The molecular mechanism of the Nisin biosynthetic pathway

The biosynthesis of Nisin is a complex process synergistically regulated by the 11-gene nisA(Z)BTCIPRKFEG gene cluster in Lactococcus lactis. It involves core steps including precursor peptide synthesis, post-translational modification, transmembrane transport, and leader sequence cleavage, accompanied by self-regulation and autoimmunity protection mechanisms. The specific molecular mechanisms are as follows:

Precursor Peptide Synthesis

Synthesis initiates with the transcription and translation of the structural gene nisA/Z. This gene encodes a 57-amino-acid Nisin precursor peptide, consisting of a 23-residue N-terminal leader sequence and a 34-residue C-terminal mature peptide domain. The leader sequence plays a key role in guiding subsequent enzymatic modification of the mature peptide domain and preventing the precursor peptide from prematurely forming an active structure in the cell that would damage the host itself. Additionally, nisA and nisZ are two variant forms of the gene, corresponding to Nisin A and Nisin Z variants, respectively. They differ only at the 27th amino acid (histidine in Nisin A vs. asparagine in Nisin Z).

Post-Translational Modification (Core Modification Step)

This step is critical for forming Nisin’s unique cyclic structure, which is collaboratively accomplished by enzymes encoded by nisB and nisC. These two enzymes form a complex with the transport protein encoded by nisT to exert their functions.

Dehydration Reaction

The dehydratase NisB, encoded by nisB, catalyzes the conversion of serine in the precursor peptide to dehydroalanine and threonine to β-methyldehydroalanine via a tRNA-dependent glutamylation mechanism. These two dehydrated amino acids are core precursors for subsequent thioether ring formation.

Cyclization Reaction

The cyclase NisC, encoded by nisC, relies on zinc ions in its active center to activate cysteine for nucleophilic attack. This enzyme catalyzes the formation of lanthionine between dehydroalanine and adjacent cysteine, and β-methylanthionine between β-methyldehydroalanine and adjacent cysteine, ultimately generating 5 thioether rings— the core structural basis for Nisin’s antimicrobial activity.

Transmembrane Transport and Mature Peptide Formation

The modified precursor peptide requires transport and processing to become an active molecule, a process executed by proteins encoded by nisT and nisP.

NisT, an ABC transporter encoded by nisT, is a membrane protein that recognizes the modified precursor peptide and transports it extracellularly using ATP as energy.

Once the modified precursor peptide is transported outside the cell, the protease NisP, encoded by nisP, specifically recognizes and cleaves the 23-residue leader sequence. The remaining 34 amino acid residues fold into the mature Nisin molecule with full antimicrobial activity.

Self-Regulation Mechanism of Synthesis

Nisin synthesis is autoregulated through a two-component regulatory system encoded by nisRK, ensuring the synthesis process is initiated on demand.

When the environmental concentration of Nisin is low, synthesis remains at a basal expression level.

As Nisin is continuously synthesized and accumulated, it acts as a signaling molecule to bind to the histidine kinase NisK encoded by nisK on the cell membrane.

This binding induces autophosphorylation of NisK, which then transfers the phosphate group to the response regulator protein NisR encoded by nisR. Phosphorylated NisR acts as a transcriptional activator, binding to the promoter region of the nis gene cluster to initiate efficient transcription of three operons (nisA(Z)BTCIP, nisRK, nisFEG), thereby amplifying Nisin synthesis efficiency.

Host Autoimmunity Protection Mechanism

To avoid damage to Lactococcus lactis by endogenously synthesized Nisin, nisI and nisFEG in the gene cluster construct a dual immune barrier:

On one hand, the immunity protein NisI encoded by nisI specifically binds to intracellular or extracellular Nisin, blocking its interaction with the host cell membrane and inhibiting Nisin from forming membrane-damaging pores.

On the other hand, the ABC transporter complex NisFEG encoded by nisFEG actively pumps Nisin that has entered the host cell out of the cell, reducing intracellular Nisin concentration and further enhancing the host’s tolerance to its own product.

Additionally, studies have found that enzyme complexes involved in Nisin synthesis have specific subcellular localization. For example, NisB dimers accumulate at the "old pole" of dividing cells, then assemble with NisC to form a modification complex that binds the precursor peptide. In contrast, NisT transporters are initially dispersed on the cell membrane and dissociate from the complex after completing transport. This spatial organization enhances the efficiency of synthesis and transport.