Biosurfactants are amphiphilic molecules, meaning they possess both water-loving and water-fearing regions, and this dual nature allows them to dramatically reduce surface and interfacial tension between immiscible liquids such as oil and water. Unlike their petroleum-derived synthetic counterparts, biosurfactants are biodegradable, exhibit low toxicity, and maintain high surface activity across a range of industrial conditions. These properties make them attractive for applications spanning environmental remediation of oil spills, enhanced petroleum recovery, food preservation, pharmaceutical formulations, and cosmetic products. The most extensively studied and commercialized classes are lipopeptides like surfactin, produced by Bacillus species, and glycolipids like rhamnolipids, produced by Pseudomonas species. However, the search for novel biosurfactant producers has increasingly turned toward lactic acid bacteria, a group generally recognized as safe (GRAS) and widely employed in food fermentation.

Lactobacillus species are particularly compelling candidates because they are already embedded in human food systems, carry a long history of safe use, and produce bioactive metabolites with both antimicrobial and surface-active properties. Prior research has shown that biosurfactants from Lactobacillus can inhibit pathogens, preserve foods, and support probiotic preparations. The biosynthetic machinery behind these molecules typically involves non-ribosomal peptide synthetases, which are large multidomain enzymes that assemble peptide structures without the ribosomal translation machinery. These NRPS systems contain conserved catalytic domains, including adenylation (A), condensation (C), and peptidyl carrier protein (PCP) domains, each responsible for specific steps in substrate recognition, activation, and peptide elongation. What makes the current study distinctive is its focus on whether the adenylation domain alone, without the full NRPS assembly line, could still contribute meaningfully to biosurfactant production.

To answer this question, the research team first turned to comparative bioinformatics. They retrieved the NRPS protein sequence from Bacillus velezensis FZB42, a well-characterized commercial biosurfactant producer with extensively studied NRPS domains, and used it as a query against the NCBI non-redundant protein database. The most significant hit was a non-ribosomal peptide synthetase from Lactococcus lactis subsp. lactis KF147, with 99% query coverage, a BLAST score of 1,052, and an E-value of 0.0, indicating a highly significant alignment despite only moderate sequence identity of approximately 26%. This combination of high coverage and low E-value strongly suggested functional relatedness between the NRPS systems of these taxonomically distant bacteria. Further BLASTp analysis using the Lactococcus sequence as query revealed homologous proteins across multiple Lactobacillus species, including hybrid NRPS-polyketide synthase proteins and adenylate-forming enzymes.

Domain architecture analysis using InterProScan confirmed the presence of conserved functional modules across the analyzed sequences. The team identified the NRPS domain (IPR010060), the PCP domain (IPR009081), and the PKS domain (IPR020806), confirming the hybrid modular nature of these proteins. The condensation domain (IPR001242), responsible for peptide bond formation between activated amino acid substrates, appeared at multiple positions. Critically, the adenylation AMP-binding domain (IPR000873) and the amino acid adenylation domain (IPR010071) were highly conserved across all analyzed strains, underscoring their central role in substrate recognition and activation. The AMP-binding C-terminal domain (IPR025110) was also detected, providing structural stability to the adenylation modules. Notably, the number and organization of these domains varied considerably between strains. Lactobacillus helveticus MTCC 5463 and L. helveticus DPC 4571 each carried two conserved domains, representing a relatively streamlined biosynthetic organization. In contrast, Lactococcus lactis subsp. lactis KF147 harbored seven conserved domains, suggesting a more complex biosynthetic architecture and potentially greater catalytic capacity.

With the bioinformatic foundation in place, the researchers moved to the laboratory. They extracted genomic DNA from L. helveticus MTCC 5463 and used gene-specific primers to amplify the AMP-binding domain by polymerase chain reaction. The resulting fragment, approximately 353 base pairs in length, was directionally cloned into the pET-28a expression vector using XhoI and EcoRI restriction enzymes. Successful cloning was verified through restriction digestion analysis, which released an insert fragment of the expected size in positive clones. The recombinant plasmid was then transformed into Escherichia coli BL21(DE3) for heterologous protein expression. Induction with isopropyl 2-D-1-thiogalactopyranoside (IPTG) at concentrations ranging from 0.2 to 1.0 mM, followed by time-course analysis from 1 to 7 hours, revealed a clear recombinant protein band at approximately 14 kilodaltons on SDS-polyacrylamide gel electrophoresis. Expression intensity increased progressively with both inducer concentration and induction time, reaching maximum accumulation between 5 and 7 hours post-induction.

The recombinant biosurfactant was then extracted from the E. coli culture using chloroform:methanol solvent systems. The researchers tested two ratios, 1:1 and 2:1, and found that the 2:1 ratio produced significantly more visible precipitation, with the greatest accumulation observed at the 5-hour extraction time point. This enhanced recovery is attributed to the higher chloroform content, which improves solubilization of lipid-rich amphiphilic molecules. To rule out the possibility that the observed activity originated from endogenous E. coli membrane lipids, the team conducted a critical empty-vector control experiment. An E. coli BL21(DE3) strain carrying the empty pET-28a vector was processed under identical conditions, and the resulting extract showed no measurable emulsification activity against any of the tested oils and no detectable bands on thin-layer chromatography. This control definitively linked the biosurfactant phenotype to the recombinant AMP-binding domain construct rather than host-derived lipids.

Functional evaluation of the extracted biosurfactant revealed impressive emulsification performance across multiple substrates. Using the emulsification index (E24) assay, which measures the percentage of emulsified layer height relative to total liquid column height after 24 hours, the recombinant product achieved approximately 75% to 82% emulsification against petrol and diesel oil, maintaining stability for a full week with minimal variation. Against mustard oil, the performance was even more striking, reaching 88% to 95%, far exceeding the 10% to 12% achieved by rhamnolipid and the 30% to 38% achieved by surfactin under the same conditions. For refined oil, the recombinant biosurfactant delivered 65% to 75% emulsification at 24 hours, sustaining approximately 65% to 70% even after one week, while rhamnolipid showed only 4% to 6% and surfactin showed moderate activity of 46% to 50%. These results demonstrate that the recombinant glycolipopeptide consistently outperformed both benchmark biosurfactants across hydrocarbon and vegetable oil substrates.

Analytical characterization provided detailed structural insights into the recombinant molecule. Thin-layer chromatography using iodine vapor revealed a yellow-brown spot with an Rf value of 0.80, indicating a relatively non-polar lipid-rich compound, while anisaldehyde staining produced a spot at Rf 0.75, consistent with glycolipid or lipopeptide chemistry. Fourier-transform infrared spectroscopy identified key functional groups: a broad band at 3,325 cm⁻¹ corresponding to hydrogen-bonded O-H and N-H stretching from carbohydrate and protein moieties, aliphatic C-H stretching at 2,916 cm⁻¹ confirming long hydrocarbon chains, a band at 1,649 cm⁻¹ assigned to amide I (C=O stretching) indicating peptide bonds, and C-O stretching at 1,021 cm⁻¹ pointing to carbohydrate or ester linkages. Together, these spectral features are characteristic of a glycolipopeptide structure containing lipid, peptide, and carbohydrate components.

Gas chromatography-mass spectrometry profiling further confirmed the hydrophobic lipid architecture of the biosurfactant. The most abundant compound identified was 9-octadecenamide, accounting for 20.50% of the total relative area, followed by significant amounts of C16 and C18 long-chain fatty acid derivatives and amides. These hydrophobic moieties form the structural basis for surface tension reduction, emulsification, and micelle formation. Nuclear magnetic resonance spectroscopy, conducted on a Bruker Avance NEO spectrometer at 400 MHz, provided complementary evidence. Proton NMR revealed strong signals at δ 1.23 to 1.58 ppm from methylene protons of long-chain fatty acyl groups, glyceryl and carbohydrate-associated signals at δ 3.72 ppm, and a broad singlet at δ 7.3 ppm assigned to exchangeable amide N-H protons, confirming peptide-associated functionalities. Carbon-13 NMR showed resonances at δ 18 ppm for terminal methyl carbons of branched-chain fatty acids, δ 66 and 76 ppm for O-CH₂ and CH-O carbons of glycerol or carbohydrate moieties, δ 56 and 58 ppm for alpha-methine carbons of amino acid residues, and carbonyl carbons at δ 173 to 175 ppm for amide or ester linkages. Collectively, the spectroscopic data confirm a multicomponent amphiphilic glycolipopeptide structure. The authors note that while the isolated adenylation domain alone is unlikely to catalyze complete glycolipopeptide biosynthesis, it may influence production indirectly through interactions with endogenous metabolic pathways in the E. coli host, and they plan future transcriptomic, proteomic, and metabolomic investigations to elucidate the precise molecular mechanism underlying this observation.

Subject of Research: Biosurfactant production mediated by the NRPS adenylation domain of Lactobacillus helveticus MTCC 5463

Article Title: In silico and experimental characterization of NRPS adenylation domain-mediated biosurfactant production in Lactobacillus helveticus MTCC 5463

Article References: In silico and experimental characterization of NRPS adenylation domain-mediated biosurfactant production in Lactobacillus helveticus MTCC 5463. (n.d.). https://doi.org/10.3389/fbrio.2026.1895576

Image Credits: AI Generated

DOI: 10.3389/fbrio.2026.1895576

Keywords: biosurfactant, Lactobacillus helveticus, NRPS, adenylation domain, glycolipopeptide, emulsification, probiotics, non-ribosomal peptide synthetase, heterologous expression, FTIR, GC-MS, NMR