Biosynthetic studies of the cyclocarbamate SB Yvonne Schmidt 1, Jos Raaijmakers 2, Harald Gross 1 1 University of Bonn, Institute for Pharmaceutical Biology, Nussallee 6, Bonn, Germany 2 Wageningen University, Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands Lipoprotein associated phospholipase A2 (LpPLA2) is responsible for hydrolysis of modified oxidized phospholipids from low density lipoprotein causing the release of pro-inflammatory lyso-phosphatidyl choline and oxidatively modified fatty acids. Inhibition of LpPLA2 is therefore considered a novel therapeutic strategy in the treatment of diseases that have an inflammatory component such as atherosclerosis. One of these metabolites is SB , a glycosylated lipopeptide produced by Pseudomonas fluorescens. The structure consists of a 5,5-bicyclic carbamate moiety, connected via two carbons and one nitrogen atom to myristic acid, which is in turn glycosylated with rhamnose. During a genomic-driven screening for lipopeptide gene clusters, we recently came across the corresponding gene cluster of SB which involves unexpectedly a two modular NRPS gene cluster. Both, its unique structure and its powerful Lp-PLA2-inhibition prompted our study of its biosynthesis. Of particular interest is hereby the modification of the putative involved second amino acid aside proline and the formation of the cyclocarbamate structure. Proof of the cluster Comparative metabolite Screening by LC-MS (Knockout vs Wildtype) Feeding studies Fig. 1 Fosmid C52 5-1contig 6 RC bp GT: Glycosyltransferase 1 Type B (Rhamnosyl-transferase) MO: FAD-dependent Monooxygenase LuxR: Transcription-regulator LuxR Type ES: Effluxsystem RND-Type SB Test organismsSB Mycobacterium smegmatis50 µg/ml Staphylococcus aureus SG µg/ml Bacillus subtilis12,5 µg/ml Listeria welchimeri25 µg/ml Staphylococcus aureus SG 51112,5 µg/ml Bacillus subtilis12,5 µg/ml Gram positive strains strain number Inhibition zone Methicillin-sensitive S. aureus51853 mm Staphylococcus aureusSG 5113 mm Bacillus subtilis1684 mm Listeria welchimeriDSM mm Corynebacterium pseudodiphtheriticum 3 mm Corynebacterium diphtheriae 3 mm Mycobacterium smegmatisATCC mm Corynebacterium xerosisVa mm Wildtype Knockout Further Bioactivity extracted mass: 554 – 556 m/z Predicted structure Isolated structures new SB congener NRPS GT According to bioinformatics, the predicted structure should be either a lipodipeptide including proline and serine or would result in a diketopiperazine moiety bearing a 3-hydroxy fatty acid. Introduction Labeling studies were performed in order to unravel the biosynthetic mechanism(s) involved in the massive rearrangement of the peptidic moiety leading to the cyclocarbamate skeleton. Feeding of single and double labeled sodium acetate provided an indirect proof for proline incorporation and confirmed the biosynthetic origin of the fatty acid. SB Gene cluster Determination of the MIC (minimal inhibition concentration) Bacterial inhibition assay DFG Research Unit 854 Post-Genomic Strategies for New Antibiotic Drugs and Targets LuxR MOES SerineProline