Alina Zimmermann (Tübingen / DE; Brunswick / DE), Julia Moschny (Tübingen / DE), Juan Pablo Gomez Escribano (Brunswick / DE), Shuning Xia (Tübingen / DE), Judith Boldt (Brunswick / DE; Hannover / DE), Ulrich Nübel (Brunswick / DE; Hannover / DE), Wolfgang Wohlleben (Tübingen / DE), Chambers C. Hughes (Tübingen / DE), Yvonne Mast (Tübingen / DE; Brunswick / DE)
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 > 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 & Biology. 15, 765–770 (2008).[2] Zimmermann et al., IJSEM. 73, 006137 (2023).