Biology subjects

Gruen, P.

Publications and source records attributed to Gruen, P..

3 recordsLinked to original sources

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↗

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↗

bioRxiv2022

Global analysis of biosynthetic gene clusters reveals conserved and unique natural products in entomopathogenic nematode-symbiotic bacteria

Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus (XP) living in mutualistic symbiosis with entomopathogenic nematodes produce a myriad of natural products to mediate bacteria-nematode-insect interactions. However, a lack of systematic analysis of the biosynthetic gene clusters (BGCs) has limited the understanding of how natural products justify the bacterial niche specificity. Here we combine pangenome and sequence similarity networks to analyze BGCs from 45 XP species. The identified 1,000 BGCs belong to 176 families, over half of which are unknown. Eleven BGCs represent the most conserved families. We then homologously express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, insect virulence factors, or insect immune suppressors. These findings account for the functional basis of bacterial natural products in this tripartite relationship.

biochemistry↗