Natural Product Discovery at the Intersection of Genomics and Synthetic Biology: Insights from Bacteroidota and Acidobacteriota

Abstract

Microbial natural products remain a valuable source of bioactive compounds, such as antibiotics; however, discovery pipelines often encounter issues such as rediscovery, culture bias, and natural product (NP)-encoding but silent biosynthetic gene clusters (BGCs). This dissertation connects and applies an omics-guided, cultivation-dependent workflow for underexplored Bacteroidota and Acidobacteriota. It also establishes a heterologous expression toolkit for optimising the expression of silent biosynthetic gene clusters by applying synthetic biology principles. The section “Genome Mining of Bacteroidota” employed genome mining tools, including quality control, taxonomy, BGC prediction, and similarity networking, to profile and describe newly isolated marine Bacteroidota. These expanded genomic reference resources support future rational strain prioritisation for natural product discovery. Thus, the work establishes a foundation for systematic genomics documentation and reporting, enabling portfolio-style reporting of novel strains that are being included in the Fraunhofer (FHG) strain collection. A genus-wide analysis of the Pedobacter (phylum Bacteroidota) identified a branch genetically enriched in multimodular non-ribosomal peptide BGCs. Genetic validation connected one NRP cluster of interest to the novel cryopeptin lipopeptide family, positioning this clade as a genetically tractable hotspot for novel scaffolds. The section “Profiling of Novel Acidobacteriota” investigates the biosynthetic potential of the Acidobacteriota. High-throughput selective cultivation yielded novel representatives of the Acidobacteriaceae, which were profiled for their BGC composition in a comparative study of curated public Acidobacteriota genomes. Metabolomics revealed the production of the phytohormones indole-3-acetic acid (IAA) and the cytokinin N6-(Δ2-isopentenyl)adenine (iP). Subsequent tryptophan supplementation drove a global metabolic shift, also increasing IAA production while generally decreasing iP. Absolute quantification of phytohormones placed amounts within functionally relevant ranges, but crude-extract testing did not increase barley seedling biomass under the tested conditions. Furthermore, tryptophan-driven metabolic reprogramming induced the production of indole-derived antifungal metabolites that inhibited the growth of the phytopathogen Septoria tritici. These results refine the ecological and functional potential of Acidobacteriota and map the distributions of plant growth-promoting traits (PGPTs) across families. The last section presents HEL, a modular, heterologous expression and library-style cloning platform that retrofits BGC clones with reusable, exchangeable expression cassettes (e.g., promoter libraries) and optional host-specific compatibility cassettes for broad chassis transfer. By doing so, HEL enables the tuning of expression by linking a prioritised BGC to standard, library-style phenotypes using modular cloning (MoClo), a Golden Gate-like cloning method. Collectively, this dissertation provides a reproducible genomic workflow for underexplored phyla and the FHG strain collection (i); new genomic resources, genetic prioritisation, and tractability in Pedobacter (ii); integrated genomic–metabolomic insights into tryptophan-induced metabolic dynamics, phytohormone biology, and PGPT architecture in Acidobacteriaceae, and (iii) a flexible genetic toolkit (HEL) to induce activation or accelerate the expression of prioritised BGCs across different heterologous hosts.