Overexpression and process optimization for enhancing L-alanine production in E. coli BL21 (DE3).

The L-alanine boasts a plethora of industrial applications; however, its commercial utilization is hindered by high production costs, scarcity of raw materials, compromised fermentative productivity, and low enantiomeric purity. To address these challenges in amino acid biosynthesis, the present study employed a synthetic metabolic engineering strategy to enhance L-alanine production in the industrially robust Escherichia coli BL21 (DE3) strain. A synthetic alaD gene cassette, computationally designed and cloned into the pUC57 vector, was expressed under the control of a high-efficiency T7 promoter. Initial expression in recombinant E. coli BL21 (DE3) (alaD⁺) under unoptimized conditions resulted in modest L-alanine titres of 54.32 mM (4.84 g/L). However, through statistical optimization using Response Surface Methodology (RSM) and cultivation under oxygen-limited batch fermentation, the L-alanine yield increased markedly to 440.47 mM (39.24 g/L) within 24 h. Given its broad industrial applicability, this biosynthetic approach offers a promising and sustainable alternative to conventional chemical synthesis, positioning recombinant E. coli BL21 (DE3) (alaD⁺) as a viable microbial platform for commercial production of L-alanine.