Diversification of Lipopeptide Analogues Drives Versatility in Biological Activities

Abstract

Cyclic lipopeptides (CLPs) are potent secondary metabolites with diverse biological functions. Bacillus strains primarily produce CLPs of three key families, namely, iturins, fengycins, and surfactins, each comprising structural variants characterized by a cyclic peptide linked to a fatty acid chain. Despite extensive research on CLPs, the individual roles of these analogues and their proportion in driving biological activity have remained largely overlooked. In this study, we purified and chemically characterized CLP variants from Bacillus velezensis UMAF6639 and tested them individually for their antifungal and plant growth-promoting effects. We isolated 5 fractions containing iturin A analogues (from C₁₃ to C₁₇), 5 fengycin fractions (containing C₁₆, C₁₇, and C₁₈ fengycin A and C₁₄, C₁₅, C₁₆, and C₁₇ fengycin B), and 5 surfactin fractions (from C₁₂ to C₁₆). We show how antifungal activity and seed radicle growth promotion relied on the lipopeptide structural variant and concentration based on the physiological ratio calculated for each lipopeptide variant. Notably, we found that the most toxic variants were the least abundant, which likely minimized autotoxicity while preserving bioactivity. This balance is achieved through synergistic interactions with more abundant, less aggressive analogues. Furthermore, certain fengycin and surfactin variants were shown to increase bacterial population density and exopolysaccharide production, crucial strategies for microbial competition with significant ecological impacts. In addition to advancing basic knowledge, our findings will support the development of precision biotechnological innovations, offering targeted solutions to drive sustainable food production and preservation strategies.

Keywords: Bacillus velezensis; analogues; antifungal; biotechnology; cyclic lipopeptides; food control.; plant growth promotion; structural variants; sustainable agriculture.

Figure 1

Representative secondary metabolites synthesized by Bacillus velezensis. (A) Chemical structure of the most commonly synthesized secondary metabolites. (B) Mass spectra of the three major lipopeptide families found in the B. velezensis UMAF6639 supernatant, including iturins, fengycins, and surfactins.

Figure 2

Production dynamics of cyclic lipopeptides in B. velezensis UMAF6639. (A) Accumulation dynamics of the lipopeptide analogues. Heatmaps illustrating the mean peak intensity obtained via MALDI-TOF mass spectrometry analysis, where color intensity reflects the relative abundance of each structural variant across time. (B) Expression pattern of the first gene of each biosynthetic operon (ituD for iturin, fenA for fengycin. and srfAA for surfactin biosynthesis), normalized to expression levels at 24 h.

Figure 3

Analytical HPLC chromatograms representing the three major cyclic lipopeptide families found in B. velezensis UMAF6639 after SPE separation. Peak profiles corresponding to (A) iturins, (B) fengycins, and (C) surfactins. The upper labels represent the retention time (RT) for each peak, grouped peaks in the same fraction, and the corresponding lipopeptide analogue detected by mass spectrometry.

Figure 4

CLP analogues show strong differences in antifungal activity when applied separately. The OD₆₀₀ was used as an indicator of fungal growth in the presence of (A) iturin, (B) fengycin, and (C) surfactin analogues. Fungal growth was measured at the physiological concentration of each analogue and at the mixture concentration. The error bars represent the standard error of the mean (SEM), and one-way ANOVA was used to analyze the statistical significance of the results (p < 0.05). The letters above the columns represent the group conditions without statistically significant differences.

Figure 5

Chemical structure and concentration impact the plant growth promotion potential of CLP analogues used to treat melon seeds. Representative image of the radicle development of melon seedlings 5 days after treatment with (A) iturins, (B) fengycins, and (C) surfactins and the corresponding quantitative measurements of root areas. Radicle area measurements of melon seedlings 5 days after treatment with separate structural variants. The error bars represent the SEM, and one-way ANOVA was used to analyze the statistical significance of the results (p < 0.05). The letters above the columns represent the group conditions without statistically significant differences.

Figure 6

Effect of naturally produced lipopeptide analogues on B. velezensis UMAF6639 during cultivation in LB medium. Iturins (A–E), fengycins (F–J), and surfactins (K–O) were tested for their effects on Bacillus growth. Cell growth was monitored every 20 min for 24 h. In each experiment, the maximum concentration of methanol used for the treatment with analogues was used as a control (purple), and analogues were tested separately at the concentration of the whole mixture (green) and at the physiological concentrations (blue) and all together at the same proportion as that produced by the bacteria (yellow). Measurement at each time point was performed in triplicate. The graphs represent the mean values, and the error bars (shadows over the lines) represent the SEM.

Figure 7

Increase in the OD₆₀₀ values of Bacillus cultures treated with different lipopeptide analogues corresponds to an increase in the bacterial count or increased extracellular matrix production, depending on the analogue. (A) Log₁₀ (CFU/mL) corresponding to Bacillus velezensis UMAF6639 cultures treated with analogues causing an increase in OD₆₀₀ values in the toxicity assay. The highest methanol concentration was used as the control condition (purple), each structural variant mixture was used at the physiological concentration calculated from the previous calibration (yellow, see Tables S2 and S3), and each analogue was used separately at its physiological concentration (blue). The error bars represent the SEM, and one-way ANOVA was used to analyze the statistical significance of the results (p < 0.05). The letters above the columns represent the group conditions without statistically significant differences. (B) Impact of fengycin structural variants on Bacillus exopolysaccharide production. The reporter strain Bacillus subtilis NCIB3610 carrying the transcriptional fusion Peps-YFP was used to monitor extracellular matrix production in response to the C₁₆/C₁₇–FengA and C₁₆/C₁₈–Feng A-C₁₇–Feng B treatments. Scale bars correspond to 10 μm. (C) Fluorescence intensity measurements corresponding to eps expression. Each dot represents an individual measurement, and horizontal lines indicate the mean fluorescence intensity with the SEM. Statistical significance was determined via the Kruskal–Wallis test, followed by Dunn’s multiple comparison test. Nonsignificant (ns), ** p < 0.01, and **** p < 0.0001. (D) Monitoring of the tasA expression, encoding the major extracellular matrix protein TasA in the B. subtilis strain P_tasA-YFP under different fengycin analogue treatments. Scale bars correspond to 10 μm. (E) Fluorescence intensity measurements corresponding to tasA expression. Data analysis and representation were performed with the same parameters as those used for the eps assay.