Pseudomonas CLiPs are ubiquitous specialized metabolites, impacting the producer’s lifestyle and interactions with the (a)biotic environment. Consequently, they generate interest for agricultural and clinical applications.

In Pseudomonas lipopeptides, the D-configuration of amino acids is generated by dedicated, dual-function epimerization/condensation (E/C) domains. The increasing attention to stereochemistry in lipopeptide structure elucidation efforts has revealed multiple examples where epimerization does not occur, even though an E/C-type domain is present. While the origin of the idle epimerization in those E/C-domains remains elusive, epimerization activity has so far shown a binary profile: it is either ‘on’ (active) or ‘off’ (inactive). Here, we report the unprecedented observation of an E/C-domain that acts ‘on and off’, giving rise to the production of two diastereoisomeric lipopeptides by a single non-ribosomal peptide synthetase system. Using dereplication based on solid-phase peptide synthesis and NMR fingerprinting, we first show that the two cyclic lipopeptides produced by Pseudomonas entomophila COR5 correspond to entolysin A and B originally described for P. entomophila L48. Next, we prove that both are diastereoisomeric homologues differing only in the configuration of a single amino acid. This configurational variability is maintained in multiple Pseudomonas strains and typically occurs in a 3:2 ratio. Bioinformatic analysis reveals a possible correlation with the composition of the flanking sequence of the N-terminal secondary histidine motif characteristic for dual-function E/C-type domains. In permeabilization assays, using propidium iodide entolysin B has a higher antifungal activity compared to entolysin A against Botrytis cinerea and Pyricularia oryzae spores. The fact that configurational homologues are produced by the same NRPS system in a Pseudomonas strain adds a new level of structural and functional diversification to those already known from substrate flexibility during the recruitment of the amino acids and fatty acids and underscores the importance of complete stereochemical elucidation of non-ribosomal lipopeptide structures.

We previously described NMR based fingerprint matching using peptide backbone (CHα) resonances as a fast and reliable way to perform structural dereplication of Pseudomonas cyclic lipodepsipeptides (CLiPs). In addition, its combination with total synthesis of a small library of CLiPs was shown to afford unambiguous determination of the stereochemistry, further opening these compounds for structure-activity relationship studies and three-dimensional structure determination. However, the need to include on-resin macrocycle formation in the synthetic workflow results in considerable burden and limits universal applicability to CLiPs. Here we show that this drawback can be removed by first converting the native CLiP of interest into its linearized analogue via controlled hydrolysis of the depsi bond through saponification. By avoiding the macrocycle formation altogether, the required synthesis effort is strongly reduced as it now only requires production of linear peptide analogues. The NMR fingerprints of the linear peptide analogues display a sufficiently distinctive chemical shift fingerprint to act as effective discriminators. The approach is developed using viscosin group CLiPs and subsequently demonstrated on putisolvin, leading to a structural revision, and tanniamide, a newly isolated compound defining a new group in the Pseudomonas CLiP portfolio. These examples demonstrate the effectiveness of the saponification- enhanced approach that broadens applicability of NMR fingerprint matching for the determination of the stereochemistry of CLiPs.