An Nuclear Magnetic Resonance Fingerprint Matching Approach for the Identification and Structural Re-Evaluation of Pseudomonas Lipopeptides

(i) Isolation, bioinformatics, and initial chemical structure characterization. P. azadiae SWRI103 was originally collected from the rhizosphere of wheat in Iran, as part of a campaign to assess the plant growth stimulating potential of fluorescent pseudomonads (50). Initial PCR screening targeting initiation and termination domains indicated the presence of a lipopeptide-specific NRPS system (51). More recently, whole genome sequencing revealed a biosynthetic gene cluster (BGC) (52) with similarities to that of several Bananamide (8:6) producers, such as Pseudomonas bananamidigenes BW11P2 (37), Pseudomonas botevensis COW3 (53) and Pseudomonas prosekii LMG26867^T (54). Here, the (8:6) refers to the (l:m) notation we introduce to provide a simple but effective classification of CLiPs belonging to the same group from a chemical structure perspective, as explained in “Materials and Methods”. As the stereochemistry of all these bananamides was unknown, we used strain SWRI103 to attempt full structure elucidation, which could only be achieved by the development of our expanded workflow, with all steps in the process described in detail hereafter (Fig. S1).

Firstly, analysis of the retrieved BGC predicted a Leu – Asp – Thr – Leu – Leu – Ser – Leu – Ile octapeptide sequence from the associated NRPS (Table 1). The total peptide sequence length and amphipathic profile matches that of previously reported and characterized bananamides (37, 53) but differs in amino acid composition. Therefore, it may represent a novel member of the Bananamide group (8:6). Next, bioinformatic analysis of the condensation/epimerization (C/E) domains was applied for the configurational analysis of the amino acids. The initial C_start domain is responsible for the incorporation of a fatty acid (FA) moiety at the N-terminus of the peptide, the exact nature of which cannot be predicted. This domain is followed by six dual activity C/E domains, responsible for the condensation of the newly recruited l-amino acid to the growing peptide chain along with by l to d epimerization of the preceding residue in the sequence (55, 56). This is followed by one ^LC_L-type domain, which lacks the epimerization functionality thus retaining the l-configuration of the preceding residue while incorporating an l-amino acid as final residue before cyclisation by a tandem of transesterification (TE) domains. Assuming that all dual C/E domains have functional epimerization activity, the combined A- and C-domain analysis predicts a FA – d-Leu – d-Asp – d-aThr – d-Leu – d-Leu – d-Ser – l-Leu – l-Ile sequence as the most likely lipopeptide biosynthesized by P. azadiae SWRI103 (Table 1).

Secondly, and independently from the bioinformatic analysis workflow, we engaged into the chemical analysis workflow of the putative novel bananamide. Following incubation of P. azadiae SWRI-103 in M9 minimal salt medium, a single CLiP-containing fraction could be extracted and purified. From high resolution MS analysis, a molecular mass of 1053.67 Da could be established i.e., within the expected range for an (8:6) CLiP. Next, NMR was used to elucidate the planar structure. That is, combined COSY/TOCSY analysis showed the presence of a single glutamic acid, serine, threonine, isoleucine, and four leucine residues in the peptide sequence while NOESY and ¹H-{¹³C} HMBC experiments independently allowed to place these in the same order as predicted from the A-domain analysis, validating the latter (Table 1). Note however that while an aspartic acid residue was predicted at position 2 from the bioinformatic analysis, the presence of a glutamic acid was observed here using NMR spectroscopy. This is not surprising, as it was previously shown that Asp/Glu selectivity from genomic predictions is not always clear-cut (37). Additionally, the ¹H-{¹³C} HMBC spectrum showed a clear ³J_CH correlation between Thr3 H^β and Ile8 C’ thereby unambiguously establishing that the ester bond occurs between the hydroxyl side chain of threonine and the isoleucine carboxyl end, thus revealing the six-residue macrocycle expected for a member of the Bananamide group. Taking into account that C₅₂H₉₂N₈O₁₄ was derived as molecular formula from the HR-MS data, a 3-hydroxydecanoic moiety was inferred and confirmed from the NMR data while its linkage to the N-terminus resulted from characteristic ³J_CH contacts with Leu1 in the ¹H-{¹³C} HMBC spectrum. As neither MS nor NMR analysis allows establishing the configuration of the individual amino acid residues, we proceeded to Marfey’s method for the analysis of amino acid configuration. Following total hydrolysis of the CLiP, Marfey’s analysis allowed to unambiguously determine the configuration of all uniquely occurring residues. For the 6 leucines however, a 2:2 d:l ratio was found, indicating configurational heterogeneity and, as a result, the exact distribution of d and l-Leu residues within the oligopeptide sequence remained hidden. Therefore, at the end of the chemical analysis workflow, a total of 6 distinct sequences which all satisfy the 2:2 ratio but differ in the distribution of d- and l-leucines should therefore be considered. As a result, the definitive elucidation of the stereochemistry and, therefore, the chemical structure thus reaches a dead-end.

When information regarding a CLiP sequence is available from both bioinformatic and chemical analysis workflows, the configurational ambiguity is sometimes resolved by proposing that one of the sequences issued from chemical analysis matches the bioinformatic prediction (Fig. S1). However, such proposals should always be treated with caution since the epimerization functionality of C/E domains can be inactive in a currently unpredictable manner. Moreover, as aptly illustrated by all cases reported here, the predicted configuration from C-domain analysis may not even match those originating from the chemical analysis workflow. Here, for instance, the bioinformatic analysis proposes a 3:1 d:l ratio for the 4 leucines rather than the 2:2 found experimentally. Nevertheless, the bioinformatic analysis can still provide guidance to narrow down the number of possible diastereomeric sequences by noting that the ^LC_L domains, which lack the epimerization functionality, will maintain the l-configuration of the amino acid introduced at the preceding position (see above). Since such ^LC_L domain is present in the final NRPS module of P. azadiae SWRI103, the penultimate Leu7 residue will invariably retain its l-configuration upon recruitment. This narrows down the number of possible sequences from 6 to 3: (8:6)-L1 (1), (8:6)-L4 (2) and (8:6)-L5 (3) (Table 1), depending on which leucine position features the remaining l-configuration. In all cases, the 3-hydroxy fatty acid tail is assumed to be (R)-configured, as is generally observed for Pseudomonas CLiPs. Notwithstanding the combination of bioinformatic and chemical analysis workflows, the stereochemistry remains ill-defined.

(ii) Extending the chemical analysis workflow by a synthesis add-on. As recently reviewed by Götze and Stallforth, several approaches exist to tackle the issue of configurational ambiguity (24). In the absence of suitable crystals for X-ray diffraction-based structure elucidation, the most common one resorts to mild acid catalyzed hydrolysis conditions or enzymatic degradation of linearized lipopeptides to yield oligopeptide fragments. These then need to be isolated, characterized by NMR or MS, and subjected to Marfey’s method to identify the correct position of the corresponding d- or l-amino acids, introducing an extensive workload without guarantee that the matter will be settled (26). An alternative solution using MS makes clever use of deuterated amino acids and the fact that d-amino acids are, with very few exceptions, generated during biosynthesis through epimerization from l-amino acids (23, 57). In all cases, a separate analysis should be performed to elucidate the configuration of the 3-hydroxy fatty acid moiety (58). Here, we rather build on our previously developed total chemical synthesis route for Viscosin (9:7) group CLiPs (48, 59) to synthesize the 3 remaining (8:6)-Lx (x = 1, 4, 5) sequences (Table 1). The total chemical synthesis strategy mostly relies on solid phase peptide synthesis, affording considerable automation and rapid access to multiple homologous sequences through parallel synthesis (48, 59, 60). With one residue less in the macrocycle and a d-Glu2 rather than d-Gln2 as main differences, the synthesis of the (8:6)-Lx sequences could proceed with minimal change to the original strategy. A more extensive discussion of the applied synthesis route and its key features can be found in the Supplementary Materials section.

Next, the exact stereochemistry of the natural compound is revealed by matching the NMR spectral fingerprint to those of the synthetic compounds. The NMR matching approach relies on previous observations where, using the ¹H-{¹³C} HSQC experiment, we established that the inversion in the configuration of a single Cα atom introduces prominent changes in the ¹H and ¹³C chemical shifts of the corresponding (C–H)α group, even when the three-dimensional structure of the CLiP was retained (47). The same trend was found for the configuration of the 3-hydroxy fatty acid moiety (48). Thus, the (C–H)α fingerprint region of the HSQC spectra of the natural compound and its synthetic (8:6)-Lx analogue are expected to be identical. In contrast, the 2 other sequences will display clearly distinct HSQC spectra in general and (C–H)α fingerprint regions in particular as they feature 2 d:l inversions compared to the natural compound (Table 1).

By individually overlaying the ¹H-{¹³C} HSQC spectra of each synthesized (8:6)-Lx variant with that of the natural compound from P. azadiae SWRI103, a straightforward visual assessment concerning similarities and differences in the ¹H and ¹³C chemical shifts can be made (Fig. 1). Being the most sensitive reporters of backbone stereochemistry, we focus here on the cross-peaks in the (C-H)α fingerprint regions (Fig. 1). Accordingly, the (8:6)-L4 (2) variant shows an excellent match with the (C–H)α fingerprint of the natural compound since all (C-H)α cross peak pairs belonging to the same residue in the sequence show excellent overlap (Fig. 1A). In contrast, a considerable mismatch exists with (8:6)-L1 (1) as visualized by five clearly non-overlapping cross-peaks (Fig. 2B). This mismatch becomes even more pronounced for (8:6)-L5 (3), where essentially none of the cross-peaks overlap (Fig. 1C). Based on this, we can establish the stereochemistry of the P. azadiae SWRI103 bananamide as identical to that of the (8:6)-L4 (2) sequence (i.e., 3R-OH C10:0 – d-Leu – d-Glu – d-aThr – l-Leu – d-Leu – d-Ser – l-Leu – l-Ile), revealing that the C/E domain in the fifth module of the NRPS system is non-functional for epimerization. A more quantitative evaluation of spectral similarity is provided in the supplementary material section.

In addition, to avoid any confusion regarding the absolute configuration of the 3-hydroxy decanoic acid, an analogue identical to L4 (2), but with a 3-(S)-hydroxydecanoic acid as fatty tail was synthetized as well (compound 2b). The overlay of this peptide with the natural compound revealed that inverting the configuration at this position clear affects the fingerprint region (supplementary information, S19). Indeed, the HSQC cross peak of the CH^β of the fatty acid is shifted to the largest extent while additional shifts were also noticed at the level of the CH^α cross-peaks of Leu1, Glu2, and Thr3. This not only confirms the possibility to discriminate the configuration at this position, but also the use of NMR based fingerprinting for configurational assignment.

(iii) Matching our reference compound with literature data. The effort to elucidate the stereochemistry of the P. azadiae SWRI103 (8:6) bananamide also allowed to settle that of MDN-0066, an (8:6) CLiP produced by Pseudomonas granadensis F-278,770^T, which showed distinct bioactivity in a renal carcinoma cell model (17). Using a chemical analysis methodology similar to the one described above, Cautain et al. (17) elucidated the peptide sequence by relying on MS analysis and NMR spectral assignment. However, the detailed stereochemical elucidation of MDN-0066 was left incomplete as Marfey’s analysis revealed configurational heterogeneity given the presence of 2 d-Leu and 2x l-Leu, a result similar to that of the P. azadiae SWRI103 bananamide. Using the tabulated ¹H and ¹³C NMR chemical shifts reported for MDN-0066 in DMSO-d6, the spectral fingerprint matching could be performed against the data of the SWRI103 bananamide recorded under identical conditions. The result, shown in Fig. 1D, shows a near identical match with (8:6)-L4 (2). This proves that MDN-0066 is identical to bananamide SWRI103, thereby disambiguating the stereochemistry of MDN-0066 and illustrating the potential of our approach for dereplication purposes. In order to assess the general applicability of our approach to Pseudomonas CLiPs, we then turned to 2 additional case studies, involving the stereochemical elucidation of orfamide B and xantholysin A.

Orfamides are important for bacterial motility, cause lysis of oomycete zoospores, and play a role in biocontrol activity against fungal pathogens and insects (6, 49, 61–63). The name-sake of the Orfamide (10:8) group, orfamide A, was extracted from Pseudomonas protegens Pf-5 and fully characterized including its complete stereochemistry by mass spectrometry, NMR spectroscopy and chiral gas chromatography (49). Orfamides B and C were extracted as minors, and their planar structures were characterized as well. Since its original discovery, orfamide B is often found as the major compound produced by newly isolated bacterial strains (35). Additionally, the orfamide group has expanded with additional orfamide-homologues (30, 35, 64, 65). However, in many cases, the stereochemistry of orfamide B or that of other orfamide homologues from newly isolated bacterial sources remained unconfirmed as it was derived from sequence similarity with the original orfamide A as extracted from P. protegens Pf-5. To unlock conformational analysis and structure-activity evaluations for the Orfamide group, we therefore proceeded to an explicit stereochemical verification.

(i) Isolation, bioinformatics, and initial chemical structure characterization of orfamide B. P. aestus CMR5c was originally isolated from the rhizosphere of red cocoyam in Cameroon in a screen for biocontrol agents against the cocoyam root rot disease caused by Pythium myriotylum (11). We already reported the initial genome mining and bioinformatic analysis of the P. aestus CMR5c BGC which revealed the presence of 3 genes with ~80% similarity to the ofaA, ofaB, and ofaC NRPS genes from P. protegens Pf-5 (35). MS and NMR analysis further confirmed the predicted primary sequence and evidenced the incorporation of a 3-hydroxy-tetradecanoic acid moiety at the N-terminus. Thus, the major CLiP produced by P. aestus CMR5c has a planar structure identical to the originally published orfamide B. Based on this similarity in primary sequence and the genomic similarity between the respective BGCs, orfamide B from P. aestus CMR5c was proposed to also possess only l-leucines (35), as was previously also postulated for the original orfamide A (49).

C-domain analysis showed that the initial C_start type domain involved in acylating the first amino acid is followed by 6 dual activity C/E domains (modules 2–7), 2 non-epimerizing ^LC_L type domains (modules 8 and 9), and a final C/E domain in the last module (Table 2). Taking into account the distribution of ^LC_L and C/E domains, the bioinformatic analysis predicts the stereochemistry as shown in Table 2, with d-Leu residues occurring at positions 1 and 5 (Table 2) contradicting the all-l-Leu configuration proposed earlier. Marfey’s analysis confirmed the configuration predictions for singly occurring amino acids and the 1:1 d:l ratio for both valines. For the leucines however, a 1:3 d:l ratio was found, invalidating the 2:2 ratio predicted from bioinformatic analysis as well as the all-l configuration proposed by Ma et al. (35).

(ii) Stereochemical elucidation using chemical synthesis and NMR fingerprint analysis. Considering the experimental evidence from the chemical analysis workflow, a total of 8 sequences should effectively be considered since the configurational d:l heterogeneity of the Leu and Val residues are combinatorically independent. Conveniently, the bioinformatic analysis allows to trim this down to 2 sequences only, strongly reducing the synthetic effort required. Indeed, the ambiguity regarding the valines can be settled by noting that, as the final residue, Val10 is not subjected to any epimerization activity. This pins the valine configurations down as d-Val4 end l-Val10. Next, the presence of ^LC_L domains in modules 8 and 9 allows to unequivocally attribute the l-configuration to Leu7 and l-Leu8. The remaining d and l configured leucines are to be distributed over positions 1 and 5. While this constitutes an apparent dead-end, configurational assignment could be finalized using fingerprint matching of the natural compound against the (10:8)-L1 (4) and (10:8)-L5 (5) sequences obtained by synthesis (Table 2) using the same strategy as discussed above for the bananamide analogues. This included the incorporation of a 3R-hydroxydecanoic moiety (3-OH C10:0) at the N-terminus rather than a 3R-hydroxytetradecanoic one (3-OH C14:0) mainly because of synthetic availability of the precursor. Previous investigation of C₁₀, C₁₂ and C₁₄ pseudodesmin analogues evidenced that lengthening of the acyl chain did not affect the ¹H and ¹³C chemical shifts of the peptide moiety in any way (60).

Fig. 2 shows the overlay of the (C–H)α fingerprint region of each synthesized (10:8)-Lx sequence with that of natural orfamide B from P. aestus CMR5c. A straightforward visual assessment clearly indicates that the (10:8)-L1 (4) variant displays a near identical fingerprint match while none of the (C–H)α cross-peaks of (10:8)-L5 (5) overlap with those of the CMR5c orfamide. The latter probably indicates a major conformational effect caused by the l to d inversion at Leu5. To remove any doubts and independently exclude the all-l-Leu configuration originally proposed from sequence similarity with orfamide B from P. protegens Pf-5, we also committed to synthesize the corresponding (10:8)-L1L5 (6) analogue which again showed significant mismatch with the natural CMR5c orfamide. (Fig. S38) All data together establish that the stereochemistry of orfamide B from P. aestus CMR5c corresponds to that of the (10:8)-L5 (5) sequence (3R-OH C14:0 – l-Leu – d-Glu – d-aThr – d-Val – d-Leu – d-Ser – l-Leu – l-Leu – d-Ser – l-Val), indicating the C/E domain in the second module is non-functional for epimerization.

(iii) Stereochemical reassessment and dereplication of the Orfamide group using literature NMR data. The results presented above appear to indicate that orfamide B from P. aestus CMR5c is a d-Leu5 diastereoisomer of orfamide B from P. protegens Pf-5 (49), since the latter is reported to possess a l-Leu5. So far, reports of CLiPs from the same (l:m) group that feature a configurational difference are few, possibly due to the lack of extensive configurational assignment among Pseudomonas CLiPs. The best-known example occurs within the Viscosin (9:7) group where CLiPs can be divided in a L-subgroup (14 sequences) and d-subgroup (5 sequences) depending on the configuration of the leucine, also at position 5 (66). Given that orfamides have been reported from multiple bacterial sources since their initial discovery, the question raised as to whether such division is also present for the Orfamide (10:8) group. Settling this matter would allow assessing whether stereochemical variation within a group is a more general feature present in the NRPS of Pseudomonas CLiPs. Conveniently, the major orfamides reported from other bacterial sources in literature feature planar structures identical to either orfamide A from P. protegens Pf-5 or orfamide B from P. aestus CMR5c, thus allowing the use of NMR fingerprinting for stereochemical evaluation. To be able to screen directly against a reference spectrum of orfamide A under standardized conditions, P. protegens Pf-5 was cultured to obtain orfamide A, together with a series of minor compounds including orfamide B and 4 previously unreported ones, which we named orfamides J – M (See supplementary information).

For the orfamide B producer Pseudomonas sessilinigenes CMR12a (67), the ¹H-{¹³C} HSQC spectral fingerprint proved identical to the spectrum of the orfamide B reference from P. aestus CMR5c. This indicated that these molecules have identical stereochemistry and feature a d-Leu5. Next, we turn to Pseudomonas sp. PH1b where genome exploration revealed an orfamide-type BGC (68). Characterization of orfamides from this isolate presents an interesting case as it produces a major and a minor orfamide with sequence identical to orfamide A and orfamide B, respectively, thus only differing by a Ile4/Val4 substitution resulting from A-domain substrate flexibility in module 4 of the NRPS. The (C–H)α spectral fingerprint of its major orfamide matched with that of orfamide A produced by P. protegens Pf-5 indicating an l-configuration for Leu5. Surprisingly, however, the spectrum of the minor orfamide of Pseudomonas sp. PH1b matched with that of orfamide B from P. aestus CMR5c, therefore indicating a d-Leu5 configuration. This result was highly unexpected as it implies that in Pseudomonas sp. PH1b, the same NRPS assembly line would yield different configurations for Leu5. More specifically, it would require the occurrence of epimerization by the C/E domain of module 6 to correlate with the sequence composition of the growing peptide chain as recruited by module 4. Since the all-l Leu configuration of the original orfamide A from P. protegens Pf-5 was derived from extensive chemical analysis but not confirmed through total synthesis, it appeared more likely that an error was made in establishing the configuration at this position. To settle this matter, we reinvestigated the stereochemistry of the original orfamide A, as extracted from P. protegens Pf-5. In contradiction with the report by Gross et al., where chiral GC revealed a 0:4 d:l ratio for the leucines, our Marfey’s analysis yielded a 1:3 d:l ratio, definitively settling the case in favor of a d-Leu5. As a result, in this case, configurational variability within one NRPS assembly line can be invalidated, and a revision is required for the configuration of Pf-5 based orfamides as shown in Table 2. Gratifyingly, this outcome was recently also arrived at using a completely independent approach involving total synthesis of orfamide A by Bando et al. (69), whereby the original stereochemistry led to loss of green algal deflagellation activity, while the corrected one was as active as the natural compound (70). Finally, we could establish that the orfamide produced by Pseudomonas sp. F6 (8) is identical to the revised orfamide A, by only making use of the tabulated chemical shift values of the published compound, and matching this to our reference spectrum of orfamide A (Fig. S43).

An additional example illustrating the strength of our combined synthesis and spectral matching approach concerns xantholysin A. First reported as the main CLiP produced by Pseudomonas mosselii BW11M1, together with minor congeners B to D, it is the prototype that defines the Xantholysin (14:8) group (71). Since no configurational analysis was performed at the time, NMR and MS analysis only provided the planar structure. Subsequent reports proposed the production of xantholysins by other Pseudomonas strains, uncovering a diverse portfolio of biological functions in the process. Pascual et al. (16) reported the production of xantholysins A to D by Pseudomonas soli F-279,208^T solely based on HR-MS of the isolates and characterized their cytotoxic activity against the RCC4 kidney carcinoma cell line. Lim et al. (9) reported HR-MS and ¹H NMR identical to those of xantholysin A and B for two lipopeptides from Pseudomonas sp. DJ15 and demonstrated their insecticidal activity against the green peach aphid, a major peach tree pest also serving as plant virus vector. In the context of exploring the tolerance of cocoyam plants against Pythium myriotylum in the field, Oni et al. (44) showed that Pseudomonas sp. COR51 produces a lipopeptide with identical planar structure to that of xantholysin A. It was also extracted and characterized from Pseudomonas xantholysinigenes in the framework of the development of a diagnostic bioinformatics tool that allows the assignment of a CLiP to a particular lipopeptide group based on the phylogeny of the MacB transporter of its producing bacterium. (54) In other work, a large-scale bacterial screening for antibiotic activity proposed the identification of xantholysins A-D in Pseudomonas sp. 250J, based on HR-MS data and high similarity in the NRPS genomic make-up to that of P. mosselii BW11M1 (72). Later on, combination of Marfey’s analysis with mapping of the C/E and ^LC_L domains in its NRPS led to propose a stereochemistry for xantholysin A, although the distribution of the remaining d-Leu and l-Leu over positions 9 and 11 remained tentative (73). Shimura et al. (74) reported the total synthesis of MA026, a xantholysin-like CLiP from Pseudomonas sp. RtlB026 with potent anti-hepatitis C activity. Discrepancies in physicochemical properties of synthetic and natural MA026 led Uchiyama et al. (75) to revise its structure by exchanging residues at position 10 and 11, as previously suggested by Li et al. (71), thus leading to a planar structure identical to xantholysin A. With more than 90% sequence similarity between their respective BGC, it was proposed that the stereochemistry of xantholysin A would be identical to that of MA026. However, Uchiyama et al. (75) also noted that the stereochemical assignment for xantholysin A from Pseudomonas sp. 250J did not match with MA026, the latter containing l-Gln6 and d-Gln13, while the reverse configuration was proposed for the former.

To resolve this on-going characterization issue, we first considered applying our combined approach to the original xantholysin A from P. mosselii BW11M1. However, the presence of configurational heterogeneity for the leucine (d:l 2:1) and glutamine/glutamic acid (Glx) residues (d:l 4:1) independently act to generate 50 possible sequences, that can be trimmed down to 15 sequences when taking into account the genomic data. However, no further prioritization is possible after combining the chemical and genomic data. Comparison of the (C–H)α fingerprint of MA026 (with known stereochemistry) to that of originally isolated xantholysin A should in principle allow to either quickly establish configurational identity or eliminate one sequence. However, NMR data for MA026 was listed without explicit assignment. To circumvent this, we adapted our own synthesis scheme to produce MA026 and subsequently established that its (C–H)α fingerprint is indeed identical with that of the original xantholysin A (Fig. 3), thus avoiding the need for further synthesis. Used as reference compound, simple comparison of its (C–H)α fingerprint with that of putative xantholysin A lipopeptides allowed to also extend the assigned stereochemistry to the major xantholysin extracted from Pseudomonas. sp. COR51 (44), P. xantholysinigenes (54) and Pseudomonas sp. 250J (73), for which the NMR data were available or provided for comparison by the original authors, respectively. (Fig. S57, S58, and S59). In conclusion, the structure of xantholysin A was determined to be 3R-OH C10:0 – l-Leu – d-Glu – d-Gln – d-Val – d-Leu – l-Gln – d-Ser – d-Val – d-Leu – d-Gln – l-Leu – l-Leu – d-Gln – l-Ile. This reveals that two modules (2 in XtlA and 7 in XltB) lack the epimerization capability expected for the respective C/E-classified domains.

Incomplete (or erroneous) structural characterization currently generates ‘scientific noise’ in the field of lipopeptide research, hampering a concerted research action for the generation of structure-activity relationships (18). The (C–H)α NMR fingerprint matching approach as described here will prevent this by offering a rapid, unambiguous characterization of CLiP structures. By standardizing the relevant experimental conditions as much as possible and by making reference data publicly available, a rapid elucidation of the complete structure of newly extracted lipopeptides, including stereochemical make-up of both the oligopeptide and the fatty acid moieties comes within reach (Fig. 4). In addition, our user friendly NMR matching methodology allowed to quickly supplement incomplete literature and characterization data with NMR fingerprint data, thereby elucidating the complete structure (including stereochemical make-up) of previously extracted CLiPs (Fig. S2). While limited to Pseudomonas CLiPs, our results support the (C–H)α NMR fingerprint matching as a tool for dereplication beyond this genus, such as those produced by other bacterial phyla, including Bacillus, Burkholderia, and Streptomyces spp. Indeed, without resorting to synthesis or elaborate chemical analysis, the identity of novel CLiPs from natural sources with previously, even only partially, characterized ones, can be established through matching of their spectra. Obviously, access to the desired NMR data for a sufficiently large collection of CLiPs, preferably with annotated fingerprints, is essential for this approach to become of use as dereplication tool for the research community. Only then can the lack of a match be interpreted as indicative of compound novelty with high confidence. While awaiting the development of a (dedicated) NMR data sharing and dereplication platform – most likely by integration of the reference spectra into existing (lipopeptide) databases, such as Norine (81, 82) – a publicly available NMR data repository accompanied by a ‘knowledge base’ has been made available at https://www.rhizoclip.be. It contains an overview of downloadable (C–H)α NMR fingerprint data of characterized CLiPs (both original spectra and tabulated chemical shift values), together with literature data on the originally determined structures. The latter includes a description of the CLiPs’ original description, molecular mass, three-dimensional structures (if available), and a summary of published antimicrobial activities. Moreover, a detailed protocol is available for researchers that wish to record NMR data of their newly extracted lipopeptides to compare them to the publicly available reference data. Finally, we invite all researchers to submit NMR data of new CLiPs, such that they can be included in this knowledge base or contact us to explore possibilities to provide such data for their compounds.

All study data are included in the article and SI Appendix. NMR data for (C–H)α spectral fingerprint matching can be found at https://www.rhizoclip.be.