7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr.

Chair

Professor

Biosynthesis of bacterial tropone natural products through enzymatic salvaging of catabolic shunt products

Secondary metabolites and natural products

Question. Various bacterial tropone natural products such as the antibiotic sulphur-containing tropodithietic acid (TDA) or (hydroxy)tropolones adopt crucial roles in symbiotic interactions with eukaryotic hosts. Their biosynthesis relies on an unusual intertwining of primary and secondary metabolism, in which the initial steps are shared for the various tropones and rely on enzymes from phenylacetic acid (paa) catabolism. In this pathway, a distinct reactive open-chain aldehyde intermediate is formed, which is usually further degraded to central metabolites. In the case of tropone biosynthesis, however, this compound is instead cyclized to the characteristic tropone scaffold. Yet, it remains largely unknown how tailoring enzymes in certain bacteria further convert the tropone scaffold into the mature natural products.Methods. We employed in vitro reconstitution of diverse tropone biosynthetic pathways from Gram-negative and Gram-positive bacteria using heterologously produced enzymes from paa catabolism as well as enzymes encoded by different tropone biosynthetic gene clusters. Key enzymes were furthermore characterized mechanistically by enzyme assays and structurally by X-ray crystallography to obtain insights into their reaction mechanisms.Results. We unravelled an unexpected mechanistic diversity for tropone functionalization involving distinct key flavoenzymes, which drastically modify the tropone scaffolds in the late-stage biosynthesis of TDA from Gram-negative Phaeobacter sp. and 3,7-dihydroxytropolone from Streptomyces sp., respectively. In both cases, these enzymes mediate several unanticipated reactions en route to the mature natural products such as a so far unique flavoenzyme-catalyzed dioxygenation in TDA biosynthesis involving oxygenolytic CoA-thioester cleavage followed by a regioselective ring epoxidation.Conclusions. The biosynthesis of 3,7-dihydroxytropolone could be completely reconstituted and functions assigned to all involved enzymes. Similarly TDA biosynthesis was scrutinized, allowing for the discovery of a novel flavoprotein dioxygenase archetype, while final pathway steps involving the sulphur incorporation are subject to further investigation.

Oral Presentation

Genome sequence-based screening for novel phosphonate producers

Phosphonates are a unique class of natural products with diverse chemical structures and bioactivities. Numerous phosphonate natural compounds found their way into the market as for example the herbicide bialaphos, the antimalarial agent fosmidomycin or the antibiotic fosfomycin. An initial biosynthetic isomerisation step catalysed by the enzyme phosphoenolpyruvate mutase (PepM) forms the characteristic carbon-phosphorus bond. Due to the conservation of the PepM enzymatic reaction in the vast majority of phosphonate producers, the respective biosynthetic gene pepM can be used as a molecular marker to screen for potential phosphonate producer strains.The aim of the study was to screen the DSMZ Actinomycetales strain collection for novel phosphonate producers based on a pepM-guided genome mining attempt and analyse their biosynthetic products for potential novel phosphonates.The DSMZ Actinomycetales strain collection harbours &gt; 4.600 actinomycetes, many of which have already been genome-sequenced. A bioinformatic analysis of 600 genome sequences yielded 50 strains containing a pepM gene and thus a potential phosphonate biosynthetic gene cluster (BGC). The identified actinomycetes were cultivated on 8-10 different media following the OSMAC (one-strain-many-compounds) approach and were screened for phosphonate antibiotic production with bioassays using the phosphonate-sensitive test strain E. coli strain WM6242 [1], whereby 17 strains showed phosphonate-specific bioactivities. Phylogenetic analysis of the PepM amino acid sequences revealed a BGC-specific cladding. Cluster networking analysis were performed in order to prioritise strains with unique clusters for further analysis. Two strains, Kitasatospora fiedleri DSM 114396 [2] and Streptomyces iranensis DSM 41954, were prioritised for further analysis. Phosphonate production of the two strains was verified by 31P-NMR analysis, whereby inactivation of the respective pepM genes abolished phosphonate production. Antibiotic compound isolation and characterisation is ongoing.[1] C. Eliot et al., Chemistry &amp; Biology. 15, 765–770 (2008).[2] Zimmermann et al., IJSEM. 73, 006137 (2023).

Discovery of a new cinnamycin analog from an Actinomadura sp. isolated from meerkat feces

The rapid global spread of antibiotic resistance emphasizes the need for new effective therapeutics. The isolation of strains, e.g. of the actinomycetes, from unexplored habitats could potentially provide access to new or endemic species as the new sources for novel secondary metabolites. Here, we investigate an antibiotic-producing isolate from a meerkat (Suricata suricatta) fecal sample with regard to its antimicrobial activity.Diluted fecal samples were plated on MYM agar. Actinobacteria were isolated based on colony and cell morphology. The isolate was phylogenetically classified based on the 16S rDNA sequence. Mature colonies were overlayed with soft agar containing Gram-negative, Gram-positive or fungal species. Cell extracts from liquid cultures as well as colonies were analyzed regarding their antibiotic spectrum by applying a panel of whole cell biosensors, in which an antibiotic-inducible promoter is fused to the luciferase cassette. The potential antibiotic biosynthetic gene clusters (BGC) were identified by genome mining. During the plate-based biosensor screens, we identified naturally resistant Bacillus subtilis colonies growing in the zone of inhibition. After repeating rounds of selection, highly resistant spontaneous mutant strains were subjected to whole genome sequencing.16S rDNA sequencing identified the isolate as Actinomadura coerulea. The isolate showed a strong and broad range of antibacterial activity against Gram-positive bacteria, including pathogens, as well as an antifungal effect. The secondary metabolites of the isolate potentially inhibit replication, block translation, and interfere with bacterial cell wall integrity. Amongst others, we identified loss of function mutations in pssA, encoding a phosphatidylserine synthase. Based on the function of PssA and genome mining, the BGC for a novel cinnamycin analog was identified. Detailed follow-up studies are currently ongoing.Our approach demonstrates that combining the exploration of niche habitats for actinomycetes with whole-cell biosensor screening and characterization of natural resistance development provides a promising strategy for identifying novel antibiotics.

Raum 5-6

Oral Session

Discovery and Biosynthesis of Bioactive Natural Products

Oral Sessions

Oral Session
Discovery and Biosynthesis of Bioactive Natural Products

Appointment

Chair

Topic

Programme

Oral SessionDiscovery and Biosynthesis of Bioactive Natural Products

Appointment

Chair

Topic

Programme

Oral Session
Discovery and Biosynthesis of Bioactive Natural Products