The biosynthesis optimization strategy of Nisin

As a natural antimicrobial peptide produced by Lactococcus lactis, nisin is widely used in food preservation, pharmaceutical antibacterial applications, and other fields due to its advantages of efficiently inhibiting Gram-positive bacteria, high safety, and degradability by human digestion. Its biosynthesis efficiency and yield are constrained by multiple factors such as strain characteristics, fermentation process, and metabolic regulation. Current research achieves high efficiency and low cost in nisin biosynthesis through four key directions: "strain modification, fermentation process optimization, metabolic pathway regulation, and synthetic biology technology application". The specific optimization strategies are as follows:

I. Screening and Directional Modification of High-Yield Strains

Strains are the core of nisin biosynthesis. Enhancing synthetic capacity from the source can be achieved through screening natural high-yield strains and genetic engineering modification, with main strategies including:

1. Screening and Domestication of Natural Strains

Isolate Lactococcus lactis from traditional fermented foods (e.g., cheese, fermented milk), soil, or plant surfaces, and screen wild strains with relatively high nisin yield using the "inhibition zone method" combined with high-performance liquid chromatography (HPLC). For example, Lactococcus lactis subsp. lactis ATCC 11454, isolated from traditional Nordic cheese, undergoes repeated subculture domestication (gradual adaptation in a medium containing low-concentration nisin to avoid growth inhibition caused by its own sensitivity to nisin), which can increase nisin yield by 20%–30%. In addition, treating strains with mutagenic agents such as ultraviolet (UV) radiation and nitrosoguanidine (NTG) to induce gene mutations enables screening of mutant strains with strong stress resistance (e.g., high sugar tolerance, acid tolerance) and enhanced nisin synthesis capacity. Some mutant strains exhibit increased sensitivity to environmental signals due to mutations in regulatory genes (e.g., nisR/nisK) that control nisin synthesis, allowing nisin synthesis to start early in fermentation and extending the synthesis cycle.

2. Genetic Engineering Modification to Improve Synthesis Efficiency

Strengthen the expression of genes related to nisin biosynthesis or knock out negative regulatory genes through gene cloning and expression regulation, including:

Strengthening the expression of nisin synthesis gene clusters: Nisin biosynthesis is regulated by gene clusters such as nisA/B/C/T/U/R/K. Among them, nisA is a structural gene (encoding the nisin precursor peptide), nisB/C is responsible for post-translational modification (dehydration, cyclization) of the precursor peptide, and nisT participates in the transmembrane transport of nisin. Cloning these gene clusters into high-efficiency expression vectors (e.g., pMG36e), introducing them into Lactococcus lactis, and optimizing promoters (e.g., replacing with the strong promoter P32) can increase the transcription level of the nisA gene by 1.5–2 times, with a corresponding increase in nisin yield by 40%–60%.

Knocking out negative regulatory genes and genes involved in competitive metabolic pathways: There are certain genes in Lactococcus lactis that negatively regulate nisin synthesis (e.g., abrB, which inhibits the transcription of the nis gene cluster). Knocking out abrB using CRISPR/Cas9 gene editing technology can relieve the inhibition of nisin synthesis. Meanwhile, knocking out the lactate dehydrogenase gene (ldh) reduces the conversion of pyruvate to lactic acid (lactic acid is the main metabolite of Lactococcus lactis and competes with nisin synthesis for carbon sources and energy), directing more carbon sources to the nisin synthesis pathway. Experimental data show that the nisin yield of ldh-knockout strains can increase by 35%–50%, and the lactic acid concentration in the fermentation broth decreases, reducing the difficulty of subsequent extraction and purification.

Heterologous expression to improve host adaptability: Introduce the nisin synthesis gene cluster into other hosts with more controllable metabolic regulation (e.g., Bacillus subtilis, Escherichia coli) and utilize the efficient metabolic capacity of the host for nisin synthesis. For example, after introducing the nis gene cluster into Bacillus subtilis, nisin can be directly secreted into the medium by virtue of the host’s strong protein secretion capacity, avoiding toxic inhibition caused by intracellular accumulation in the host. Additionally, the fermentation cycle is shortened from 24–36 hours (for Lactococcus lactis) to 18–24 hours.

II. Systematic Optimization of Fermentation Processes

Fermentation process parameters directly affect strain growth and nisin synthesis rate. Creating a suitable microenvironment for nisin synthesis can be achieved by regulating parameters such as carbon-nitrogen sources, temperature, pH, and dissolved oxygen, with specific optimization directions including:

1. Selection and Ratio Optimization of Carbon-Nitrogen Sources

Carbon sources provide energy and carbon skeletons for nisin synthesis, so carbon sources that are easily utilized by strains and can supply energy continuously should be selected. Although glucose is easily utilized, excessive glucose causes the "glucose effect" (prioritizing glucose metabolism and inhibiting nis gene expression). Therefore, a mixed carbon source of glucose, sucrose, and lactose (e.g., glucose:lactose = 1:2) is used to extend the carbon source supply cycle and avoid the glucose effect. Meanwhile, lactose acts as an inducer that can activate the promoter of the nis gene cluster and promote nisin synthesis.

In terms of nitrogen sources, since nisin is essentially a polypeptide, sufficient amino acid supply is required. Choosing complex nitrogen sources such as yeast extract, peptone, and soy protein hydrolysate (e.g., yeast extract:peptone = 1:1.5) not only provides amino acids but also contains growth factors (e.g., B vitamins), which can promote strain growth and nis gene expression. Experiments show that the optimized carbon-nitrogen ratio can increase nisin yield by 25%–40%, and the cell density in the fermentation broth increases, reducing nisin degradation caused by cell autolysis.

2. Dynamic Regulation of Temperature and pH

The optimal growth temperature of Lactococcus lactis is 30–32°C, while the optimal temperature for nisin synthesis is slightly lower (28–30°C):

Early fermentation stage (0–8 hours, logarithmic growth phase of cells): Control the temperature at 32°C to promote rapid cell proliferation.

Mid-fermentation stage (8–24 hours, nisin synthesis phase): Reduce the temperature to 29°C to avoid high-temperature inhibition of the activity of nisB/C modification enzymes.

Late fermentation stage (24–36 hours, stable synthesis phase): Maintain the temperature at 28°C to extend the nisin synthesis period.

In terms of pH, lactic acid produced by strain metabolism lowers the pH of the fermentation broth (nisin synthesis is inhibited when pH < 5.0). Automatic supplementation of ammonia water or sodium hydroxide is required to dynamically regulate the pH within 5.5–6.0: a pH that is too high (>6.5) promotes excessive cell growth and consumes excessive carbon-nitrogen sources; a pH that is too low (<5.5) inactivates the nisR/K two-component regulatory system, preventing nisin synthesis. Dynamic pH regulation can increase nisin yield by 30%–50% and reduce nisin degradation under acidic conditions (Nisin is prone to cyclic structure damage when pH < 4.0).

3. Synergistic Optimization of Dissolved Oxygen and Agitation Rate

Although Lactococcus lactis is a facultative anaerobe, a small amount of oxygen is required for nisin synthesis (to maintain the activity of the nisT transport protein):

Early fermentation stage (cell proliferation phase): Control dissolved oxygen (DO) at 15%–20% to promote cellular respiratory metabolism.

Mid-fermentation stage (synthesis phase): Reduce DO to 5%–10% to avoid cell damage caused by reactive oxygen species (ROS) under high oxygen conditions.

Late fermentation stage (stable phase): Maintain DO at approximately 8% to ensure continuous nisin transport.

The agitation rate needs to match the dissolved oxygen level:

Early stage: Agitation rate of 200–250 r/min to ensure sufficient contact between cells and nutrients.

Mid-stage: Reduce the agitation rate to 150–200 r/min to reduce shear damage to cells caused by agitation.

Late stage: Maintain the agitation rate at 180 r/min to avoid stratification of the fermentation broth.

Synergistic optimization of dissolved oxygen and agitation can increase nisin yield by 15%–25%, and the uniformity of nisin in the fermentation broth is enhanced, facilitating subsequent extraction.

III. Regulation of nisin Biosynthesis Metabolic Pathways

By regulating the synergistic relationship between the central metabolism of Lactococcus lactis and the nisin synthesis pathway, metabolic flux waste is reduced, and carbon sources and energy are directionally directed to nisin synthesis. The main strategies include:

1. Strengthening Precursor Substance Supply

The nisin precursor peptide is composed of 11 types of amino acids (e.g., alanine, valine, isoleucine). Precursor supply can be improved by supplementing key amino acids or strengthening amino acid synthesis pathways: adding 0.1%–0.3% alanine and valine to the fermentation medium to directly provide raw materials for nisin synthesis; or strengthening amino acid synthesis genes (e.g., alaC, which encodes alanine transaminase) in strains through genetic engineering to increase the intracellular concentration of key amino acids. Experiments show that strengthening precursor supply can increase nisin yield by 20%–30% and improve nisin purity (reducing short peptide impurities caused by insufficient precursors).

2. Regulating the Balance of ATP and NADPH

Nisin synthesis (especially post-translational modification and transmembrane transport) consumes large amounts of ATP and NADPH: increase intracellular ATP levels by adding creatine phosphate (an ATP regenerator) or strengthening the phosphoglycerate kinase gene (pgk) in the glycolytic pathway; simultaneously, strengthen the glucose-6-phosphate dehydrogenase gene (zwf) in the pentose phosphate pathway to promote NADPH production. Regulation of ATP and NADPH balance can provide more sufficient energy for nisin synthesis, increasing yield by 18%–28% and reducing premature cell death caused by insufficient energy.

3. Inhibiting the Activity of Nisin-Degrading Enzymes

In the late fermentation stage, cell autolysis releases proteases (e.g., lactococcal protease), leading to nisin degradation. Nisin degradation can be reduced by adding protease inhibitors (e.g., benzamidine, EDTA, at concentrations of 0.05%–0.1%) or knocking out protease genes (e.g., prtP): protease inhibitors can inhibit the activity of serine proteases and metalloproteases, reducing the nisin degradation rate from 30%–40% to 10%–15% in the late fermentation stage; prtP-knockout strains can completely avoid the production of lactococcal protease, significantly improving the stability of nisin in the fermentation broth and extending the window period for subsequent extraction.

IV. Innovative Application of Synthetic Biology Technology

Using the concept of "modular design and precise regulation" in synthetic biology, an artificial metabolic network for efficient nisin synthesis is constructed to break through the limitations of traditional optimization:

1. Constructing a "Gene Switch" for Nisin Synthesis

Design a controllable nis gene expression switch using the quorum sensing (QS) system: concatenate the native nisin quorum sensing promoter (PnisA) of Lactococcus lactis with a reporter gene and the nis gene cluster to construct a "self-inducible expression system". In the early fermentation stage, the cell density is low, and PnisA is not activated; when the cell density reaches a threshold (OD₆₀₀ ≈ 1.5), a small amount of secreted nisin activates PnisA, initiating high-efficiency expression of the nis gene cluster and realizing "on-demand synthesis" of nisin. This avoids inhibition of cell growth by early nisin synthesis and increases yield by 35%–55%.

2. Multi-Host Synergistic Synthesis System

Introduce different modules of nisin synthesis (precursor peptide synthesis, post-translational modification, transport) into different hosts (e.g., Escherichia coli for precursor peptide synthesis, Lactococcus lactis for post-translational modification and transport) to achieve synergistic synthesis through co-cultivation: Escherichia coli uses its rapid growth characteristics to synthesize large amounts of nisin precursor peptides and releases them into the medium via secretion signal peptides; Lactococcus lactis absorbs the precursor peptides, completes dehydration and cyclization modification, and transports them outside the cell. This multi-host system avoids metabolic burden in a single host, increases nisin yield by 40%–60%, and can optimize synthesis efficiency by regulating the ratio of the two hosts.

3. Efficient Pathway for Artificially Synthesizing Nisin Analogs

Modify the nisA gene through site-directed mutagenesis to design nisin analogs with higher antibacterial activity (e.g., Nisin Z, Nisin F), while optimizing their synthesis pathways: for example, mutating the 27th amino acid in the nisA gene from histidine to lysine results in a nisin analog with 2–3 times higher antibacterial activity against Listeria; introducing heterologous modification enzyme genes (e.g., cyclases from other antimicrobial peptides) into the synthesis pathway enhances structural stability, allowing it to maintain activity in neutral or weakly alkaline environments and expanding application scenarios.

V. Challenges and Future Directions of Optimization Strategies

Current optimization of nisin biosynthesis still faces certain challenges: in genetic engineering modification, the transformation efficiency of Lactococcus lactis is low (compared to Escherichia coli), requiring optimization of electroporation conditions or development of new vectors; in fermentation process optimization, controlling the uniformity of dissolved oxygen and pH during large-scale production is difficult, requiring the combination of bioreactor design (e.g., airlift reactors) to improve mass transfer efficiency; the cost of synthetic biology technology is high, requiring reduction in the cost of gene editing and artificial network construction.

Future research directions will focus on "green synthesis" and "function expansion": on one hand, using agricultural waste (e.g., straw hydrolysate, soybean residue protein) as low-cost carbon-nitrogen sources to reduce fermentation costs and achieve sustainable production; on the other hand, endowing nisin with new functions (e.g., targeted antibacterial activity, synergistic action with other antibacterial substances) through synthetic biology technology, further expanding its applications in food and pharmaceutical fields and promoting its upgrade from a "traditional preservative" to a "multifunctional bioactive molecule".