The synthetic biology application of Nisin

Synthetic biology provides core solutions for high-yield and green production of Nisin. By reconstructing biosynthetic pathways, optimizing chassis cells, and designing efficient regulatory systems, it enables doubling Nisin yield while achieving low energy consumption and low pollution in production processes. This represents a key direction to replace traditional fermentation technologies.

I. Synthetic Biology Strategies for Constructing High-Yield Nisin Strains

1. Optimization of Core Biosynthetic Pathways

Enhancing Key Gene Expression: Integrate the Nisin biosynthetic gene cluster (nisA/B/C/T/I/K/R/P) downstream of strong promoters (e.g., p43 promoter, T7 promoter) or amplify copy numbers (via multi-copy plasmids) to improve precursor peptide synthesis and modification efficiency.

Relieving Feedback Inhibition: Knock out Nisin’s self-regulatory repressor genes (e.g., nisI) or mutate binding sites of regulatory proteins (NisR) to avoid product accumulation-induced inhibition of the synthetic pathway, sustaining biosynthesis.

Supplying Precursor Metabolic Flux: Overexpress alanine dehydrogenase, serine dehydratase, etc., to enhance supply of precursor amino acids (e.g., alanine, serine), providing sufficient raw materials for Nisin peptide chain synthesis and increasing yield.

2. Modification and Adaptation of Chassis Cells

Selecting Optimal Chassis Strains: Use Lactococcus lactis (natural Nisin-producing strain) as a basis, knocking out redundant metabolic pathways (e.g., lactate dehydrogenase gene ldh) to reduce byproduct consumption and redirect more carbon sources to Nisin synthesis. Alternatively, use E. coli or Bacillus subtilis as heterologous chassis, achieving efficient heterologous expression by introducing complete synthetic gene clusters and modification enzyme systems.

Enhancing Cellular Tolerance: Use CRISPR-Cas9 to knock out repressors of Nisin efflux pump genes (e.g., nisT) or express stress-resistant proteins (e.g., heat shock protein Hsp70) to improve chassis cell tolerance to high Nisin concentrations, avoiding product toxicity-induced growth inhibition.

3. Design of Dynamic Regulatory Systems

Application of Responsive Promoters: Construct dynamic expression systems regulated by Nisin-inducible promoters (e.g., PnisA). Low-level expression of synthetic genes in early fermentation avoids premature product accumulation; mid-stage auto-induction by Nisin triggers high-efficiency synthesis, balancing growth and productivity.

Precise Metabolic Flux Distribution: Use RNA switches or ribosome binding site (RBS) engineering to regulate expression timing of synthetic pathway genes and cell growth-related genes, prioritizing cell proliferation in the logarithmic phase and concentrating resources on Nisin synthesis in the stationary phase.

II. Synthetic Biology Optimization for Green Production Processes

1. Reducing Energy Consumption and Raw Material Costs

Utilizing Low-Cost Carbon Sources: Modify chassis cell carbon utilization pathways to enable utilization of cellulose, galactose, etc., from agricultural waste (e.g., corn stover hydrolysate, beet pulp), replacing traditional glucose feedstocks to reduce costs and achieve resource recycling.

Optimizing Fermentation Conditions: Engineer strains to adapt to mild fermentation conditions (25–30°C, low oxygen) via synthetic biology, reducing energy consumption in heating and aeration; shorten fermentation cycles (from 48 hours to 24–36 hours) to improve production efficiency.

2. Reducing Pollutant Emissions

Simplifying Downstream Separation: Fuse signal peptides (e.g., SEC signal peptide) to the Nisin synthetic gene terminus for direct secretion into fermentation broth, avoiding pollutants from cell lysis. Alternatively, express affinity tags (e.g., His-tag) for rapid purification via affinity chromatography, reducing organic solvent use and pollution.

Byproduct Resource Utilization: Engineer strains to convert fermentation byproducts (e.g., lactic acid) into high-value products (e.g., 3-hydroxypropionate) or reduce byproduct formation via metabolic engineering, achieving "zero-waste" fermentation and lowering environmental pressure.

3. Constructing Continuous Fermentation Processes

Developing Stable Production Strains: Use chromosomal integration instead of plasmid expression to avoid plasmid loss during fermentation, ensuring genetic stability. Combine with immobilized cell technology to enable continuous batch fermentation, reducing water consumption and pollution from equipment cleaning and sterilization.

III. Application Effects and Industrialization Potential

1. Improvements in Yield and Efficiency

Laboratory Scale: Modified high-yield strains achieve Nisin yields of 5–10 g/L, 3–5 times higher than wild-type strains (0.5–2 g/L); heterologous expression strains (e.g., E. coli) reach 3–6 g/L, breaking the yield bottleneck of natural strains.

Process Optimization Effects: Green processes improve carbon source utilization by 20%–30%, reduce energy consumption by 15%–25%, and cut pollutant emissions by over 40%, meeting environmental protection requirements.

2. Industrial Application Scenarios

Food Preservative Production: Combining high-yield strains with green processes significantly reduces industrial production costs of Nisin, promoting its widespread use in meat products, dairy products, beverages, etc., as a substitute for chemical preservatives.

Pharmaceutical Intermediate Synthesis: Provides an efficient production platform for high-activity Nisin mutants (e.g., genetically engineered broad-spectrum mutants), meeting medical demands for high-purity, low-cost antimicrobial peptides.

IV. Challenges and Solutions

1. Core Challenges

Low Modification Efficiency in Heterologous Expression: Insufficient efficiency of thioether ring modification (mediated by NisB/C enzymes) in heterologous chassis cells results in low proportions of active products.

Stability in Large-Scale Fermentation: Lab-constructed high-yield strains tend to exhibit metabolic imbalance and reduced product tolerance during large-scale fermentation.

Regulatory and Safety Verification: Synthetic biology strains require biosafety assessments to ensure no escape risks and no harmful substances in products.

2. Solutions

Co-Expressing Modification Enzyme Systems: Co-express modification and processing enzymes (NisB, NisC, NisP, etc.) in heterologous chassis, optimizing expression ratios to improve active product conversion.

Scaling Up Fermentation Processes: Combine metabolomics and transcriptomics data with AI algorithms to optimize pH, dissolved oxygen, and feeding strategies in large-scale fermentation, maintaining stable strain productivity.

Standardizing Safety Assessments: Establish safety evaluation protocols for synthetic biology strains to ensure genetic stability, no horizontal gene transfer risks, and product compliance with food or pharmaceutical standards.