Optimize the biosynthetic pathway of Nisin

Nisin is a class of lantibiotics produced by Lactococcus lactis, which exhibits potent activity against Gram-positive bacteria (e.g., Bacillus and Staphylococcus) and is widely used in food preservation and medical fields. Its biosynthesis is a complex process involving gene regulation, post-translational modification, and transport processing. Optimizing the synthetic pathway to improve yield and reduce costs is a core requirement for its industrial application. The following elaborates on key optimization strategies and research progress starting from the analysis of biosynthetic mechanisms.

I. Core Pathways and Rate-Limiting Steps of Nisin Biosynthesis

Nisin biosynthesis relies on the coordinated expression of the nis gene cluster (approximately 11 genes, such as nisA/B/C/T/P/K/R/I/F/E), with the specific process divided into four steps:

Precursor peptide synthesis: The structural gene nisA encodes a precursor peptide (pre-nisin), consisting of an N-terminal leader peptide (guiding modification and transport) and a C-terminal core peptide (the final active sequence).

Post-translational modification: The core peptide undergoes dehydration catalyzed by the dehydratase NisB (converting serine/threonine residues to dehydroalanine/dehydrobutyrine) and cyclization catalyzed by the cyclase NisC (forming lanthionine structures via thioether bond cyclization), resulting in a modified peptide with an active conformation.

Transport and processing: The modified precursor peptide is transported extracellularly by the ABC transporter NisT, and the leader peptide is then cleaved by the protease NisP to release mature nisin.

Self-protection and regulation: Immune genes nisI/F/E encode transporters and resistance proteins to protect the producing bacteria from self-produced nisin. Meanwhile, mature nisin acts as a signaling molecule, activating transcription of the nis gene cluster through a two-component system composed of the sensor kinase NisK and response regulator NisR, forming a "synthesis-induction" positive feedback loop.

The rate-limiting steps of this pathway mainly include: efficiency of post-translational modification (catalytic capacity of NisB/NisC), precursor peptide transport and processing (synergistic effect of NisT/NisP), and transcriptional regulation intensity of the gene cluster (signal transduction mediated by NisK/NisR).

II. Key Optimization Strategies for the Biosynthetic Pathway

Targeting the above rate-limiting steps, research has achieved optimization through genetic engineering, metabolic engineering, and synthetic biology approaches, focusing on three levels: "gene expression regulation", "metabolic flux distribution", and "enzyme function enhancement".

Precise Regulation of Gene Cluster Expression

The transcriptional efficiency of the nis gene cluster directly determines nisin synthesis, with the core being enhancing promoter activity and signal transduction efficiency.

Promoter engineering: The native nisA promoter (PnisA) is regulated by nisin induction but has low basal expression. Replacing it with strong constitutive promoters (e.g., P32, PnisZ from Lactococcus lactis) or optimizing the -10/-35 region sequences of PnisA (to enhance binding to RNA polymerase) can increase the transcriptional level of nisA/B/C by 2-3 fold. For example, optimizing the -35 region of PnisA from "TTGACA" to "TTGATA" increased precursor peptide synthesis by 40%.

Two-component system enhancement: NisK/NisR is a key signaling pathway activating nis gene transcription. Overexpressing nisK/nisR or enhancing NisK affinity for nisin via site-directed mutagenesis (e.g., mutating serine at position 234 of the histidine kinase domain of NisK to aspartic acid) can improve signal transduction efficiency, increasing gene cluster transcriptional activity by 1.5-2 fold and reducing the induction threshold (from 5 ng/mL to 2 ng/mL).

Optimization of gene copy number: Increasing the copy number of nisA (the precursor peptide gene) via plasmid or genomic integration can directly improve precursor peptide yield, but "metabolic burden" must be avoided—when the copy number exceeds 3, cell growth rate decreases by 15%, which instead reduces total yield. The current optimal strategy is integrating 1-2 additional copies into the genome, combined with promoter optimization, which can increase precursor peptide synthesis by 60%.

Synergistic Enhancement of Post-Translational Modification and Transport Processing

The modification efficiency of NisB/NisC and the transport/processing capacity of NisT/NisP are core bottlenecks in nisin maturation, requiring breakthroughs through enzyme function optimization and coordinated expression.

Functional enhancement of modification enzymes: The dehydration reaction of NisB depends on ATP and Mn²⁺. Site-directed mutagenesis to enhance its ATP-binding domain (e.g., mutating glycine at position 456 to alanine) can improve catalytic efficiency (with kcat/Km increased by 30%). The cyclization reaction of NisC is affected by substrate conformation; co-expression with NisB (rather than overexpression alone) can increase modification efficiency by 50%, as the two form a complex facilitating substrate transfer.

Coordination of transport and processing: NisT must bind to the leader peptide of the precursor peptide to initiate transport, while NisP must recognize and cleave the leader peptide extracellularly. Insufficient NisT expression leads to intracellular accumulation of modified peptides (inhibiting cell activity); weak NisP activity blocks the release of mature nisin. Constructing a co-expression module of nisT and nisP (using the same promoter and RBS sequence) can increase transport-processing efficiency by 40%, raising the proportion of extracellular mature nisin from 65% to 85%.

Directional Distribution of Metabolic Flux

Nisin synthesis requires large amounts of precursors (e.g., amino acids, ATP, NADPH). Optimizing carbon source and energy metabolism to allocate more resources to the synthetic pathway is key to yield improvement.

Supply of precursor amino acids: The nisin core peptide contains alanine, serine, threonine, etc. Overexpressing enzymes involved in their synthesis (e.g., alanine transaminase alaT, serine synthase serA) can increase intracellular amino acid concentrations by 20-30% and nisin yield by 15-25%. Additionally, supplementing exogenous amino acids (e.g., 0.5 g/L L-serine) can further alleviate precursor limitations.

Optimization of energy and reducing power: During fermentation of Lactococcus lactis, glucose is mainly converted to lactic acid via glycolysis (consuming NADH), leading to insufficient supply of ATP and NADPH. Knocking out the lactate dehydrogenase gene (ldh) and overexpressing NADH kinase (converting NADH to NADPH) can increase intracellular ATP levels by 40% and NADPH by 50%, while reducing lactic acid accumulation-induced cell inhibition, improving nisin yield by 30-40%.

Relief of carbon catabolite repression: The carbon catabolite repressor protein (CcpA) inhibits nis gene expression (when glucose is excessive). Knocking out ccpA or mutating its binding site (e.g., the cre sequence on the nisA promoter) can relieve repression, increasing nisin yield under high glucose concentrations (50 g/L) by 25%.

Synergistic Optimization of Fermentation Conditions

Genetic optimization must be combined with fermentation conditions to maximize yield, with key parameters including:

pH control: Lactic acid accumulation during fermentation lowers pH (<5.0), inhibiting NisB/NisC activity. Maintaining pH at 6.0-6.5 by supplementing NaOH can retain over 80% of modification enzyme activity (only 40% retained at pH 5.0).

Temperature and dissolved oxygen: The optimal growth temperature of Lactococcus lactis is 30°C, but the optimal temperature for nisin synthesis is 25°C (maximizing NisK signal transduction efficiency). Moderate aeration (dissolved oxygen 10-15%) can promote ATP production via the respiratory chain, increasing yield by 20% compared to anaerobic conditions.

Induction timing: Adding low-concentration nisin (1 ng/mL) for induction when cell density reaches OD₆₀₀=0.6 avoids growth inhibition from early induction, with final yield 35% higher than induction at the early logarithmic phase.

III. Existing Challenges and Future Directions

Current optimization still faces bottlenecks: first, balancing multi-gene co-expression (e.g., overexpression of nisA may lead to insufficient modification capacity of NisB), requiring dynamic regulation systems (e.g., quorum-sensing-based promoters) to achieve "on-demand expression"; second, the complexity of post-translational modification (substrate specificity of NisB/NisC) makes significant improvements via simple mutations difficult; third, stability in industrial scale-up (e.g., uneven pH and temperature in fermenters affecting expression consistency).

Future research should combine synthetic biology to design "artificial gene circuits", such as coupling the nis gene cluster with cell growth (e.g., driven by growth phase-specific promoters) or constructing "self-induction-self-secretion" systems (mature nisin automatically enhances synthesis via feedback). Additionally, genome editing (e.g., CRISPR-Cas9) to knock out redundant genes (e.g., protease genes degrading nisin) can further reduce product loss.

The optimization of nisin's biosynthetic pathway is the result of synergy between gene regulation, metabolic engineering, and fermentation processes. By enhancing key gene expression, improving modification and transport efficiency, and directionally allocating metabolic resources, laboratory-scale yields have been increased from 1000-2000 IU/mL to 5000-8000 IU/mL. Future efforts need to address multi-target coordination and industrial stability issues to promote its broader application in food and medical fields.