Metabolomics-Guided Discovery and Characterization of five new Cyclic Lipopeptides from Freshwater Isolate Pseudomonas sp.

Abstract

In order to fill the discovery void of novel anti Gram-negative substances, a selection of metabolomic tools was established for industrial routine application. The herein described platform was tested with a set of Streptomycetes strains, before environmental isolates were analyzed. First, two methods for quality control and chemical diversity assessment were compared. It appeared that a vector space model reduces the effect of outliers, compared to a principle component analysis, and is thereby most suitable to describe heterogeneous data such as metabolome comparisons. Second, the pipeline of data generation yielded unsupervised annotations of many literature known as well as structurally related, yet not described, molecules (variable dereplication) in the bacterial compound mixtures. The majority of compounds was detected by both applied methods, mass spectrometry (MS) based bucket annotation and tandem MS based molecular networking.In the following, the survey for new anti Gram negative compound producers was extended to environmental isolates. In that sense, the potential of the bacterial community of Lake Stechlin (Brandenburg, Germany) was evaluated. Different sample types, namely plankton associated and free living microorganisms, were retrieved from the lake and analyzed on the basis of their microbiome composition. The observed OTU distribution pattern was comparable to previously published observations, thus was considered to genuinely reflect the natural bacterial composition of the lake. The different samples types were shown contain distinct bacterial communities, thus made a great overall biodiversity available for further experiments.Prioritization of isolated bacteria from Lake Stechlin was carried out by cell free su- pernatant assays against E.coli DH5alpha. A metabolom analysis led to the discovery of a undescribed group of cyclic lipopeptides (CLPs) in the culture broth of Pseu- domonas sp. FhG100052. Culture condition optimization facilitated the isolation and subsequent structure elucidation of the five new compounds. Characterization comprised extensive MS/MS and NMR experiments in combination with Marfey s analysis. The data were in agreement with in silico analysis of the corresponding biosynthetic gene cluster (BGC). The new compounds resemble members of the Amphisin group [147] as they are constructed of a 3-hydroxy fatty acid linked to the N-terminus of an undecapeptide core. Most strikingly, the length of the incorporated fatty acid seems to define the moderate growth inhibitory effects against the Gram negative pathogen Moraxella catarrhalis FH6810 as observed by MIC values ranging from no inhibition (> 128 myg/mL) to 4 myg/mL.