L-valine during the fermentation process

In the fermentation of L-valine, feedback inhibition is a key bottleneck restricting efficient product synthesis. Its core mechanism is that the end product, L-valine, inhibits key enzymes in its own synthetic pathway (such as acetohydroxyacid synthase, AHAS) through allosteric effects, leading to blocked metabolic flux. Relieving feedback inhibition through gene editing technology is an important strategy to improve L-valine yield, with specific approaches as follows:

I. Site-directed Mutagenesis of Target Key Enzyme Genes to Relieve Allosteric Inhibition

In the L-valine synthesis pathway, acetohydroxyacid synthase (AHAS) is the first rate-limiting enzyme and the main target of feedback inhibition—high concentrations of L-valine bind to the allosteric site of AHAS, inhibiting its catalytic activity (conversion of pyruvate to acetolactate). Modifying the gene encoding AHAS (e.g., ilvBN) using gene editing technologies (such as CRISPR/Cas9 or site-directed mutagenesis PCR) can disrupt its allosteric site and reduce sensitivity to L-valine.

For example, in Escherichia coli, mutating specific amino acid sites of the ilvBN gene (e.g., positions 48 and 52, such as G48S and A52T) can increase the inhibition constant (Ki) of AHAS for L-valine by 10-50 times. Even when the concentration of L-valine in the fermentation broth reaches 20-30 g/L, the enzyme activity can still be maintained above 80%, significantly promoting the flow of metabolic flux toward product synthesis.

II. Strengthening Branched Metabolic Flux Regulation to Reduce Competitive Feedback Inhibition

L-valine, L-isoleucine, and L-leucine belong to branched-chain amino acids and share part of the synthetic pathway (e.g., the initial step catalyzed by AHAS). All three may exert synergistic feedback inhibition on AHAS. Blocking competitive branched metabolism through gene editing can reduce the accumulation of other branched-chain amino acids, indirectly relieving inhibition on L-valine synthesis.

Knocking out key genes for isoleucine synthesis: For instance, knocking out the ilvA gene encoding threonine deaminase blocks the production of the L-isoleucine precursor (α-ketobutyrate), reducing the inhibition of AHAS by L-isoleucine.

Weakening the leucine synthesis pathway: Using RNA interference or promoter attenuation technology (e.g., replacing the promoter of the leuABCD operon with a weak promoter) reduces the synthesis rate of L-leucine, avoiding its combined inhibition of AHAS activity with L-valine.

These strategies can redirect the branched-chain amino acid metabolic flux toward L-valine, reducing metabolic blockages caused by competitive feedback inhibition.

III. Modifying Transporter Genes to Enhance Product Efflux

High intracellular concentrations of L-valine are a direct trigger for feedback inhibition. Enhancing its extracellular transport capacity through gene editing can reduce intracellular accumulation and alleviate inhibition at the source.

Studies have found that branched-chain amino acid transporters in Escherichia coli (e.g., BrnQ) are responsible for pumping intracellular L-valine out of the cell. Overexpressing the brnQ gene (e.g., driven by strong promoters T7 or lacUV5) or mutating it to improve transport efficiency (e.g., enhancing binding affinity with L-valine) can reduce intracellular L-valine concentration by 30%-50% while increasing product accumulation in the fermentation broth. Additionally, knocking out genes that may mediate L-valine reverse transport (e.g., ygaH) can reduce product reabsorption, further maintaining a low intracellular concentration environment.

IV. Synergistic Editing of Global Metabolic Networks to Optimize Precursor Supply and Energy Balance

Relieving feedback inhibition must be combined with overall optimization of metabolic flux to avoid the effects of insufficient precursors or by-product accumulation offsetting improved enzyme activity.

Strengthening precursor pyruvate supply: Knocking out key genes in the pyruvate decomposition pathway (e.g., ldhA encoding lactate dehydrogenase, ackA encoding acetate kinase) reduces the shunting of pyruvate to lactate and acetate; simultaneously, overexpressing the phosphoenolpyruvate carboxylase gene (ppc) promotes the conversion of glycolytic intermediates to pyruvate, providing sufficient precursors for L-valine synthesis.

Balancing NADH regeneration: L-valine synthesis requires NADH participation. Editing genes related to the oxidative respiratory chain (e.g., ndh, encoding NADH dehydrogenase) optimizes NADH regeneration efficiency, avoiding metabolic flux stagnation due to coenzyme deficiency.

Relieving feedback inhibition in L-valine fermentation requires multi-dimensional synergy through gene editing: "targeted enzyme activity enhancement - enhanced product efflux - metabolic flux optimization". Site-directed mutagenesis relieves allosteric inhibition of key enzymes, transporter modification reduces intracellular accumulation, branched pathway knockout minimizes competitive inhibition, and optimization of precursor and coenzyme networks further enhances efficiency. Currently, multi-gene synergistic editing using tools like CRISPR-Cas9 has enabled Escherichia coli fermentation to achieve L-valine yields exceeding 80 g/L, providing core technical support for industrial production.