Manuel Einsiedler

Maculalactones are bioactive γ-butyrolactone natural products with promising antifouling properties, yet their limited availability constrains further development. Here, we report the characterization of the key biosynthetic enzymes MacE and MacF from the maculalactone (mac) biosynthetic gene cluster in Nodularia sp. NIES-3585. Exploiting the substrate flexibility of these biocatalysts, we developed a one-pot chemo-enzymatic platform that enabled the synthesis of 13 maculalactone derivatives in isolated yields of up to 96%. Furthermore, a plug-and-play combinatorial biosynthesis strategy with predictable product outcomes was established by assembling an artificial biosynthetic gene cluster from three distinct pathways, enabling the biosynthesis of previously inaccessible maculalactone analogs, including indole-containing derivatives, in a heterologous host (E. coli). Biological evaluation reveals that maculalactone derivatives exhibit cancer-type selective cytotoxicity, with several compounds displaying potent cytotoxicity against colorectal cancer cells (HCT116) with the best IC50 values reaching 2.7 μM. In contrast, only moderate or negligible activity was observed against lung and breast cancer cell lines, indicating pronounced selectivity. Initial mechanistic studies show that these compounds induce DNA damage, leading to apoptosis and inhibition of cancer cell proliferation. In addition, several derivatives exhibited antibacterial activity against Gram-positive pathogens, including Streptococcus pneumoniae. Collectively, our work establishes a general and programmable strategy for maculalactone diversification, expands their chemical and biological space, and highlights their potential as a promising scaffold for the development of selective anticancer agents.

The glycopeptide antibiotics (GPAs) are clinically relevant antibiotics defined by their highly cross-linked structure, which is required for their antimicrobial activity. While GPAs are typically characterized as type I–IV GPAs – heptapeptides that bind to the d-Ala-d-Ala terminus of Lipid II – an expansion of these subtypes has recently been seen for type V GPAs that possess altered structural characteristics and mechanisms of action. The major structural changes seen with this class are due to varied cross-linking occurring between the aromatic side chain residues, with these cross-links installed by cytochrome P450 (Oxy) enzymes that are recruited to the peptide substrate during nonribosomal peptide synthesis by a specialized recruitment domain, known as the X-domain. Given the importance of these cross-links for the structural rigidity and activity of GPAs, in vitro characterization of their activity remains a priority for the field. In this study, we synthesized a range of peptides, including an authentic complestatin precursor, to explore the cross-linking of these peptides by a selection of Oxy enzymes. Our results show the importance of stereochemistry in the C-terminal portion of the peptide substrate, revealing alternate cross-linking patterns and emphasizing the challenge of working with complete type V GPA peptides in vitro.

Terpenes make up a structurally and functionally highly diverse class of natural products found across all living organisms. Pseudomonas species possess significant potential for the biosynthetic assembly of farnesyl pyrophosphate (FPP)-derived terpenes with unusual carbon skeletons. However, their structural and biosynthetic diversity has been largely unexplored. Here, we report the discovery of grimophan, a C₁₇ terpene featuring a rare deltacyclane skeleton. The compound was accessed by heterologous expression of the pgr biosynthetic gene cluster from Pseudomonas grimontii DSM 17515 in E. coli. The roles of the enzymes involved in grimophan biosynthesis were elucidated through in vitro reconstitution of the entire biosynthetic pathway. In-depth functional studies on the involved SAM-dependent methyltransferases led to the discovery of methyltransferase-like enzymes that do not perform functional group transfer, but rather significantly enhance the production titer of noncanonical terpenes. These enzymes thus constitute valuable tools for enhancing biotechnological production levels of noncanonical C₁₆ and C₁₇ terpenes. The function of the cognate terpene synthase PgrE was evaluated by targeted point mutations within the observed Asp-rich motif D₉₂DMPLG₉₇ to map the effects of active-site residues on product formation. These investigations facilitated redirecting product selectivity, leading to alternative products. Overall, our work sheds light on the general biosynthetic logic leading to noncanonical C₁₇ terpenes and provides a basis for their targeted discovery by genome mining and heterologous expression and for engineering C₁₇ terpene structural frameworks.

Maculalactones are bioactive γ-butyrolactone natural products with promising antifouling properties, yet their limited availability constrains further development. Here, we report the identification and characterization of the key biosynthetic enzymes MacE and MacF from the maculalactone (mac) biosynthetic gene cluster in Nodularia sp. NIES-3585. In vitro reconstitution confirmed that MacE catalyzes acyloin condensation between phenylpyruvic acids, while MacF mediates subsequent fusion of the MacE product with cinnamoyl-CoA derivatives. Structure-guided engineering improved enzyme solubility, enabling systematic substrate scope expansion. Through a one-pot enzymatic synthesis, we synthesized 13 maculalactone derivatives with yields reaching more than 96%, while a complementary combinatorial biosynthesis approach enabled the incorporation of sterically demanding indole moieties, thereby significantly expanding the maculalactone structural space. Bioactivity screening against A549 lung adenocarcinoma cells revealed moderate cytotoxicity (IC₅₀ = 20.82–56.64 μM), with methylation of phenol groups significantly enhancing activity. Several analogs exhibited notable antibacterial activity against clinically relevant Gram-positive pathogens, including Streptococcus pneumoniae. Our work provides a sustainable platform for producing structurally diverse maculalactones, laying the foundation for future biomedical applications.

Terpenes are a structurally and functionally highly diverse class of natural products found across all living organisms. Pseudomonas species possess significant potential for the biosynthetic assembly of farnesyl pyrophosphate (FPP)-derived terpenes with unusual carbon skeletons. However, their structural and biosynthetic diversity is largely unexplored. Here, we report the discovery of grimophan, an unprecedented C17 terpene featuring a deltacyclane skeleton. The compound was accessed by heterologous expression of the pgr biosynthetic gene cluster from Pseudomonas grimontii DSM 17515 in E. coli. The roles of the enzymes involved in grimophan biosynthesis were elucidated through in vitro reconstitution of the entire biosynthetic pathway. In-depth functional studies on the involved SAM-dependent methyltransferases led to the discovery of novel methyltransferase-like enzymes that do not perform functional group transfer, but rather significantly enhance the production titer of non-canonical terpenes. These enzymes thus constitute valuable tools for increasing biotechnological production levels of terpenes. The function of the cognate terpene synthase PgrE was evaluated by targeted point mutations within the conserved Asp-rich motif D92DMPLG97 to map effects of active-site residues on product formation. These investigations facilitated redirecting product selectivity, leading to alternative products. Overall, our work sheds light on the general biosynthetic logic leading to non-canonical C17 terpenes, provides a basis for their targeted discovery by genome mining and heterologous expression, and for engineering C17 terpene structural frameworks.

Antimicrobial resistance remains a formidable challenge to modern medicine, with bacterial resistance mechanisms increasingly eroding the utility of clinically important antibiotics. While recent efforts have expanded the antibacterial pipeline, the development of resistance in priority pathogens continues to exceed the pace of new drug development. One emerging strategy to overcome resistance is the rational design of hybrid antibiotics that engage multiple binding sites. Here we describe the design, synthesis, and microbiological and structural characterization of hybrid antibiotics of azithromycin, tedizolid, and chloramphenicol that span the peptidyltransferase center (PTC) and nascent peptide exit tunnel (NPET) in the bacterial ribosome. We characterize the binding of four such hybrids by cryo-electron microscopy, granting insight into their molecular mechanisms of action. We identify a hybrid of azithromycin and tedizolid that is active against a diverse panel of multidrug-resistant Gram-positive bacteria and is minimally affected by ribosomal protection (ABC-F) resistance mechanisms. These results extend our understanding of ribosome inhibition and provide a pipeline for the rational design of dual-action antibiotics that target the ribosome. In a broader context, this work offers a framework for developing bifunctional inhibitors that engage adjacent binding sites by means of a rational cycle of synthetic optimization, biological evaluation, and structural characterization.

Furanolides represent an emerging class of natural products known for their structural diversity and potent bioactivities, including antibiotic, cytotoxic, or algicidal effects. The systematic exploration of their biological activity profiles for the discovery of new hits for drug development thus constitutes a promising endeavor. However, their low natural abundance and the resulting difficulty in obtaining sufficient quantities have limited further in-depth investigations into their biological activity and structure-activity relationships (SARs). Building on our recent discovery of biosynthetic enzymes catalyzing furanolide core-structure assembly, we herein developed a cost-effective, one-pot enzymatic toolbox that enables the fast generation of hundreds of furanolide structural analogs. We systematically evaluated antimicrobial activities and cytotoxicity against the A549 lung cancer cell line for a representative selection of library congeners and explored their SARs. Several derivatives demonstrated significant cytotoxicity, particularly against lung cancer stem cells, offering promising insights into the development of furanolides as potential anticancer agents. Additionally, some analogs displayed promising antibacterial activity against important Gram-positive pathogens such asStaphylococcus aureus.

Furanolides represent an emerging class of natural products known for their structural diversity and potent bioactivities, including antibiotic, cytotoxic, or algicidal effects. The systematic exploration of their biological activity profiles for the discovery of new hits for drug development thus constitutes a promising endeavor. However, their low natural abundance and the resulting difficul-ty in obtaining sufficient quantities have limited further in-depth investigations into their biological activity and structure-activity relationships (SAR). Building on our recent discovery of biosynthetic enzymes catalyzing furanolide core-structure assembly, we herein developed a cost-effective, one-pot enzymatic toolbox that enables the fast generation of hundreds of furanolide structural analogs. We systematically evaluated antimicrobial activities and cytotoxicity against the A549 lung cancer cell line for a repre-sentative selection of library congeners and explored their SAR. Several derivatives demonstrated significant cytotoxicity, particu-larly against lung cancer stem cells, offering promising insights into the development of furanolides as potential anticancer agents. Additionally, some analogs displayed promising antibacterial activity against important Gram-positive pathogens such as Staphylo-coccus aureus.

Antimicrobial resistance remains a formidable challenge to modern medicine, with bacterial resistance mechanisms increasingly eroding the utility of clinically important antibiotics. While recent efforts have expanded the antibacterial pipeline, the development of resistance in priority pathogens continues to exceed the pace of new drug development. One emerging strategy to overcome resistance is the rational design of hybrid antibiotics that engage multiple binding sites. Here we describe the design, synthesis, and microbiological and structural characterization of hybrid antibiotics of azithromycin, tedizolid, and chloramphenicol that span the peptidyltransferase center (PTC) and nascent peptide exit tunnel (NPET). We characterize the binding of five such hybrids to the bacterial ribosome by cryo-electron microscopy, granting insight into their molecular mechanisms of action. We identify a hybrid of azithromycin and tedizolid that is active against a diverse panel of Gram-positive bacteria, including strains with both macrolide and oxazolidinone resistance mechanisms. These results extend our understanding of ribosome inhibition and demonstrate that the rational design of dual-action antibiotics can restore or even enhance antibacterial efficacy against resistant strains. In a broader context, this work offers a promising framework for developing next-generation therapeutics to combat multidrug-resistant pathogens and reinvigorate the dwindling antibiotic pipeline.

Polycyclic tetramate macrolactams (PoTeMs) represent a growing class of bioactive natural products that are derived from a common tetramate polyene precursor, lysobacterene A, produced by an unusual bacterial iterative polyketide synthase (PKS)/non‐ribosomal peptide synthetase (NRPS). The structural and functional diversity of PoTeMs is biosynthetically elaborated from lysobacterene A by pathway‐specific cyclizing and modifying enzymes. This results in diverse core structure decoration and cyclization patterns. However, approaches to directly edit the PoTeM carbon skeleton do currently not exist. We thus set out to modify the PoTeM core structure by exchanging the natural l‐ornithine‐derived building block by l‐lysine, hence extending macrocycle size by an additional CH₂ group. We developed streamlined synthetic access to lysobacterene A and the corresponding extended analog and achieved cyclization of both precursors by the cognate PoTeM cyclases IkaBC in vitro. This chemo‐enzymatic approach corroborated the catalytic competence of IkaBC to produce a larger macrolactam yielding homo‐ikarugamycin. We thus engineered the adenylation domain active site of IkaA to directly accept l‐lysine, which upon co‐expression with IkaBC delivered a recombinant bacterial homo‐ikarugamycin producer. Our work establishes an entirely new PoTeM structural framework and sets the stage for the biotechnological diversification of the PoTeM natural product class in general.