Rational design and screening of L-valine production strains

L-valine is an important essential amino acid, widely used in food, medicine, feed, and other fields. Currently, microbial fermentation is the main method for its production. The following is relevant content about the rational design and screening of L-valine-producing strains:

I. Rational Design

Metabolic pathway optimization: The biosynthetic pathway of L-valine is relatively complex, involving multiple key enzymes. The synthetic pathway can be enhanced by replacing promoters of key genes such as ilvCDE. In Corynebacterium glutamicum, introducing an acetohydroxyacid synthase mutant from industrial L-valine production sources can significantly increase L-valine yield. Meanwhile, pathways competing with its synthesis can be weakened; for example, replacing the start codon of the isocitrate dehydrogenase-encoding gene icd can weaken the tricarboxylic acid cycle, redirecting more metabolic flux toward L-valine synthesis.

Transport system modification: Knocking out genes encoding transport proteins that are unfavorable for L-valine accumulation (e.g., knocking out the brnQ gene in Escherichia coli) can reduce its extracellular export and promote intracellular accumulation. Additionally, overexpressing operons encoding transport proteins (e.g., overexpressing the brnFE operon from Corynebacterium glutamicum) can enhance extracellular secretion of L-valine, reduce intracellular product concentration, relieve feedback inhibition, and thereby increase yield.

Cofactor balance regulation: The synthesis of L-valine involves multiple cofactors. Optimizing cofactor balance pathways by introducing NADH-preferring acetohydroxyacid synthase mutants and heterologous NADH-dependent leucine dehydrogenases can improve L-valine yield. Furthermore, introducing the exogenous Entner-Doudoroff pathway helps enhance cofactor supply and promote L-valine synthesis.

Utilization of transcriptional regulators: Studying transcription factors related to L-valine biosynthesis—for example, in Escherichia coli, overexpressing pdhR and inhibiting rpoS expression—can promote its synthesis. Additionally, genetic circuits based on L-valine biosensors can be established to dynamically inhibit citrate synthase expression, balancing bacterial growth and L-valine production, and improving strain production efficiency.

II. Strain Screening

Traditional mutagenesis screening: Microorganisms such as Corynebacterium glutamicum and Escherichia coli are treated with physical (e.g., ultraviolet rays) or chemical (e.g., nitrosoguanidine) mutagens to induce gene mutations. Then, using screening media combined with methods such as fluorescence-activated cell sorting (FACS), 96-well plate screening, and shake-flask fermentation, strains with increased L-valine yield are selected from a large number of mutants.

Metabolic engineering-based screening: Engineering strains constructed according to rational design ideas need to be evaluated for performance through a series of screening methods. Preliminary screening can first be conducted in small shake flasks by determining L-valine content in fermentation broth to select strains with higher yields. The selected strains are then subjected to fed-batch fermentation in fermenters to further evaluate indicators such as yield, sugar-acid conversion rate, and production intensity, ultimately obtaining high-efficiency strains suitable for industrial production.

Systems biology-based screening: With the development of omics technologies, transcriptomics, proteomics, and metabolomics can be used to comprehensively analyze changes in gene expression, protein levels, and metabolites of strains under different conditions. This helps gain in-depth understanding of the metabolic network and regulatory mechanisms of L-valine synthesis, providing more molecular-level information for strain screening and enabling more precise selection of strains with high synthetic capacity.