All chemicals utilized in this study were of analytical grade. Sephadex LH-20 was obtained from GE Biotechnology. Medium-pressure liquid chromatography (MPLC) separations were carried out on a Biotage Isolera One system equipped with a Biotage SNAP Cartridge Ultra C18 column (120 g). Further purification was performed by semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC) using an Agilent 1200 HPLC system fitted with an Agilent Eclipse XDB-C18 column (5 μm, 250 × 9.4 mm). NMR spectra were acquired on a Bruker Avance 400 spectrometer operating at 400 MHz for ¹H and 100 MHz for ¹³C. High-resolution electrospray ionization mass spectrometry (HRESIMS) data were collected on an Agilent 6530 TOF LC/MS instrument.

Genome mining was conducted to identify potential strains producing novel citrulassin-type lasso peptides from the IFB database, following a targeted two-step screening strategy integrated with bioinformatic structural prediction. First, the lasso cyclase (citC) gene from the citrulassin A BGC was used as the query sequence for BLASTP searches against the genomic sequences of strains in the IFB database. A sequence identity cutoff of >45% was set to retrieve strains harboring homologous citC, suggesting the presence of potential lasso peptide BGCs. Second, to enrich for citrulassin-producing candidates, a secondary probe was used: the PAD gene (homologous to WP_064069847.1). This enzyme is distally encoded and critical for citrulline formation during citrulassin A biosynthesis. BLASTP analysis was independently conducted on the genomic data of first-round positive strains to identify those harboring PAD homologs. Strains co-harboring both a lasso peptide BGC with a lasso cyclase C homolog and an extra-cluster PAD homolog were prioritized as potential producers. These dual genetic features are hallmark characteristics of citrulassin biosynthesis, as PAD-mediated arginine deimination is essential for converting arginine to citrulline in mature citrulassins. Finally, the precursor peptides encoded within the candidate BGCs were analyzed using the RiPPMiner tool. This bioinformatic platform was utilized to predict core peptide sequences (following leader peptide cleavage) and tentative structural features of the mature lasso peptides, thereby facilitating the selection of strains with the potential to produce novel citrulassin-type compounds.

The 15 candidate strains were inoculated into 250-ml flasks containing 50 ml TSB medium and cultivated at 30 °C, 220 rpm for 24 h to obtain seed cultures. These seed cultures were then transferred to various screening media and fermented at 30 °C with shaking at 150 rpm. After 7 days of cultivation, XAD-16 resin was added to the fermentation broth to absorb the target products. The resin was washed with deionized water and then extracted three times with methanol. The combined methanol extracts were concentrated under reduced pressure to yield a crude extract. This crude material was initially fractionated by medium-pressure liquid chromatography (MPLC) using a step gradient of CH₃OH/H₂O (10–100%, v/v; flow rate 5 ml/min). Collected fractions were analyzed by analytical HPLC and pooled based on their chromatographic profiles, or subjected to further purification via gel column chromatography. Fractions purified by gel column chromatography were re-analyzed. Those containing the target compound were concentrated under reduced pressure, dissolved in methanol, and centrifuged to collect the supernatant. Final separation and purification were achieved by semi-preparative HPLC to obtain pure compounds.

To evaluate the inhibitory bioactivity of the lasso peptide citrulassin N (1), agar-based inhibition assays were carried out following a previously reported protocol with minor adjustments (Palmer et al., 2018). In brief, bacterial strains were first grown overnight on LB agar plates and then inoculated into LB broth. The cultures were incubated at 37 °C until reaching an OD600 of 0.8. Subsequently, the culture was diluted 1:1,000 into sterilized LB medium containing 0.5% agar that had been pre-warmed to 40 °C, and the mixture was poured into plates. Onto the surface of each solidified soft-agar plate, 10 μl of purified lasso peptide was applied at varying concentrations across a tested gradient. Plates were then incubated at 37 °C for 24 h. Antibacterial activity was determined by measuring the clear inhibition zones formed around the peptide spots. All assays were performed in three independent replicates.

A two-step targeted genome mining strategy was employed to identify potential citrulassin-producing strains from the IFB bacterial genome database. In the first round, the lasso peptide cyclase gene citC (A4V12_08815) from the citrulassin A biosynthetic gene cluster (BGC) was used as a BLASTP query, applying a sequence identity cutoff of >45%. This search yielded 34 putative lasso peptide BGCs distributed across 34 distinct bacterial strains (Figure 2 and Table 1).

To evaluate whether these BGCs encoded citrulassin-type precursors, the corresponding precursor peptides were analyzed using the RiPPMiner platform, which enables accurate prediction of core peptide sequences and lasso peptide cross-links. Notably, all predicted core peptides shared a conserved arginine residue at position 9, corresponding to the first amino acid immediately outside the macrolactam ring in the lasso topology. This positional conservation is a defining feature of citrulassin-type lasso peptides, as this arginine residue serves as the substrate for post-translational citrullination. In the second round of screening, a confirmed peptidylarginine deiminase (PAD) homolog (WP_064069847.1) was employed as a secondary probe for BLASTP analysis against the 34 first-round positive strains. This analysis identified 15 strains that co-harbored both a citrulassin-like lasso peptide BGC and an extra-cluster PAD homolog (Figure 2 and Table 1). The co-occurrence of a conserved Arg9-containing precursor and a putative PAD enzyme strongly suggests the biosynthetic potential for citrulline-modified lasso peptide production, thereby substantially narrowing the pool of candidate producers.

The 15 candidate strains identified through dual-probe genome mining were subjected to fermentation, followed by metabolite profiling using liquid chromatography–mass spectrometry (LC–MS). Among these strains, Streptomyces sp. NAX00255 exhibited a prominent molecular ion peak at m/z 820.9511 corresponding to [M+2H]²⁺, which closely matched the molecular weight predicted for a citrulline-modified lasso peptide based on RiPPMiner analysis. Encouraged by this result, large-scale fermentation of Streptomyces sp. NAX00255 was carried out to facilitate compound isolation. Subsequent purification using a combination of ODS medium-pressure liquid chromatography (MPLC) and semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC) afforded 10 mg of the target compound, designated citrulassin N (1).

Citrulassin N (1) was isolated as a white amorphous powder. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) established its molecular formula as C₇₀H₁₂₁N₂₁O₂₄, based on the observed [M+2H]²⁺ ion at m/z 820.9511 (calcd for C₇₀H₁₂₃N₂₁O₂₄, 820.9519), corresponding to 21 degrees of unsaturation. Although the limited quantity of compound resulted in reduced signal-to-noise ratios for certain NMR spectra, the molecular formula was fully consistent with predictions derived from the associated biosynthetic gene cluster using RiPPMiner. Comprehensive analysis of ¹H NMR, ¹³C NMR, ¹H–¹H COSY, HSQC, HMBC, and MS/MS data enabled unambiguous structural assignment of compound 1.

The ¹H NMR (Supplementary Figure S4) and ¹H–¹H HSQC (Supplementary Figure S6) spectra showed typical features of a peptide-derived compound, including 16 amide NH signals (δ_H 6.80, 6.90, 7.06, 7.16, 7.20, 7.24, 7.28, 7.56, 7.70, 7.80, 7.88, 8.01,8.23, 8.34, 8.42, and 8.92). The ¹³C NMR spectrum (Supplementary Figure S5) also displayed 20 amide/acid carbonyl signals (158.3, 158.4, 159.2, 167.7, 169.4, 169.5, 170.1, 170.8, 171.0, 171.2, 171.6, 172.0, 172.5, 172.8, 173.0, 173.3, 173.6, 173.7, 173.8, and 174.4). Comprehensive analysis of the 1D (¹H, ¹³C) and 2D (¹H-¹H COSY, HSQC, and HMBC) NMR spectra (Supplementary Figures S4–S8) revealed compound 1 contained 15 amino acid residues, including four Leucines (Leu), a Glutamine (Gln), three Serines (Ser), a Glycine (Gly), two Asparagines (Asn), an Aspartic acid (Asp), a Citrulline (Cit), an Isoleucine (Ile), and a Lysine (Lys). Given that 20 carbonyl signals account for 20 of the 21 degrees of unsaturation, we infer the presence of one additional ring structure in the peptide, which aligns with the predicted cyclic architecture of a lasso peptide.

MS/MS analysis serves as a critical tool for providing direct evidence of amino acid connectivity within peptide chains. LC-MS/MS fragmentation data (Supplementary Figure S1) clearly established the sequence of the C-terminal heptapeptide (Cit9-Leu10-Ile11-Leu12-Ser13-Lys14-Asn15) and corroborated the presence of the macrolactam ring formed by the eight-residue segment (Leu1-Leu2-Gln3-Ser4-Ser5-Gly6-Asn7-Asp8). These results conclusively demonstrate that the planar structure of compound 1 consists of an eight-membered macrolactam ring linked to a seven-residue C-terminal tail, thereby defining its architecture as a lasso peptide (Figure 3).

Based on genomic analysis of Streptomyces sp. NAX00255 and the conserved biosynthetic logic of citrulassins, a plausible biosynthetic pathway of citrulassin N (1) was proposed (Figure 4). The precursor peptide encoded within the lasso peptide BGC consists of a leader peptide followed by a 15-residue core peptide (LLQSSGNDRLILSKN), which is consistent with the typical modular architecture characteristic of lasso peptide precursors. The biosynthesis initiates with the proteolytic cleavage of the leader peptide by a leader peptidase, releasing the mature core peptide. Subsequently, the lasso cyclase (a CitC homolog) catalyzes the formation of an eight-membered N-terminal macrolactam ring via amide bond formation between the α-amino group of Leu1 and the side-chain carboxylate of Asp8, a key step in constructing the lasso topology. Notably, the ninth residue in the precursor core peptide is arginine, which is positioned immediately outside the macrolactam ring and serves as the substrate for citrullination. Deimination of Arg9 to citrulline is proposed to be catalyzed by an extra-cluster PAD homolog (WP_064069847.1), yielding the signature citrulline modification that defines citrulassin N. Finally, the C-terminal tail (Leu10-Asn15) threads through the macrolactam ring to form the mechanically interlocked lasso structure, which is consistent with the structural features deduced from NMR and MS/MS data. This biosynthetic pathway closely mirrors that of citrulassin A, underscoring that the conserved combination of a citrulassin-type lasso peptide BGC and an extra-cluster PAD gene is sufficient to direct the biosynthesis of citrulline-modified lasso peptides (Figure 1). These findings further validate the effectiveness of the dual-probe genome-mining strategy for targeted discovery of structurally unique RiPPs.

Given that several lasso peptides have been reported to exhibit antimicrobial activities (Carson et al., 2023; King et al., 2023; Mucha et al., 2025), the antibacterial potential of citrulassin N (1) was evaluated against a panel of Gram-positive (Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 9372) and Gram-negative (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922) bacterial strains. The results indicated that the compound exhibited only weak inhibitory activity against Staphylococcus aureus ATCC 6538, with a minimum inhibitory concentration (MIC) of 40 μg/ml, and showed no significant activity against the other tested strains (Table 2).