NRPS enzymes operate like molecular assembly lines. Each module of the enzyme carries out a single step in peptide synthesis, and the three core domains of a module divide the labor precisely. The adenylation domain selects and activates an amino acid in an ATP-driven reaction, releasing pyrophosphate as an aminoacyl-adenylate forms. The thiolation domain, also called the peptidyl carrier protein or PCP domain, must be modified by a 4′-phosphopantetheinyl transferase that attaches a 4′-phosphopantetheine group to a conserved serine residue, converting the inactive apo form into the active holo form essential for tethering intermediates. The condensation domain then catalyzes the peptide bond between the newly loaded amino acid and the growing chain carried by the previous module. Because the adenylation domain can accept both proteinogenic and non-proteinogenic amino acids, including D-amino acids generated by epimerization domains and methylated residues added by methylation domains, NRPS products can adopt chemical shapes that ribosomal synthesis cannot produce. A terminal thioesterase domain releases the finished peptide, often cyclizing it into a ring that improves stability, protects the molecule from enzymatic degradation, and enhances surface activity.

The flagship NRPS product in the biosurfactant world is surfactin, a cyclic lipopeptide synthesized by Bacillus subtilis. Its peptide moiety follows the sequence Glu–Leu–D-Leu–Val–Asp–D-Leu–Leu and is linked to a β-hydroxy fatty acid chain of twelve to sixteen carbons. The fatty-acid component contributes substantially to the amphiphilic character that makes surfactin so effective: it can reduce water’s surface tension to 27 mN/m at concentrations of just 10 mg/L, among the strongest effects recorded for any biosurfactant. Surfactin also displays antimicrobial, antiviral, anti-inflammatory, and antitumor bioactivities, and it can penetrate the phospholipid bilayer of bacterial membranes so thoroughly that the cells rupture. Iturin and fengycin, two other Bacillus lipopeptides, follow related NRPS or hybrid PKS–NRPS pathways and are prized for antifungal activity against plant pathogens such as Fusarium and Aspergillus.

Biosurfactants come in several structural classes beyond lipopeptides. Glycolipids, the most extensively studied category, pair a lipid with a carbohydrate: rhamnolipids from Pseudomonas aeruginosa link up to two rhamnose sugars with hydroxy fatty acids and can drop surface tension from roughly 72 mN/m to 25–30 mN/m at concentrations between 20 and 50 mg/L; sophorolipids from the yeast Candida bombicola attach a sophorose sugar to hydroxy fatty acids and occur in acidic and lactonic forms with different solubility and emulsification properties; trehalolipids from Mycobacteria and Rhodococcus pair trehalose with long-chain fatty acids and show both surface activity and immunomodulatory effects. Polymeric biosurfactants such as emulsan, produced by Acinetobacter calcoaceticus, stabilize oil-in-water emulsions so effectively that they remain stable even when temperature, pH, or salinity changes. Rhamnolipids, notably, are synthesized through glycosyltransferase and acyltransferase pathways rather than a typical NRPS assembly line, so they cannot be termed NRPS products.

Probiotic bacteria, particularly those classified as Generally Recognized As Safe, or GRAS, have long been known to produce biosurfactants with lower toxicity than synthetic alternatives. Lactic acid bacteria from the Lactobacillaceae family and Bifidobacteria attach to mucus, interact with harmful bacteria, and produce beneficial substances, and their biosurfactants display antimicrobial, antiadhesive, and antibiofilm functions. Rhamnolipids can stop Pseudomonas aeruginosa and other bacteria from forming biofilms on hospital devices, and surfactin and related biosurfactants eliminate biofilms during and after formation, reducing infections and contamination in healthcare and food industries. Yet the molecular mechanisms behind biosurfactant production in probiotics remained unclear, and natural isolates produce such small quantities that industrial scale-up has been difficult.

The Indian team compiled a list of commercialized probiotics from e-commerce and brand websites, covering products from Bio-K Plus in Canada, Nature Made and Now Foods in the United States, Florastor from Biocodex, Culturelle, Danone’s DanActive and Activia yogurts from France, ProViva from Sweden, and Zbiotics from California, among others. They retrieved genome sequences from the NCBI database for the strains used in these products and screened them with Sempi 2.0, a web server that predicts NRPS and polyketide synthase sequences in bacterial and fungal genomes. Domain annotation with InterProScan identified conserved adenylation and AMP-dependent synthetase/ligase regions, and DOG 2.0 illustrated the arrangement of the domains within each protein.

Of twenty-nine commercialized probiotic strains examined, genome sequences were successfully retrieved for only fifteen, and twenty-five unique NRPS sequences were identified across nine different strains. Every probiotic strain examined contained the AMP-dependent synthetase/ligase domain, and only Lactobacillus paracasei and Lactobacillus rhamnosus also carried an additional adenylation domain. The researchers modeled the three-dimensional structures of these domains with Swiss-Model, achieving sequence coverage often above 97 percent and template similarity ranging from 22 to 57 percent. Alpha helices made up between 11 and 39 percent of the predicted structures, while beta sheets accounted for 17 to 37 percent, indicating a versatile fold suited to the protein’s activity. Validation with the SAVES server, using ERRAT, VERIFY3D, and PROCHECK Ramachandran analysis, showed that most models passed the stereochemical quality tests. Models from Bifidobacterium lactis Bi-07 and Lactobacillus acidophilus NCFM scored highest, with VERIFY3D values near 79 percent and Ramachandran favored-region occupancy of 96 and 95 percent respectively.

The most consequential comparison came from superimposing the probiotic adenylation-domain models against the crystal structure of the surfactin synthetase termination module SrfA-C from Bacillus subtilis, restricted to the adenylation-domain residue range. The team built a rigorous reference panel: three positive outgroups from known non-probiotic biosurfactant producers, Bacillus subtilis ATCC 21332, Bacillus velezensis FZB42, and Pseudomonas aeruginosa PAO1, all showed their lowest RMSD to SrfA-C, between 0.312 and 0.842 Å, far below their RMSD to an unrelated acyl-CoA synthetase reference. A deliberate negative outgroup, Escherichia coli K-12, matched best to the acyl-CoA synthetase reference rather than SrfA-C, confirming that the comparison correctly distinguishes genuine NRPS adenylation domains from housekeeping ANL-superfamily folds. Of the ten probiotic models, seven showed their lowest RMSD to SrfA-C, overlapping the range seen in the positive outgroups. One model, from Bifidobacterium lactis 35624, exhibited the smallest RMSD with surfactin at just 0.490 Å, making it a strong candidate for biosurfactant applications.

Three of the ten probiotic models, however, matched best to different ANL-superfamily references rather than SrfA-C, suggesting they are not structurally distinct from housekeeping enzymes and should not be carried forward as NRPS candidates. The authors caution that the predominance of the AMP-dependent synthetase/ligase domain, in the absence of a complete C–A–PCP/TE domain organization, indicates these proteins should not be interpreted as complete NRPS modules solely from domain annotation. The candidate sequences also lacked any identifiable Sfp-type phosphopantetheinyl transferase, making it impossible to assign them to a functional canonical NRPS biosynthetic pathway without experimental validation. Future work, the researchers argue, should employ genome mining, comparative genomics, transcriptomics, proteomics, heterologous expression, and LC-MS/MS structural analysis to confirm the molecular identity of any metabolites formed and their relationship to NRPS-predicted products.

If validated, the implications are substantial. The genes responsible for surfactin-like biosurfactant production could be cloned and optimized in probiotic hosts, and Bifidobacterium lactis could be engineered to produce more biosurfactants or to impart unique features to the substances it creates. Gene overexpression of NRPS clusters, promoter engineering that responds to signals like nutrient depletion or stress, and heterologous expression in robust industrial microbes such as E. coli and Saccharomyces cerevisiae have all been analyzed as strategies to improve biosurfactant formation in GRAS organisms. Modular synthetic biology platforms could accelerate the optimization of NRPS pathways for high yield, broader substrate scope, and novel products, transferring domains between NRPS systems to create hybrid enzymes that synthesize biosurfactants with specific desired features. The convergence of probiotic science, computational structural biology, and synthetic biology could turn the microbes already trusted for gut health into factories for the sustainable surface-active molecules that industry increasingly demands.

Subject of Research: Unlocking probiotic potential: Non-ribosomal peptide synthetase (NRPS)-driven biosurfactant synthesis for industrial innovation

Article Title: Unlocking probiotic potential: Non-ribosomal peptide synthetase (NRPS)-driven biosurfactant synthesis for industrial innovation

Article References: Pandey, K., Kumari, M., & Singhal, B. (2026). Unlocking probiotic potential: Non-ribosomal peptide synthetase (NRPS)-driven biosurfactant synthesis for industrial innovation. Frontiers in Industrial Microbiology, 4, Article 1964028. https://doi.org/10.3389/finmi.2026.1964028

Image Credits: AI Generated

DOI: 10.3389/finmi.2026.1964028

Keywords: Unlocking, probiotic, potential, Non-ribosomal, peptide, synthetase, NRPS, driven, biosurfactant, synthesis, industrial, innovation