The function of the Nisin biosynthetic gene cluster

The biosynthesis of Nisin is regulated by a ~14 kb gene cluster (nisABTCIPRKFEG) on the genome of Lactococcus lactis. This cluster contains 11 core genes, functionally divided into four modules: precursor peptide synthesis and post-translational modification, transport, regulation, and host immune protection. All genes act synergistically to complete Nisin synthesis, secretion, and host self-protection. The specific functions of each gene are analyzed as follows:

Precursor Peptide Synthesis and Post-Translational Modification Module

Genes in this module are primarily responsible for synthesizing the Nisin precursor peptide and forming the mature, antimicrobial active peptide structure through a series of modification reactions—core steps of Nisin biosynthesis.

nisA: Its core function is to encode the Nisin precursor peptide. This precursor peptide includes a leader sequence and a mature peptide domain, which is inactive initially and requires subsequent modification and processing to become functional Nisin.

nisB and nisC: These are key genes for post-translational modification, jointly participating in the formation of Nisin’s characteristic lanthionine rings. NisB catalyzes the dehydration of specific serine and threonine residues in the precursor peptide, while NisC catalyzes the formation of lanthionine and methyllanthionine cross-links between cysteine residues and dehydrated amino acids. These cross-links are critical for Nisin’s antimicrobial activity and structural stability. Deletion of either nisB or nisC completely blocks Nisin modification, preventing the host strain from producing active Nisin.

nisP: Encodes a subtilisin-like serine protease. When the precursor peptide modified by nisB and nisC is transported near the cell membrane, NisP specifically cleaves the leader sequence, releasing the mature Nisin peptide—one of the final key steps for Nisin to gain antimicrobial activity. In vitro experiments show that cell extracts of Escherichia coli overexpressing nisP can directly cleave the Nisin precursor peptide to generate the active mature peptide.

Transport Module

This module contains only the nisT gene, which encodes an ABC transporter protein. After Nisin completes post-translational modification, NisT actively transports the intracellular Nisin precursor peptide (pre-cleavage) or mature peptide to the extracellular space. Deletion of nisT not only causes intracellular Nisin accumulation but also significantly reduces host immunity and abolishes the strain’s Nisin-producing ability, indicating a synergistic link between its transport function and subsequent immune regulation.

Regulation Module

The nisRK genes in this module form a two-component regulatory system, which precisely controls the expression of the entire gene cluster through an autoregulatory mechanism—acting as the "switch" for Nisin synthesis.

nisK: Encodes a membrane-localized receptor protein with two transmembrane domains. Its extracellular region specifically senses extracellular Nisin concentration signals. When Nisin is secreted extracellularly and reaches a certain concentration, NisK is activated and undergoes autophosphorylation.

nisR: Encodes an intracellular regulatory protein belonging to the response regulator family. Phosphorylated NisK transfers the phosphate group to NisR; activated NisR binds to the promoter regions of relevant genes in the cluster, initiating transcription of core genes such as nisA and forming a positive feedback loop for Nisin synthesis. Experiments confirm that overexpression of nisRK can increase Nisin yield by approximately 45%, while deletion of nisR completely halts transcription of Nisin biosynthesis-related genes.

Host Immune Protection Module

This module includes two sets of genes—nisI and nisFEG—which form two independent immune systems to prevent the host from being killed by endogenously synthesized Nisin, serving as the key for Lactococcus lactis to tolerate its own product.

nisI: Encodes a membrane-bound protein that specifically binds extracellular Nisin molecules, reducing Nisin’s destructive effect on the host cell membrane. Strains lacking nisI can still produce Nisin but show significantly decreased tolerance to Nisin and reduced overall Nisin yield, indicating an indirect synergistic effect with Nisin synthesis efficiency.

nisFEG: These three genes form an operon, collectively encoding an ABC transporter complex. It further enhances host resistance by actively pumping Nisin that has invaded the host cell out of the cell. Studies show that nisI and nisFEG must act synergistically to achieve efficient host tolerance to Nisin; a single gene cannot match the immune level of wild-type strains. For example, when both nisI and nisFEG are expressed, the host can tolerate high concentrations of exogenous Nisin, while deletion of either gene leads to a significant reduction in host immunity.