Genetic engineering modification of Nisin

Genetic engineering modification of Nisin, through precise modification of its structural genes or introduction of functional sequences, can significantly broaden its antimicrobial spectrum and enhance activity. The core lies in designing mutation or fusion strategies based on its antimicrobial mechanism (targeting the bacterial cell wall peptidoglycan precursor lipid Ⅱ), combined with optimization of expression systems to achieve efficient production.

I. Genetic Modification Targets Based on Structure-Function Relationships

Nisin consists of 34 amino acids with 5 thioether rings (A-E). Its antimicrobial activity depends on specific binding to lipid Ⅱ and the ability to form membrane pores. Key modification targets include:

Rings A and B: Responsible for recognizing the pyrophosphate group of lipid Ⅱ. Site-directed mutations (e.g., replacing Ser6 in ring A with Lys) enhance electrostatic interactions, improving binding affinity to Gram-positive bacteria.

Rings C and D: Involved in membrane pore formation. Introducing hydrophobic amino acids (e.g., Leu, Ile) strengthens hydrophobic interactions with bacterial cell membranes, increasing bactericidal efficiency.

C-terminal Region: Extending or replacing C-terminal amino acids (e.g., adding positively charged Arg) enhances penetration into certain drug-resistant bacteria (e.g., vancomycin-resistant enterococci).

II. Genetic Engineering Strategies to Broaden Antimicrobial Spectrum

1. Modifications Targeting Gram-Negative Bacteria

Introducing Cationic Peptides: Fusing the C-terminal of the Nisin gene with short peptides capable of penetrating Gram-negative outer membranes (e.g., cationic fragments of polymyxin B) via genetic recombination to construct fusion proteins. The modified product disrupts the outer membrane barrier of Gram-negative bacteria, allowing Nisin to access lipid Ⅱ, thereby increasing antimicrobial activity against E. coli and Salmonella by 2–3 orders of magnitude.

Overcoming Efflux Pump Resistance: Mutating Nisin’s efflux pump recognition sites (e.g., Val20 in ring D to Ala) to reduce efflux efficiency, expanding efficacy against drug-resistant strains that expel Nisin via efflux pumps.

2. Expanding Activity Against Fungi and Viruses

Fusion with Antifungal Domains: Genetically engineering hybrid peptides by fusing Nisin with fungal membrane-targeting segments (e.g., β-sheet domains of defensins). These modified products act on both bacterial lipid Ⅱ and fungal ergosterol, reducing the MIC against Candida albicans from >200 μg/mL to below 50 μg/mL.

Incorporating Viral Inhibitory Sequences: Inserting peptide segments that bind viral capsid proteins into the Nisin gene. The modified product inhibits viral adsorption to host cells, exhibiting both antimicrobial and antiviral activity (e.g., 80% inhibition rate against noroviruses).

III. Key Modification Technologies to Enhance Activity

1. Site-Directed Mutations for Improved Efficacy

Enhancing Lipid Ⅱ Binding: Mutating His27 of Nisin to Arg strengthens electrostatic interactions with the pyrophosphate group of lipid Ⅱ via increased positive charge, reducing the MIC against Staphylococcus aureus from 25 μg/mL to 8 μg/mL.

Stabilizing Spatial Structure: Introducing disulfide bonds at key positions of thioether rings (e.g., adding Cys-Cys pairing between rings A and E) improves structural stability of Nisin in high-temperature or acidic environments, extending its active half-life by 2–3 times.

2. Genetic Recombination for Optimized Expression and Modification

Codon Optimization: Modifying codons of the Nisin structural gene according to the codon preference of hosts (e.g., Lactococcus lactis, E. coli) increases expression levels by 30%–50%, providing a basis for screening highly active mutants.

Regulating Post-Translational Modifications: Overexpressing modifying enzymes required for Nisin biosynthesis (e.g., dehydratase NisB, cyclase NisC) ensures correct formation of thioether rings in mutants, avoiding activity loss due to defective modification.

IV. Expression Systems and Screening Methods

1. Selection of High-Efficiency Expression Systems

Prokaryotic Expression: Lactococcus lactis (the natural producer of Nisin) is preferred, as it can overexpress modified genes via plasmid vectors and correctly perform post-translational modifications, retaining >90% activity. E. coli expression requires solving thioether ring modification issues, typically by co-expressing NisB/C enzymes.

Eukaryotic Expression: Pichia pastoris is used to express fusion Nisin (e.g., modified products containing eukaryotic signal peptides), suitable for producing medical-grade products, but folding conditions must be optimized to maintain activity.

2. High-Throughput Screening Strategies

Antimicrobial Zone Assay: Co-culturing modified expression strains with indicator bacteria (e.g., Staphylococcus aureus, E. coli) and using zone diameter to quickly screen highly active mutants.

Fluorescent Labeling: Detecting binding efficiency between modified products and lipid Ⅱ using lipid Ⅱ fluorescent probes, combined with flow cytometry to screen mutants with enhanced affinity.

Minimum Inhibitory Concentration (MIC) Determination: Performing gradient MIC tests on candidate mutants to accurately evaluate activity against different pathogens.

V. Application Potential and Challenges

1. Expanded Application Scenarios

Food Preservation: Broad-spectrum modified Nisin can replace some chemical preservatives in foods (e.g., ready-to-eat meats, vegetables) to control both Gram-positive (e.g., Listeria) and Gram-negative (e.g., E. coli) bacteria, extending shelf life by 3–5 days.

Medical Field: Highly active mutants can treat multi-drug resistant infections (e.g., methicillin-resistant Staphylococcus aureus), and combination with antibiotics reduces the risk of drug resistance.

2. Key Challenges

Balancing Structural Stability: Some mutations may destabilize Nisin’s spatial structure; molecular dynamics simulations are needed to predict mutation effects and reduce screening blindness.

Safety Verification: Modified Nisin requires verification of toxicity, immunogenicity, and in vivo metabolic pathways to meet food or medical standards.

Industrialization Costs: Stability of genetically engineered strains and large-scale fermentation processes need optimization to reduce production costs for practical application.

Through genetic engineering modification, Nisin is expected to evolve from a traditional narrow-spectrum antimicrobial agent to a "broad-spectrum, high-efficiency, environment-tolerant" multifunctional biological preservative or antimicrobial drug. Its core lies in breaking limitations of activity range and intensity through structural optimization while retaining lipid Ⅱ targeting.