Optimization of Preparation Process of L-Threonine by Fermentation

The optimization of L-threonine production via fermentation can be approached from several aspects, including strain selection, culture medium optimization, fermentation condition control, fermentation process monitoring and control, and downstream separation and purification. The details are as follows:  

Ⅰ.Strain Selection  

1. Mutagenesis Breeding  

Mutagenesis breeding involves treating wild-type strains with physical (e.g., UV rays, X-rays) or chemical (e.g., nitrosoguanidine) mutagens to induce gene mutations. Through extensive screening, high-yield L-threonine mutant strains can be obtained. For example, Escherichia coli exposed to UV radiation may generate mutants with significantly increased L-threonine accumulation compared to the original strain.  

2. Genetic Engineering Breeding  

A thorough understanding of key enzyme genes in the L-threonine biosynthesis pathway, such as homoserine dehydrogenase and threonine synthase, enables genetic modification of strains. Techniques such as gene cloning and expression regulation can be employed to enhance production. For instance, introducing a feedback-resistant homoserine dehydrogenase gene into E. coli increases enzyme activity and reduces feedback inhibition, significantly boosting L-threonine yield. Additionally, introducing genes conferring resistance to amino acid analogs can enhance strain tolerance and production in selective environments.  

Ⅱ.Culture Medium Optimization  

1. Carbon Source Selection  

Selecting an appropriate carbon source is crucial for bacterial growth and acid production. Common choices include glucose, sucrose, and starch hydrolysates. Studies indicate that glucose, as a rapidly metabolizable carbon source, provides sufficient energy for early cell growth. However, excessive glucose concentration can trigger catabolite repression, inhibiting L-threonine synthesis. A fed-batch strategy or a combination of glucose and a slowly metabolizable carbon source (e.g., corn steep liquor) can balance cell growth and L-threonine production.  

2. Nitrogen Source Optimization  

Nitrogen sources are vital for bacterial growth and L-threonine biosynthesis. Organic nitrogen sources (e.g., yeast extract, peptone) contain abundant amino acids and vitamins, promoting cell growth, while inorganic nitrogen sources (e.g., ammonium sulfate, urea) provide essential nitrogen for L-threonine synthesis. Optimizing the ratio of organic to inorganic nitrogen sources can improve yield. For example, determining the optimal proportion of yeast extract and ammonium sulfate can significantly enhance fermentation efficiency.  

3. Addition of Precursors  

Adding precursors of the L-threonine biosynthesis pathway, such as aspartic acid, can bypass certain metabolic steps and increase substrate availability, thereby boosting production. However, precise control of precursor concentrations is necessary, as excessive amounts may inhibit bacterial growth.  

Ⅲ.Fermentation Condition Control  

1. Temperature Control  

Optimizing fermentation temperature based on bacterial growth and acid production characteristics is essential. During the growth phase, a higher temperature promotes cell proliferation, while during L-threonine synthesis, a slightly lower temperature enhances enzyme activity and yield. For example, maintaining 37°C during early cultivation and reducing to 32°C in the later fermentation stage improves L-threonine production.  

2. pH Regulation  

pH significantly influences bacterial metabolism and L-threonine biosynthesis. The fermentation process typically occurs in a neutral to slightly alkaline environment. pH can be adjusted using calcium carbonate or ammonia. Real-time monitoring and maintaining pH within an optimal range (e.g., 7.2–7.6) ensures efficient bacterial growth and L-threonine production.  

3. Oxygen Supply Control  

L-threonine fermentation is an aerobic process, requiring sufficient oxygen for bacterial metabolism and acid synthesis. Oxygen levels can be controlled by adjusting aeration rates and agitation speed. Insufficient oxygen limits respiration and metabolism, whereas excessive oxygen may cause excessive foaming, hindering fermentation and increasing energy consumption. Maintaining an optimal dissolved oxygen range (e.g., 30%–50% saturation) enhances L-threonine yield.  

Ⅳ.Fermentation Process Monitoring and Control  

1. Biomass Monitoring  

Techniques such as turbidity measurement, dry weight determination, and online biomass sensors can be used for real-time monitoring of cell growth. Adjusting fermentation conditions based on biomass changes, such as modifying feed rates, ensures optimal bacterial growth while preventing nutrient imbalances.  

2. Product Concentration Monitoring  

Regularly measuring L-threonine concentration in the fermentation broth using high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA) allows for process optimization. If production rates decline, adjustments in fermentation conditions or feed composition can extend the synthesis phase and improve overall yield.  

3. Metabolite Analysis  

Analyzing key metabolic products (e.g., organic acids, amino acids) during fermentation helps assess metabolic pathway efficiency. Metabolic flux analysis can identify bottlenecks, enabling targeted process optimizations such as increasing precursor availability or adjusting fermentation parameters to enhance L-threonine biosynthetic flow.  

Ⅴ.Downstream Separation and Purification  

1. Fermentation Broth Pretreatment  

After fermentation, initial pretreatment of the broth, such as heating or pH adjustment, can denature and precipitate proteins and other impurities, reducing broth viscosity for easier separation. Filtration or centrifugation is used to remove bacterial cells and solid residues, yielding a clarified fermentation broth.  

2. Extraction Process Optimization  

Appropriate extraction methods, such as ion-exchange resin separation or precipitation, can be selected for L-threonine purification. Ion-exchange resins exploit L-threonine’s charge properties at different pH levels to facilitate separation. Optimizing resin type, loading conditions (e.g., pH, flow rate), and elution parameters enhances adsorption efficiency and purity. Precipitation methods utilize L-threonine’s solubility characteristics under specific conditions. Adding precipitants (e.g., ethanol, acetone) can induce crystallization, with optimized conditions improving precipitation yield.  

3. Refinement and Purification  

The crude L-threonine extract undergoes further refinement using crystallization or chromatographic techniques to achieve higher purity. Optimizing crystallization conditions (e.g., temperature, pH, solvent composition) produces uniform, high-purity L-threonine crystals. Chromatographic separation (e.g., gel filtration, reverse-phase chromatography) further removes residual impurities, yielding high-purity L-threonine for diverse applications.