Research Progress on post-translation modification of Nisin

Nisin, a class of lantibiotics synthesized by Lactococcus lactis, owes its biological activity to the special thioether ring structures formed during post-translational modification (PTM). The natural Nisin precursor peptide (pre-nisin) undergoes a series of enzymatic modifications, processing, and transport steps to form the mature peptide with bacteriostatic activity. In recent years, in-depth research into the enzymatic mechanisms, structure-function relationships, and artificial modification of Nisin post-translational modification has provided critical theoretical support for expanding its application scope. The specific research progress is summarized as follows:

I. Core Pathways and Enzymatic Mechanisms of Post-translational Modification

Nisin post-translational modification is a multi-step enzymatic reaction process involving key stages such as precursor peptide synthesis, dehydration, cyclization, protease cleavage, and transport secretion. Each step is synergistically accomplished by enzyme proteins encoded by a specific gene cluster (the nis gene cluster).

Precursor Peptide Synthesis and Signal Peptide CleavageThe Nisin-encoding gene is nisA (or nisZ/nisF in some strains). The precursor peptide generated through its transcription and translation consists of three parts: an N-terminal signal peptide (containing 23 amino acids), a core peptide region (containing 34 amino acids, which forms the active structure after modification), and a C-terminal leader peptide (containing 56 amino acids, which regulates the modification process). Signal peptide cleavage is catalyzed by signal peptidase (SPase), which recognizes the junction sequence between the hydrophobic and polar regions at the C-terminus of the signal peptide. After signal peptide excision, the intermediate containing the core peptide and leader peptide is transported to the inner side of the cell membrane, preparing for subsequent modifications. Studies have found that the sequence conservation of the signal peptide directly affects transport efficiency; mutating key amino acids in the signal peptide can significantly increase the modification rate of the precursor peptide.

Dehydration Reaction: Conversion of Serine/Threonine to Dehydroalanine/DehydrobutyrineThe dehydration reaction is one of the core steps in Nisin post-translational modification, catalyzed by the dehydratase NisB. NisB is a zinc-containing metalloenzyme with two conserved catalytic domains, and its mechanism of action is as follows:

It recognizes serine (Ser) and threonine (Thr) residues at specific positions in the core peptide (the core peptide of natural Nisin A contains 5 Ser and 2 Thr residues);

It catalyzes an elimination reaction between the hydroxyl group on the side chain of Ser/Thr and the α-hydrogen of the adjacent amino acid, removing one molecule of water to generate dehydroalanine (Dha) and dehydrobutyrine (Dhb).

These two unsaturated amino acids are critical precursors for subsequent thioether ring formation. Recent studies have shown that NisB catalysis exhibits strict sequence specificity; the "Ser/Thr-X-Gly" motif in the core peptide is the key recognition site. The presence of the leader peptide enhances the binding affinity between NisB and the core peptide, ensuring the precision of the dehydration reaction.

Cyclization Reaction: Thioether Ring Formation and Spatial Structure ConstructionThe cyclization reaction is catalyzed by the cyclase NisC and constitutes the core step determining Nisin's antibacterial activity. NisC is a flavin mononucleotide (FMN)-dependent oxidoreductase, with the following mechanism of action:

It recognizes the Dha/Dhb residues generated by the dehydration reaction and the sulfhydryl groups (-SH) of adjacent cysteine (Cys) residues;

It catalyzes a nucleophilic addition reaction between the sulfhydryl groups and the double bonds of Dha/Dhb to form thioether rings. Natural Nisin A contains 5 characteristic thioether rings, including 4 monocyclic structures and 1 bicyclic structure, which together form the rigid spatial conformation of Nisin.

Recent crystal structure analysis has revealed that NisC binds the leader peptide via its N-terminal domain and the core peptide via its C-terminal domain, forming a "sandwich"-type catalytic complex that ensures the cyclization reaction proceeds sequentially from the N-terminus to the C-terminus. In addition, NisC catalysis depends on the phospholipid environment of the cell membrane; the membrane-bound state can significantly enhance its enzymatic activity.

Leader Peptide Cleavage and Mature Peptide SecretionThe intermediate that has undergone dehydration and cyclization modifications requires cleavage of the C-terminal leader peptide by the protease NisP to release the mature Nisin core peptide. NisP is a membrane-anchored serine protease, and its cleavage site is located at the junction sequence (Ala-Phe) between the core peptide and the leader peptide. Secretion of the mature peptide is mediated by the ABC transporter NisT, which hydrolyzes ATP to provide energy for transmembrane transport of mature Nisin to the extracellular space. Studies have found that NisT and NisP exhibit synergistic effects; the presence of the leader peptide can simultaneously activate the cleavage activity of NisP and the transport activity of NisT, ensuring that the modified mature peptide is rapidly secreted extracellularly to avoid degradation by intracellular proteases.

II. Regulatory Effects of Post-translational Modification on Nisin Structure and Function

Nisin's bacteriostatic activity is closely related to the special structures formed through post-translational modification. The dehydration efficiency, cyclization integrity, and residue modification types during the modification process directly determine its antibacterial spectrum, activity intensity, and stability.

Key Role of Thioether Ring Structure in Bacteriostatic ActivityThe thioether ring structure endows Nisin with a rigid spatial conformation, enabling it to target and bind lipid Ⅱ (a critical precursor for bacterial cell wall synthesis) on the cell membrane of Gram-positive bacteria. The specific manifestations are as follows:

The rigid structure of the thioether ring enhances the binding affinity between Nisin and lipid Ⅱ, with a binding constant (K_d) reaching the nanomolar level;

A complete thioether ring structure is a prerequisite for Nisin to form transmembrane pores; the absence of any single thioether ring reduces its bacteriostatic activity by more than 80%. For example, a Nisin mutant lacking the first thioether ring exhibits an increase in the minimum inhibitory concentration (MIC) against Staphylococcus aureus from 0.5 μg/mL to 16 μg/mL.

Impact of Dehydration Modification on Nisin StabilityThe Dha/Dhb residues generated by the dehydration reaction can enhance the thermal and acid stability of Nisin. Studies have shown that the unmodified Nisin precursor peptide is completely inactivated after heating at 100°C for 10 min, while the dehydrated intermediate retains more than 60% of its activity. In an acidic environment at pH 2.0, the half-life of modified Nisin is extended more than 5-fold compared with the unmodified precursor peptide, a characteristic that makes it suitable for high-temperature food processing scenarios.

Expansion of Nisin Functions Through Unnatural ModificationsUnnatural post-translational modifications of Nisin can be achieved by genetically engineering the nis gene cluster, thereby expanding its antibacterial spectrum and application range:

Introduction of unnatural amino acids: Replacing Ser/Thr in the core peptide with selenoserine generates selenodehydroamino acids after dehydration modification; the resulting selenoether rings can enhance Nisin's inhibitory effect against drug-resistant bacteria;

Glycosylation modification: Introducing glycosyltransferases into the NisB/NisC catalytic system allows sugar chains to be attached to specific residues of Nisin, improving its penetration ability against Gram-negative bacteria (requiring compounding with EDTA);

Phosphorylation modification: Phosphorylation modification of Nisin is achieved by fusing phosphatase domains, enhancing its complexation ability with heavy metal ions and expanding its application in food preservation and environmental remediation.

III. Regulatory Strategies and Application Research of Nisin Post-translational Modification

Enzymatic Modification to Improve Modification Efficiency and Product DiversityTo address the low catalytic efficiency and strong substrate specificity of natural NisB/NisC, researchers have modified these modification enzymes through directed evolution, rational design, and other methods:

Directed evolution of NisB: Mutating the catalytic domain of NisB via error-prone PCR technology yielded mutants that increase dehydration efficiency by 2–3-fold, while broadening substrate specificity to enable the catalysis of dehydration reactions of unnatural amino acids;

Rational design of NisC: Mutating the FMN binding site of NisC enhanced its binding ability to the coenzyme, increasing the rate of the cyclization reaction by 40% and significantly improving the thioether ring integrity of the product.

Construction of Efficient Modified Expression Systems via Synthetic BiologyUsing synthetic biology techniques, the nis gene cluster (nisA/B/C/P/T) has been integrated into host strains such as Escherichia coli and Bacillus subtilis to construct heterologous expression systems, addressing the low yield of natural Lactococcus lactis strains:

An integrated "secretion-modification-transport" expression system was constructed in Escherichia coli; by optimizing signal peptide and promoter sequences, the Nisin yield reached 150 mg/L, more than 5 times that of natural strains;

Nisin modification enzyme genes were introduced into Bacillus subtilis to achieve fusion expression of Nisin with other antimicrobial peptides; after modification, the fusion peptides exhibited both the bacteriostatic activity of Nisin and the functions of other peptides (e.g., antioxidant, antiviral).

Expansion of Nisin Applications Mediated by Post-translational ModificationBased on structural regulation through post-translational modification, the application scope of Nisin has expanded from food preservatives to the pharmaceutical, agricultural, and other fields:

Development of anti-drug-resistant bacterial drugs: Glycosylation modification enhances Nisin's activity against Gram-negative bacteria; the modified Nisin exhibits an MIC of 8 μg/mL against Pseudomonas aeruginosa, making it applicable for the treatment of infections caused by drug-resistant bacteria;

Agricultural biopesticides: Nisin was fused with the chitinase gene; the modified fusion peptide achieves a 90% inhibition rate against plant pathogens (e.g., Botrytis cinerea) with no toxicity to crops;

Biosensor construction: Utilizing the modification dependence of Nisin, it was fused with fluorescent proteins; rapid detection of Gram-positive bacteria in food is achieved by measuring changes in fluorescence intensity.

IV. Research Challenges and Future Directions

Current research on Nisin post-translational modification still faces several challenges: first, the enzymatic mechanisms of modification enzymes have not been fully elucidated, particularly the detailed interactions between NisB/NisC and the leader peptide, which require further analysis; second, the modification efficiency of heterologous expression systems needs to be improved, and the stability of products with unnatural modifications remains to be verified; third, the mechanisms underlying activity attenuation of modified Nisin in complex environments are not yet clear.