Analysis of the biosynthetic gene cluster of Nisin

The biosynthesis of nisin is a complex process coordinately regulated by a specific gene cluster. This gene cluster is mainly localized on the plasmid or chromosome of nisin-producing strains (e.g., Streptococcus lactis, Streptococcus cremoris). The core gene cluster typically consists of the framework genes nisA/B/C/T/I/R/K/F/E/G, encompassing structural genes, modification genes, transport genes, regulatory genes, and immunity genes. These genes complement each other in function, collectively completing the entire process of nisin from peptide chain synthesis to mature secretion, while endowing the strains with self-resistance to nisin.

I. Core Gene Composition and Functional Division

The components of the nis gene cluster exhibit distinct functional modular characteristics.

1. Structural Genes

Represented by nisA and nisB (in some strains, nisZ is present, encoding the nisin Z subtype, which differs from nisin A by only one amino acid), nisA is the key structural gene. Its transcription product is the nisin precursor peptide (pre-nisin A), which comprises a 57-amino-acid N-terminal signal peptide (responsible for subsequent transport recognition) and a 34-amino-acid mature peptide precursor sequence—this mature peptide precursor requires modification to form bacteriostatically active nisin.

Adjacent to nisA, the nisB gene encodes the NisB protein, a phosphopantetheinyl transferase-related protein. It recognizes specific serine and threonine residues in the precursor peptide, laying the foundation for subsequent dehydration reactions and serving as a key initiating protein for the "modification step" in nisin biosynthesis.

2. Modification Genes

Including nisC and nisT, these genes are responsible for the chemical modification and activity activation of the precursor peptide. The nisC gene encodes the NisC protein, a dehydratase that dehydrates the serine (Ser) and threonine (Thr) residues marked by NisB in the precursor peptide, converting them into dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively. These two unsaturated amino acids are precursors for nisin to form "lanthionine (Lan)" and "β-methyl lanthionine (MeLan)"; these thioether bonds are the core structural basis for nisin to bind to the cell membrane of Gram-positive bacteria and exert bacteriostatic activity.

The nisT gene encodes an ATP-binding cassette (ABC) transporter with dual functions: on one hand, it recognizes the precursor peptide (modified by NisB/C) carrying the signal peptide and transports it outside the cell membrane using ATP as an energy source; on the other hand, during transport, NisT also assists in cleaving the N-terminal signal peptide of the precursor peptide, releasing the initially modified mature peptide precursor and creating conditions for subsequent cyclization reactions.

3. Regulatory Genes

Acting as the "switch" controlling the efficiency and timing of synthesis in the nis gene cluster, the main regulatory genes include nisI, nisR, and nisK, which form a typical "two-component regulatory system." The nisK gene encodes a histidine kinase sensor located on the cell membrane, which can sense changes in the concentration of nisin in the environment—when nisin accumulates to a certain threshold in the environment, NisK undergoes autophosphorylation and transfers the phosphate group to the intracellular response regulator protein NisR. Phosphorylated NisR activates its function as a transcription factor, binds to the promoter regions of various genes in the nis gene cluster (e.g., nisA, nisB, nisC, nisT), and initiates or enhances the transcription of these genes, forming a positive feedback loop of "nisin-induced self-synthesis" to ensure efficient nisin synthesis in the late logarithmic phase of bacterial growth.

The nisI gene encodes the NisI protein, an immune protein located outside the cell membrane. It can bind to free nisin in the environment, preventing nisin from damaging the cell membrane of nisin-producing strains themselves. Meanwhile, it can also reduce the tolerance of surrounding non-nisin-producing strains to nisin through competitive binding, indirectly expanding the bacteriostatic range of nisin.

4. Auxiliary Functional Genes

The nis gene cluster also contains auxiliary functional genes such as nisF, nisE, and nisG. Although these genes are not essential for nisin synthesis, they play important roles in synthesis efficiency and strain adaptability. The proteins encoded by nisF and nisE form an ABC transporter complex (NisF/E), which can pump a small amount of nisin that enters the cell out of the cell, further enhancing the self-immunity of the strain and forming an "internal-external coordinated" resistance mechanism with the NisI protein.

The function of the protein encoded by nisG has not been fully clarified, but studies have found that its deletion leads to a approximately 30% decrease in nisin synthesis. It is speculated that nisG may be involved in the folding process of the precursor peptide after modification or assist NisT in the stable release of the mature peptide after signal peptide cleavage, serving as an auxiliary factor for maintaining the efficiency of nisin biosynthesis.

II. Organization and Synergy Mechanism of the Gene Cluster

The genes in the nis gene cluster are not randomly arranged but form compact transcription units based on "functional relevance." For example, nisA, nisB, nisC, and nisT often constitute a continuous transcription operon (nisA-B-C-T), ensuring that the steps of precursor peptide synthesis, modification, and transport proceed efficiently in sequence and avoiding toxicity caused by the accumulation of intermediate products in the cell.

The regulatory genes nisR and nisK are usually located upstream or downstream of this operon. Through the NisR/K two-component system, they precisely regulate the transcription of the entire operon and also affect the expression of immune genes such as nisI and nisF/E/G, realizing the synergy of "synthesis-regulation-immunity." This gene arrangement not only improves the efficiency of nisin biosynthesis but also enhances the horizontal transfer ability of the gene cluster among different strains. Studies have found that plasmids carrying the nis gene cluster can be transferred to other Streptococcus lactis strains through conjugative transfer, enabling strains that originally did not produce nisin to acquire nisin-synthesizing ability—this is also an important reason for the widespread distribution of nisin-synthesizing ability in lactic acid bacteria.

III. Diversity and Subtype Differentiation of the nis Gene Cluster

The diversity and subtype differentiation of the nis gene cluster are important characteristics of its adaptation to different environments. In addition to the most common nisA gene (encoding nisin A), there are subtype genes such as nisZ (encoding nisin Z, where the 27th amino acid is changed from histidine to asparagine, resulting in higher water solubility) and nisQ (encoding nisin Q, with a broader bacteriostatic spectrum) in nature. Differences in these subtype genes mainly stem from point mutations or fragment insertions in the nisA gene, while other components of the gene cluster (e.g., nisB, nisC, nisR/K) are highly conserved, ensuring that the biosynthesis mechanisms of different nisin subtypes are basically consistent.

In addition, some strains contain additional regulatory genes (e.g., nisL) in their nis gene clusters. The protein encoded by nisL can bind to NisR, further enhancing the transcriptional activation ability of NisR on downstream genes, allowing the strain to efficiently synthesize nisin even under low-concentration induction. This reflects the optimization of the nis gene cluster for environmental adaptability during evolution.

The nisin biosynthesis gene cluster is a highly integrated "molecular machine." Through the synergistic effect of structural genes, modification genes, transport genes, regulatory genes, and immunity genes, it completes the entire process of nisin from peptide chain synthesis, chemical modification, activity activation to secretion and release. At the same time, through precise regulatory mechanisms and self-immune protection, it ensures that the strain avoids self-damage while synthesizing bacteriostatic substances.

Analysis of the nis gene cluster not only reveals the molecular basis of nisin biosynthesis but also provides key targets for optimizing nisin yield through genetic engineering modification (e.g., enhancing nisA transcription efficiency, modifying the nisR/K regulatory system) and expanding the nisin bacteriostatic spectrum (e.g., fusing other antimicrobial peptide domains), laying a theoretical foundation for the broader application of nisin in food preservation, medicine, and other fields.