Production optimization of L-leucine in microbial fermentation

As an essential branched-chain amino acid for humans, L-leucine is widely used in pharmaceuticals, food, feed, and other fields, with a continuously growing market demand. Microbial fermentation has become the mainstream industrial production technology for L-leucine due to its low raw material cost, environmental friendliness, and high product purity. The core goal of production optimization is to improve product synthesis efficiency (yield, conversion rate, productivity) and reduce production costs through strain improvement, fermentation process regulation, metabolic flux optimization, and other means. This article systematically analyzes the production optimization strategies and technological progress of L-leucine microbial fermentation from four dimensions: strain construction, fermentation process optimization, metabolic regulation mechanisms, and downstream separation coupling.

I. Directed Construction and Improvement of High-Yield Strains

Strains are the core of fermentation production. High-yield L-leucine strains need to possess four key characteristics: "efficient carbon source utilization, directed metabolic flux distribution, smooth product secretion, and strong stress resistance." Currently, the mainstream improvement strategies use Escherichia coli and Corynebacterium glutamicum as hosts for directed modification through metabolic engineering and synthetic biology technologies:

1. Enhancing Expression of Key Enzymes in the L-Leucine Synthesis Pathway

The biosynthesis of L-leucine starts with pyruvate and generates the product through three key reactions, with the core being the enhancement of the activity of rate-limiting enzymes in the pathway:

Chorismate Mutase (CM) and Prephenate Dehydrogenase (PDH): Relieve feedback inhibition of the synthesis pathway by tyrosine and phenylalanine. Site-directed mutagenesis modifies the allosteric regulatory sites of the enzymes (e.g., the CM gene ilvE mutant of C. glutamicum), making them no longer inhibited by end products.

α-Isopropylmalate Synthase (ALS): As the first rate-limiting enzyme in L-leucine synthesis, overexpression of the wild-type alsS gene (from Bacillus subtilis) or its mutants improves the conversion efficiency of pyruvate to α-isopropylmalate. The enzyme activity can be increased by 2~3 times, directly driving metabolic flux toward L-leucine.

α-Isopropylmalate Dehydrogenase (IMDH) and Leucine Dehydrogenase (LDH): Overexpression of the ilvD and leuA genes enhances the dehydrogenation and transamination of intermediate products, reduces the accumulation of intermediate metabolites, and improves product synthesis rate.

2. Blocking Competitive Metabolic Pathways to Reduce Carbon Source Diversion

The basal metabolism of host strains consumes a large amount of carbon sources for the synthesis of other amino acids, nucleic acids, or fats. Competitive pathways need to be blocked through gene knockout:

Knock out the valB and ilvC genes to block the synthesis pathways of valine and isoleucine, avoiding the diversion of branched-chain amino acid synthesis precursors.

Knock out the pykA and pykB genes to inhibit pyruvate kinase activity, reduce the conversion of pyruvate to lactic acid and acetic acid, and increase the supply of pyruvate to the L-leucine synthesis pathway.

Knock out the aceE and aceF genes to weaken the carbon metabolic flux of the tricarboxylic acid cycle (TCA), directing more carbon sources to the glycolytic pathway to provide sufficient precursors for L-leucine synthesis.

3. Enhancing Product Secretion and Cell Permeability

Intracellular accumulation of L-leucine causes feedback inhibition, and secretion efficiency directly affects fermentation yield. The product transport system needs to be optimized:

Overexpress L-leucine efflux protein genes (e.g., the brnFE gene of C. glutamicum). These proteins mediate the transmembrane transport of L-leucine, increasing intracellular product secretion efficiency by 30%~50% and effectively relieving end-product feedback inhibition.

Moderately increase cell membrane permeability by weakening cell wall synthesis-related genes (e.g., murE, murG) or adding cell wall synthesis inhibitors such as penicillin and glycine, promoting product efflux and reducing toxic pressure caused by intracellular accumulation.

4. Improving Strain Stress Resistance and Carbon Source Utilization Capacity

In industrial fermentation, high sugar concentration, high product concentration, and acidic environments inhibit strain growth. Strain stress resistance needs to be enhanced:

Overexpress stress response genes (e.g., sigH, dnaK) to improve strain tolerance to high osmotic pressure and high product concentration, increasing cell survival rate to over 85% during fermentation.

Heterologously express glucose transporter genes (e.g., ptsG) or xylose isomerase genes (xylA) to broaden the range of usable carbon sources, enabling strains to utilize cheap carbon sources such as corn syrup and xylose, thereby reducing raw material costs.

II. Systematic Optimization and Regulation of Fermentation Processes

Fermentation processes bridge strain potential and industrial production. Parameter optimization is required to achieve "synergy between strain growth and product synthesis." Core optimization directions include carbon-nitrogen ratio adjustment, fermentation process parameter regulation, and feeding strategy design:

1. Selection and Ratio Optimization of Carbon and Nitrogen Sources

Carbon and nitrogen sources are core nutrients for fermentation, and their types and ratios directly affect yield:

Carbon Sources: Rapidly utilizable carbon sources such as glucose and sucrose are preferred, with an initial concentration controlled at 30~50 g/L (excessively high concentrations cause osmotic inhibition, while excessively low concentrations limit growth). In industrial production, corn starch hydrolysate (glucose content 60%~70%) can replace pure glucose, reducing raw material costs by over 30%. Some modified strains can utilize pentose sugars such as xylose and arabinose, adapting to lignocellulosic biomass raw materials and further reducing costs.

Nitrogen Sources: A composite nitrogen source of "organic nitrogen + inorganic nitrogen" is adopted. Inorganic nitrogen (NH₄Cl, (NH₄)₂SO₄) provides rapidly available nitrogen, with an initial concentration controlled at 5~8 g/L. Organic nitrogen (yeast extract, corn steep liquor, soybean meal hydrolysate) provides growth factors and amino acids, with an addition amount of 10~15 g/L. Biotin and B vitamins in corn steep liquor promote strain growth and enzyme activity. After optimization, the carbon-nitrogen ratio (C/N) is controlled at 10:1~15:1, increasing carbon source conversion rate to 40%~50%.

2. Precise Regulation of Key Fermentation Parameters

Temperature, pH, and dissolved oxygen (DO) during fermentation are core environmental factors affecting strain metabolism and product synthesis, requiring stage-specific precise regulation:

Temperature: A "two-stage temperature control" strategy is adopted—30~32℃ during the strain growth stage (0~12 h) to promote cell proliferation; increasing to 34~36℃ during the product synthesis stage (12~48 h) to improve the activity of key enzymes in the synthesis pathway, increasing product synthesis rate by 20%~30%.

pH: The optimal pH for the L-leucine synthase system is 6.5~7.5. During fermentation, pH stability is maintained by automatic feeding of NH₄OH or dilute H₂SO₄. pH below 6.0 or above 8.0 should be avoided, as it reduces enzyme activity, disrupts metabolic flux, and decreases product yield by over 15%.

Dissolved Oxygen (DO): A "stage-specific DO control" strategy is adopted—maintaining DO at 30%~50% (air saturation) during the growth stage to ensure aerobic respiration and rapid cell proliferation; reducing DO to 10%~20% during the product synthesis stage. A moderately hypoxic environment inhibits the TCA cycle, promotes carbon source diversion to the L-leucine synthesis pathway, and avoids lactic acid and acetic acid accumulation caused by anaerobic respiration.

3. Design of Fed-Batch Fermentation Strategies

Fed-batch fermentation avoids osmotic inhibition caused by excessively high initial nutrient concentrations and maintains continuous strain growth and product synthesis. Common strategies include:

Glucose Feeding: A combined mode of "constant-rate feeding + feedback feeding" is adopted. Constant-rate feeding (rate 1~2 g/(L·h)) is used in the initial stage. When the glucose concentration in the fermentation broth drops to 5~10 g/L, the feeding rate is adjusted through an online glucose sensor to maintain the glucose concentration at 2~5 g/L, avoiding carbon source deficiency or excess.

Nitrogen Source and Precursor Feeding: After 12~16 h of fermentation, a mixed solution of (NH₄)₂SO₄ and corn steep liquor is supplemented to provide nitrogen sources and growth factors. For strains limited by pyruvate, a small amount of pyruvate (concentration 1~2 g/L) can be supplemented to directly increase precursor supply, improving product yield by 10%~15%.

Metal Ion Feeding: Appropriate supplementation of Mg²⁺ (1~2 mmol/L) and Mn²⁺ (0.1~0.3 mmol/L) as coenzymes for key enzymes in the synthesis pathway enhances enzyme activity and promotes product synthesis.

III. Metabolic Flux Regulation and Fermentation Process Intensification

Regulating intracellular metabolic networks to achieve directed distribution of nutrients such as carbon and nitrogen sources toward the L-leucine synthesis pathway is a core means to improve yield:

1. Key Node Regulation Based on Metabolomics

Metabolomics technology is used to analyze concentration changes of intracellular intermediate metabolites (e.g., pyruvate, α-ketoglutarate, α-isopropylmalate) during fermentation, identifying metabolic bottlenecks:

If α-isopropylmalate accumulates, it indicates insufficient ALS enzyme activity, requiring further optimization of alsS gene expression or addition of enzyme activators.

If pyruvate accumulates, it indicates insufficient carbon source diversion to the L-leucine pathway, requiring weakening of competitive pathways or enhancement of ALS enzyme expression to promote directed metabolic flux distribution.

2. Synergistic Optimization of Coenzyme and Energy Metabolism

The L-leucine synthesis process consumes coenzymes such as NADPH. Regulation of coenzyme regeneration pathways is needed to improve energy supply:

Overexpress glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) to strengthen the pentose phosphate pathway (PPP), increasing NADPH production and providing sufficient coenzymes for dehydrogenase reactions such as IMDH.

Optimize DO and carbon source supply during fermentation to maintain the intracellular ATP/ADP ratio at 1.5~2.0, providing energy for amino acid synthesis and avoiding metabolic flux stagnation caused by insufficient energy.

3. Inhibiting Byproduct Formation and Improving Product Purity

Byproducts such as acetic acid, lactic acid, and succinic acid are easily produced during fermentation, which not only consume carbon sources but also affect strain growth and product separation:

Regulate the feeding rate to avoid the "Crabtree effect" (fermentative overflow metabolism) caused by excess glucose, reducing acetic acid production.

Overexpress acetic acid metabolism-related genes (e.g., acsA) to promote acetic acid reuse, or add a small amount of citric acid (0.5~1 g/L) to inhibit lactate dehydrogenase activity and reduce lactic acid accumulation.

Optimize pH and DO control to maintain stable intracellular microenvironment, reducing byproduct formation rate (byproduct ratio can be reduced from 15%~20% to below 5%).

IV. Fermentation-Separation Coupling Technology and Downstream Optimization

Traditional fermentation requires downstream separation after fermentation completion. Product accumulation easily causes feedback inhibition. Fermentation-separation coupling technology can remove products in real time, breaking the yield bottleneck:

1. Application of In Situ Product Recovery (ISPR) Technology

Membrane Separation Coupling: Ultrafiltration membranes (molecular weight cutoff 10~50 kDa) are used for real-time separation of extracellular L-leucine during fermentation. The membrane pore size can retain cells and macromolecular impurities, while the product passes through the membrane into the permeate, which is then concentrated by nanofiltration membranes. This technology can increase fermentation yield by 30%~40% and reduce downstream separation steps.

Ion Exchange Resin Adsorption: Weakly acidic cation exchange resins (e.g., Amberlite IRC-50) are added to the fermentation broth. The resin specifically adsorbs L-leucine (pI=6.0), and in situ adsorption and elution of the product are achieved by pH adjustment (pH 5.5~6.5 for adsorption, pH 8.0~9.0 for elution), avoiding intracellular product accumulation.

Solvent Extraction: Alcohol solvents (e.g., n-butanol) or ionic liquids are used as extractants for real-time separation of L-leucine from the fermentation broth through liquid-liquid extraction. The extraction rate can reach over 85% without affecting strain growth.

2. Optimization of Downstream Separation and Purification Processes

Downstream separation costs account for 30%~50% of the total cost. The optimization focus is on simplifying the process and improving yield:

Pretreatment: The fermentation broth is filtered through a plate-and-frame filter or centrifuged (8000~10000 r/min) to remove bacterial cells. Flocculants (PAC+PAM) are used to assist sedimentation, reducing subsequent separation load.

Extraction and Concentration: L-leucine is adsorbed by ion exchange chromatography (cation exchange resin) and eluted with a gradient of NH₄OH solution. The eluate is concentrated under reduced pressure (temperature 60~70℃, vacuum degree -0.08~-0.09 MPa) to a concentration of 20%~30%.

Crystallization and Drying: The concentrated solution is cooled to 10~15℃ for crystallization, and seed crystals are added to promote crystal growth, with a crystallization rate of over 90%. The crystals are separated by centrifugation and vacuum-dried (temperature 50~60℃), resulting in a final product purity ≥98.5% and a total yield increased from 70%~75% in traditional processes to 85%~90%.

V. Technical Challenges and Future Development Directions

1. Existing Challenges

There is still room for improvement in the carbon source utilization efficiency of strains, especially insufficient ability to utilize cheap biomass raw materials (e.g., straw hydrolysate) and problems with inhibitor tolerance.

The precision of metabolic flux regulation during fermentation is insufficient. Accumulation of intermediate metabolites and formation of byproducts still restrict yield.

The industrial application cost of fermentation-separation coupling technology is high, and issues such as membrane fouling and resin regeneration need further resolution.

Controlling the stability of fermentation parameters in industrial production is difficult, with batch-to-batch yield fluctuations of 10%~15%.

2. Future Optimization Directions

Precision Modification by Synthetic Biology: Use CRISPR-Cas9 gene editing technology to construct "modular" strains, achieving precise regulation of key enzyme expression; combine genome-scale metabolic models (GEM) to predict metabolic bottlenecks and guide rational strain design.

Intelligent Fermentation Control: Integrate online sensors (glucose sensors, pH sensors, DO sensors) with AI algorithms to real-time monitor fermentation parameters, dynamically optimize feeding rate, temperature, pH, etc., achieving adaptive control of the fermentation process and reducing batch fluctuations.

Development of Green and Low-Carbon Processes: Use industrial waste (e.g., sugar production wastewater, papermaking black liquor) as carbon and nitrogen sources to reduce raw material costs; develop low-energy consumption separation technologies (e.g., membrane distillation, pressure-driven membrane separation) to reduce energy consumption and pollution in downstream processes.

Co-Fermentation of Multiple Products: Construct strains that simultaneously produce L-leucine and other high-value products (e.g., glutamic acid, proline), improving resource utilization efficiency and economic benefits of the fermentation process.

The production optimization of L-leucine microbial fermentation is a systematic project involving "strain improvement, process regulation, metabolic regulation, and separation coupling." Breaking through strain yield can be achieved by strengthening synthetic pathways and blocking competitive pathways through metabolic engineering technology; maximizing strain potential can be achieved by optimizing carbon-nitrogen sources, designing feeding strategies, and precise parameter regulation; reducing production costs and improving product purity can be achieved through fermentation-separation coupling technology and downstream process optimization. Currently, in industrial production, the L-leucine yield of modified C. glutamicum strains has reached 50~70 g/L, with a conversion rate of 18%~25% and a productivity of 1.5~2.0 g/(L·h).