L-valine is produced by Escherichia coli fermentation

The optimization of the fed-batch process for L-valine production via Escherichia coli fermentation focuses on dynamically regulating the supply of nutrients such as carbon sources, nitrogen sources, and trace elements, combined with precise control of fermentation environment parameters, to achieve synergistic and efficient cell growth and product synthesis. The specific optimization directions are as follows:

I. Optimization of Feeding Strategies

The core of the fed-batch process is to dynamically supplement substrates according to the metabolic needs of different fermentation stages, avoiding substrate excess inhibition or nutrient deficiency.

Carbon source feeding: Common carbon sources include glucose, sucrose, or pyruvate. In the initial stage, the carbon source concentration needs to be maintained at a low level (e.g., 5-10 g/L) to avoid the "glucose effect" (inhibition of the activity of enzymes related to valine synthesis) caused by excessive glucose. When the carbon source is consumed to 2-3 g/L, exponential feeding or feedback feeding (based on the sudden increase signal of dissolved oxygen) is used for supplementation, controlling the carbon source concentration in the middle and late stages of fermentation at 1-5 g/L. This not only ensures the metabolic activity of bacteria but also provides sufficient precursors (pyruvate, α-ketoisovalerate) for valine synthesis. Some studies have adjusted the metabolic flux toward valine synthesis and reduced the accumulation of by-products (such as lactic acid and acetic acid) by supplementing mixed carbon sources (e.g., glucose and glycerol).

Nitrogen source feeding: Nitrogen sources (such as ammonia water, ammonium sulfate, and yeast extract) not only provide nitrogen for bacteria but also affect the pH of the fermentation broth. The initial nitrogen source concentration needs to be moderate; excessive nitrogen can lead to excessive bacterial growth and consumption of carbon sources. In the middle stage of fermentation, ammonia water or ammonium sulfate is supplemented to maintain the nitrogen source concentration at 0.5-2 g/L, and ammonia water is used to adjust the pH to 6.5-7.5 (the suitable pH range for key enzymes in valine synthesis). Studies have shown that staged supplementation of organic nitrogen sources (such as yeast extract) can promote the synthesis of enzymes related to valine synthesis (e.g., acetohydroxy acid synthase) in bacteria, thereby increasing product yield.

Precursor and cofactor feeding: The key precursors for valine synthesis are pyruvate and α-ketoisovalerate. Supplementation of a small amount of pyruvate (0.5-1 g/L) can directly promote the metabolic flux toward product accumulation. In addition, cofactors such as biotin and vitamin B1 are involved in enzyme activation. Appropriate supplementation of biotin (5-10 μg/L) in the middle and late stages of fermentation can enhance bacterial activity and reduce metabolic blockages.

II. Regulation of Fermentation Environment Parameters

Temperature control: A staged temperature control strategy is adopted. In the early stage (0-12 h), the temperature is controlled at 37℃ to promote rapid bacterial growth; in the middle and late stages (after 12 h), it is reduced to 30-34℃ to inhibit excessive bacterial reproduction, avoid enzyme activity decline caused by high temperature, and facilitate valine synthesis.

Dissolved oxygen control: Dissolved oxygen levels affect respiratory metabolism and product synthesis. The dissolved oxygen needs to be maintained at 20%-40% by adjusting the stirring speed (300-600 rpm) and aeration rate (1-2 vvm). Too low dissolved oxygen will cause bacterial metabolism to shift to anaerobic respiration, accumulating by-products such as lactic acid; too high dissolved oxygen may inhibit product synthesis due to oxidative stress. In the late stage of fermentation, appropriately reducing the dissolved oxygen to 15%-20% can reduce energy consumption and promote valine secretion into the extracellular environment.

pH control: In addition to adjusting pH through nitrogen source feeding, buffer solutions (such as phosphate buffer) can be used to stabilize the pH of the fermentation broth. Studies have found that pH fluctuations exceeding 0.5 units will significantly reduce the activity of valine synthetase. Therefore, real-time monitoring and feedback regulation are required to stabilize the pH at 6.8-7.2.

III. Mitigation of By-Product Inhibition

By-products such as acetic acid and lactic acid are easily accumulated during Escherichia coli fermentation. When their concentration exceeds 5 g/L, they will inhibit bacterial growth and valine synthesis. Optimizing the feeding strategy is the key to alleviating by-products: controlling the carbon source feeding rate to avoid "overflow metabolism" of bacteria in a high carbon source environment; supplementing a small amount of citrate (1-2 g/L) in the middle stage of fermentation can consume excess pyruvate through the tricarboxylic acid cycle, reducing by-product generation; in addition, screening Escherichia coli mutants resistant to high concentrations of by-products and combining with feeding processes can further increase valine yield.

IV. Effects and Directions of Process Optimization

Through the synergistic optimization of the above feeding strategies and environmental parameters, the yield of L-valine produced by Escherichia coli fermentation can be increased from the initial 10-20 g/L to 40-60 g/L, and the conversion rate (product/carbon source) can be increased to 0.25-0.35 g/g. Future research will focus on combining metabolic engineering to modify strains (such as knocking out by-product synthesis genes and enhancing valine transporter expression) with feeding processes to further improve productivity and product purity, promoting industrial application.