRxiv2025

High-throughput engineering and modification of non-ribosomal peptide synthetases based on Golden Gate assembly

Non-ribosomal peptide synthetases (NRPS) are multimodular enzymes that produce complex peptides with diverse biological activities, potentially being used as clinical drugs. However, the pharmaceutical applications of such natural peptides often require further derivatisation and modification of the peptide backbone, mainly performed by chemical synthesis. A sustainable alternative resembles the in vivo engineering of NRPS to change and modify the enzyme properties rationally and, thus, the produced products. The novel NRPS engineering concept, the eXchange Unit Thiolation domain (XUT), allows the efficient modular assembly of different natural NRPS fragments to form hybrid NRPS that produce defined peptides. In this study, we describe a Golden Gate assembly (GGA) method for efficient high-throughput generation of novel and engineered NRPS libraries utilising the XUT concept. This method was applied to generate over 100 novel NRPS with the possibility of changing starter, elongation, and termination modules, respectively. Additionally, we applied this method for targeted modification of the xenoamicin biosynthetic gene cluster (BGC) XabABCD from Xenorhabdus doucetiae, resulting in the generation of 25 novel xenoamicin derivatives. Graphical AbstractA Golden Gate assembly (GGA) method was developed for the efficient assembly of natural and engineered non-ribosomal peptide synthetases (NRPS). This method has enabled the creation of NRPS libraries to generate novel peptides in high-throughput as well as the targeted derivatisation of natural products (NP). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/650154v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1161805org.highwire.dtl.DTLVardef@1832a43org.highwire.dtl.DTLVardef@4bc6b8org.highwire.dtl.DTLVardef@e3908d_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Activation of the Yersinia type III secretion system induces large-scale chromosomal and virulence plasmid DNA rearrangements

The type III secretion system (T3SS) is used by Gram-negative bacteria, including important pathogens, to manipulate eukaryotic target cells by injecting effector proteins. Type III secretion and bacterial physiology are known to be tightly interconnected and influence each other. Most notably, secreting cells undergo growth arrest in the T3SS model organisms Yersinia, Salmonella and Shigella. The molecular basis of this phenotype, referred to as secretion-associated growth inhibition, is debated. In Yersinia, T3SS genes are encoded extra-chromosomally in the plasmid of Yersinia virulence (pYV), whose copy number increases upon induction of T3SS secretion. In this study, we characterize the link between T3SS activity and subcellular organization by localizing and quantifying the pYV and chromosomal DNA in Yersinia enterocolitica. We find that activation of secretion not only increases the number of pYV plasmids per bacterium, but that the plasmids also move towards the membrane and poles. This relocalization is not caused by transertion (coupled transcription, translation and translocation) of effectors, but part of a broader DNA rearrangement, leading to a distinct relocalization of chromosomal DNA to mid-cell. We hypothesize that these striking DNA rearrangements occurring during secretion are a main factor in the secretion-associated growth inhibition of pathogenic bacteria.

microbiology↗

bioRxiv2024

Exploiting the inherent promiscuity of the acyl transferase of the stambomycin polyketide synthase for the mutasynthesis of analogues

The polyketide specialized metabolites of bacteria are attractive targets for generating analogues, with the goal of improving their pharmaceutical properties. Here, we aimed to produce C-26 derivatives of the giant anti-cancer stambomycin macrolides using a mutasynthesis approach, as this position has been shown previously to directly impact bioactivity. For this, we leveraged the intrinsically broad specificity of the acyl transferase domain (AT12) of the modular polyketide synthase (PKS), which is responsible for the alkyl branching functionality at this position. Feeding of a panel of synthetic and commercially available dicarboxylic acid mutasynthons to an engineered strain of Streptomyces ambofaciens (Sa) deficient in synthesis of the native -carboxyacyl-CoA extender units, resulted in six new series of stambomycin derivatives as judged by LC-HRMS and NMR. Notably, the highest product yields were observed for substrates which were only poorly accepted when AT12 was transplanted into a different PKS module, suggesting a critical role for domain context in the overall functioning of PKS proteins. We also demonstrate the superiority of this mutasynthesis approach - both in terms of absolute titers and yields relative to the parental compounds - in comparison to the alternative precursor-directed strategy in which monoacid building blocks are supplied to the wild type strain. We further identify a malonyl-CoA synthetase, MatB_Sa, with specificity distinct from previously identified promiscuous enzymes, making it a useful addition to a mutasynthesis toolbox for generating atypical, CoA activated extender units. Finally, we show that two of the obtained (deoxy)-butyl-stambomycins exhibit antibacterial and antiproliferative activities similar to the parental stambomycins, while an unexpected butyl-demethyl congener is less potent. Overall, this works confirms the interest of biosynthetic pathways which combine a dedicated route to extender unit synthesis and a broad specificity AT domain for producing bioactive derivatives of fully-elaborated complex polyketides.

synthetic biology↗

bioRxiv2024

Polyketide synthase-derived sphingolipids determine microbiota-mediated protection against pathogens in C. elegans

Protection against pathogens is a major function of the gut microbiota. Although bacterial natural products have emerged as crucial components of host-microbiota interactions, their exact role in microbiota-mediated protection is largely unexplored. We addressed this knowledge gap with the nematode Caenorhabditis elegans and its microbiota isolate Pseudomonas fluorescens MYb115 that is known to protect against Bacillus thuringiensis (Bt) infection. We find that MYb115-mediated protection depends on sphingolipids that are derived from an iterative type I polyketide synthase (PKS), thereby describing a noncanonical pathway of bacterial sphingolipid production. We provide evidence that MYb115-derived sphingolipids affect C. elegans tolerance to Bt infection by altering host sphingolipid metabolism. This work establishes sphingolipids as structural outputs of bacterial PKS and highlights the role of microbiota-derived sphingolipids in host protection against pathogens.

molecular biology↗

bioRxiv2024

Identification, structure and function of the methyltransferase involved in the biosynthesis of the dithiolopyrrolone antibiotic xenorhabdin

Xenorhabdins (XRDs) are produced by Xenorhabdus species and are members of the dithiopyrrolone (DTP) class of natural products that have potent antibacterial, antifungal and anticancer activity. The amide moiety of their DTP core can be methylated or not to fine-tune the bioactivity properties. However, the enzyme responsible for the amide N-methylation remained elusive. Here, we identified and characterized the amide methyltransferase XrdM that is encoded nearly 600 kb away from the XRD gene cluster using proteomic analysis, methyltransferase candidate screening, gene deletion, and allied approaches. In addition, crystallographic analysis and site-directed mutagenesis proved that XrdM is completely distinct from the recently reported DTP methyltransferase DtpM, and that both have been tailored in a species-specific manner for DTP biosynthesis in Gram-negative/positive organisms. Our study expands the limited knowledge of post-NRPS amide methylation in DTP biosynthesis and reveals the evolution of two structurally completely different enzymes for the same reaction in different organisms.

microbiology↗

bioRxiv2024

Genetic Toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling

BackgroundBacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for this genus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work. ResultsIn the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications. Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743. ConclusionsThe results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.

microbiology↗

bioRxiv2023

Transertion and cell geometry organize the Escherichia coli nucleoid during rapid growth

AO_SCPLOWBSTRACTC_SCPLOWBacterial chromosomes are spatiotemporally organized and sensitive to environmental changes. However, the mechanisms underlying chromosome configuration and reorganization are currently not fully understood. Using 3D single-molecule localization microscopy and live-cell imaging, we show that the Escherichia coli nucleoid adopts a condensed, membrane-associated configuration during rapid growth. To study the influence of different biosynthetic processes on nucleoid morphology and positioning, we recorded multi-colour super-resolution images during drug treatment. After developing analysis routines for confocal and super-resolution images, we captured highly resolved snapshots which revealed the complete loss of the membrane-bound state of the nucleoid within 10 minutes of halting transcription and translation. This indicates an active role of transertion (coupled transcription, translation and membrane insertion) in nucleoid organization. In contrast, cell wall synthesis inhibition only affects nucleoid organization during morphological changes. Further, we provide evidence that the E. coli nucleoid spatially correlates with MreB in unperturbed E. coli cells, while this correlation diminishes in cells with changed cell geometry or upon inhibition of protein biosynthesis. Replication inhibition experiments, as well as multi-drug treatments highlight the role of entropic effects and transcription in nucleoid condensation and positioning. In summary, we provide experimental evidence for transertion as a principal organiser of the bacterial nucleoid, and show that an altered metabolic state and antibiotic treatment both lead to major changes in the degree of transertion and overall spatial organization of the nucleoid. Our high-resolution characterization reveals dynamics of antibiotic action and provides tools to quantify bacterial chromosome organization. This does not only provide valuable insights into the role of transertion, but can also be applied to study other cell-biological processes.

microbiology↗

bioRxiv2023

Guidelines for Optimizing Type S Non-Ribosomal Peptide Synthetases

Bacterial biosynthetic assembly lines, such as non-ribosomal peptide synthetases (NRPS) and polyketide synthases, are often subject of synthetic biology - because they produce a variety of natural products invaluable for modern pharmacotherapy. Acquiring the ability to engineer these biosynthetic assembly lines allows the production of artificial non-ribosomal peptides (NRP), polyketides, and hybrids thereof with new or improved properties. However, traditional bioengineering approaches have suffered for decades from their very limited applicability and, unlike combinatorial chemistry, are stigmatized as inefficient because they cannot be linked to the high-throughput screening platforms of the pharmaceutical industry. Although combinatorial chemistry can generate new molecules cheaper, faster, and in greater numbers than traditional natural product discovery and bioengineering approaches, it does not meet current medical needs because it covers only a limited biologically relevant chemical space. Hence, methods for high-throughput generation of new natural product-like compound libraries could provide a new avenue towards the identification of new lead compounds. To this end, prior to this work, we introduced an artificial synthetic NRPS type, referred to as type S NRPS, to provide a first-of-its-kind bicombinatorial approach to parallelized high-throughput NRP library generation. However, a bottleneck of these first two generations of type S NRPS was a significant drop in production yields. To address this issue, we applied an iterative optimization process that enabled titer increases of up to 55-fold compared to the non-optimized equivalents, restoring them to wild-type levels and beyond.

synthetic biology↗