The biosynthetic gene expression system of Nisin

As a class II lantibiotic synthesized by Lactococcus lactis, the biosynthesis of nisin is a complex process regulated by the coordination of multiple genes, essentially relying on a highly organized gene cluster (the nis gene cluster) and its corresponding expression regulatory system. In-depth analysis of the composition of this system, its regulatory mechanism, and heterologous expression strategies is key to optimizing Nisin yield and expanding its application scenarios. Relevant research can be conducted from three dimensions: gene cluster structure, expression regulatory mechanism, and heterologous expression optimization.

I. Core Composition and Functions of the Biosynthetic Gene Cluster

The nisin biosynthetic gene cluster (usually located on a plasmid or chromosome) consists of five major categories of genes: structural genes, modification genes, transport genes, immunity genes, and regulatory genes. These genes work synergistically to complete the entire process of nisin from peptide chain synthesis to mature secretion, and the orderly expression of genes is a prerequisite for the correct synthesis of the product.

Structural Genes (nisA/nisZ)

These are the starting point of nisin biosynthesis. Their expression products are Nisin precursor peptides (pre-nisin), which include an N-terminal signal peptide (guiding subsequent transport) and a C-terminal propeptide (requiring modification to form an active structure). Structural genes vary among different strains; for example, nisA and nisZ encode nisin A and Nisin Z, respectively (differing only in the 27th amino acid: histidine in A and asparagine in Z). However, their expression patterns are consistent—both require transcription initiation under the regulation of specific promoters (PnisA/PnisZ), and the activity of these promoters directly determines the synthesis efficiency of the precursor peptide.

Modification Genes (nisB, nisC, nisI, nisP)

These are responsible for the post-translational modification of the precursor peptide, a core step in nisin acquiring antimicrobial activity. Their expression must be synchronized with the expression timing of structural genes. Among them:

The dehydratase encoded by nisB dehydrates specific serine and threonine residues in the precursor peptide to form dehydroalanine (Dha) and dehydrobutyrine (Dhb);

The cyclase encoded by nisC catalyzes the formation of thioether rings (lantibiotic rings) between dehydrated amino acids and cysteine residues—these two modification steps determine nisin’s spatial structure and antimicrobial activity;

The protease encoded by nisP cleaves the N-terminal signal peptide of Nisin after it is secreted extracellularly, converting the precursor peptide into mature active Nisin;

The immunity protein encoded by nisI protects the host bacterium itself from damage by immature nisin during modification. Its expression level must be coordinated with Nisin synthesis to avoid host autotoxicity.

Transport Gene (nisT)

Its expression product is an ABC transporter, which can recognize the precursor peptide (or modified precursor peptide) with a signal peptide and transport it extracellularly using ATP as energy, completing the Nisin secretion process. The expression of this gene must be synchronized with modification genes: if the transport efficiency is lower than the modification efficiency, unsecreted nisin precursor peptides may accumulate intracellularly, which instead inhibits host growth; conversely, unmodified precursor peptides may be secreted, failing to form active products.

Regulatory Genes (nisR, nisK)

These act as the "switch" of the entire gene expression system, regulating the expression timing and intensity of other genes through a two-component regulatory system (TCS). The histidine kinase encoded by nisK (located on the cell membrane) can sense the concentration signal of extracellular nisin (or its precursor), undergo autophosphorylation, and then transfer the phosphate group to the response regulator protein encoded by nisR (located intracellularly). Phosphorylated NisR can bind to the promoter regions (e.g., PnisA) of structural, modification, and transport genes, activating the transcription of these genes. This forms a positive feedback loop of "nisin induction → gene expression → more Nisin synthesis," ensuring the efficiency and continuity of gene expression.

II. Expression Regulatory Mechanism of Nisin Biosynthetic Genes

The expression of the nis gene cluster is not constantly initiated but is regulated by three factors: signal induction, metabolic status, and environmental conditions. Among these, the positive feedback regulation of the two-component regulatory system (NisK/NisR) is the core mechanism, while other factors indirectly regulate gene expression by affecting the activity of this system.

Nisin-Mediated Positive Feedback Regulation

This is the most critical regulatory mechanism. When Lactococcus lactis enters the late logarithmic growth phase, a small amount of nisin precursor peptide begins to be synthesized intracellularly. After modification and transport to the extracellular space, part of the nNisin binds to NisK on the cell membrane, triggering conformational changes and autophosphorylation of NisK. Phosphorylated NisK transfers the phosphate group to NisR; the activated NisR acts as a transcription factor, binding to the promoter regions of genes such as nisA, nisB, nisC, and nisT (all of which contain NisR binding sites). This significantly enhances the transcription efficiency of these genes, thereby synthesizing more nisin. This positive feedback mechanism enables Nisin synthesis to be rapidly initiated and maintained at a high efficiency in a short period, avoiding the waste of nutrients in the early growth stage.

Metabolic Status Regulation

The carbon metabolic status of Lactococcus lactis affects the expression of nisin genes. Studies have shown that when carbon sources (e.g., glucose) in the medium are sufficient, cells produce large amounts of ATP through glycolysis, which supports the energy supply of ABC transporters (NisT) and the activity of RNA polymerase for gene transcription. Meanwhile, sugar metabolism intermediates (e.g., phosphoenolpyruvate) can optimize the activity of the NisK/NisR system by regulating intracellular pH. When carbon sources are depleted, cells enter the stationary phase, ATP production decreases, the transport efficiency of NisT declines, and organic acids accumulated intracellularly may inhibit the binding activity of NisR, leading to reduced Nisin gene expression. In addition, the availability of nitrogen sources (e.g., amino acids) also affects precursor peptide synthesis—insufficient nitrogen sources reduce the efficiency of ribosomal pre-nisin synthesis, indirectly inhibiting the expression of subsequent modification and transport genes.

Environmental Factor Regulation

Environmental conditions such as temperature, pH, and dissolved oxygen regulate Nisin gene expression by affecting cell activity or the function of the NisK/NisR system. For example:

The optimal growth temperature of Lactococcus lactis (30-32°C) is consistent with the optimal expression temperature of nisin genes; temperatures above 37°C reduce the kinase activity of NisK, preventing effective activation of NisR;

NisR exhibits the strongest DNA-binding ability at pH 6.0-6.5, while pH below 5.0 causes deprotonation of NisR, losing its ability to bind promoters;

Nisin synthesis requires a strictly anaerobic or microaerobic environment—under aerobic conditions, the accumulation of reactive oxygen species in cells damages the structure of modification enzymes such as NisB and NisC, leading to failure of precursor peptide modification and subsequent negative feedback inhibition of gene expression.

III. Research on Heterologous Expression Systems of Nisin Biosynthetic Genes

The Nisin yield of native Lactococcus lactis is relatively low (usually 200-500 IU/mL), and it is limited by its own metabolic characteristics, making it difficult to meet the needs of large-scale industrial production. Therefore, constructing heterologous expression systems (introducing the nis gene cluster into other microbial hosts) has become an important direction to improve nisin yield and expand its synthetic hosts. Current research focuses on three aspects: host selection, gene cluster optimization, and expression regulation adaptation.

Selection of Heterologous Hosts

An ideal heterologous host must meet the requirements of "compatibility with nis gene expression, no self-toxicity, easy cultivation, and controllable metabolism." Commonly used hosts currently include:

Other lactic acid bacteria (e.g., Bifidobacterium animalis, Lactobacillus plantarum): These hosts are closely related to Lactococcus lactis, and their intracellular metabolic environment (e.g., pH, redox potential) is similar to the natural expression environment of nis genes, reducing functional adaptation issues of modification enzymes and transport enzymes. As food-grade microorganisms, lactic acid bacteria do not require additional removal of host toxic components from the expressed Nisin, making them suitable for food-related applications.

Escherichia coli (E. coli): E. coli has the advantages of fast growth, simple genetic manipulation, and large-scale fermentation, but two key issues need to be addressed: first, the high intracellular redox potential of E. coli may affect the modification activity of NisB and NisC, requiring co-expression of reductases (e.g., glutathione reductase) to regulate the intracellular environment; second, E. coli lacks the natural immunity mechanism of Nisin, so the nisI gene must be introduced simultaneously to prevent the host from being killed by self-synthesized nisin.

Pichia pastoris: As a eukaryotic host, Pichia pastoris can achieve high-copy integration and secretory expression of foreign genes, and can perform certain post-translational modifications on proteins (although Nisin modification relies on prokaryotic enzymes, the secretory system of Pichia pastoris can optimize the extracellular release of Nisin). Current studies have achieved preliminary heterologous synthesis of Nisin by co-expressing nisA, nisB, nisC, and nisT in Pichia pastoris.

Optimization Strategies for Heterologous Expression

Streamlining and reconstruction of the gene cluster: The native nis gene cluster contains multiple auxiliary genes (some related to host adaptability). During heterologous expression, redundant genes (e.g., those related to the unique metabolism of Lactococcus lactis) can be eliminated, retaining only core genes such as nisA/nisZ, nisB, nisC, nisT, nisP, nisR, and nisK to reduce the metabolic burden on the host. At the same time, core genes are concatenated into the same expression vector, and gene sequences are optimized using host-preferred codons (e.g., nisA optimization for E. coli codon preference) to improve mRNA translation efficiency.

Promoter replacement and modification: The native PnisA promoter relies on Nisin induction and may have insufficient activity in heterologous hosts due to the lack of effective activation by NisK/NisR. Therefore, studies often replace PnisA with host constitutive promoters (e.g., the T7 promoter of E. coli, the AOX1 promoter of Pichia pastoris) or modify the NisR binding site of the PnisA promoter to enhance its binding ability with heterologous host transcription factors, achieving efficient and controllable gene expression.

Synergistic optimization of metabolic pathways: The metabolic status of heterologous hosts directly affects nisin synthesis. For example, when expressing nisin in E. coli, it is necessary to knock out the lactate dehydrogenase gene (reducing lactic acid production) and enhance the expression of ATP synthesis-related genes (e.g., atpABC) to provide sufficient energy for NisT transport. In lactic acid bacteria hosts, the logarithmic growth phase can be extended by optimizing carbon source supply (e.g., using a glucose-lactose mixed carbon source) to provide continuous metabolic support for Nisin gene expression.

IV. Research Significance and Future Directions

Research on the nisin biosynthetic gene expression system not only reveals the precise regulatory mechanism of antibiotic synthesis in prokaryotes but also provides a typical model for the application of microbial synthetic biology. By analyzing the expression rules of nis genes, a systematic engineering strategy of "gene regulation → metabolic optimization → product synthesis" can be constructed, providing references for the biosynthetic research of other lantibiotics (e.g., epidermin, salivaricin). Future research directions will focus on three areas:

Using technologies such as single-cell sequencing and protein interaction analysis to further clarify the signal transduction details of the NisK/NisR system and identify new regulatory factors;

Developing universal expression vectors compatible with multiple hosts to achieve efficient adaptation of nis genes in different microorganisms;

Combining synthetic biology methods to construct engineered strains integrating "Nisin synthesis → product separation," promoting breakthroughs in industrial nisin yield to meet the large-scale application needs of the food, pharmaceutical, and other fields.