Impact of stringent stress response and amino acid supplementation on recombinant protein production in Escherichia coli

Abstract

The metabolic response of Escherichia coli BL21 DE3 strains upon amino acid supplementation and stringent stress was examined to assess its effects on growth, protein production, metabolic efficiency, and cellular stress. In the absence of amino acids, E. coli prioritized amino acid synthesis, essential for recombinant protein production. Supplementation led to faster growth, higher cell densities, and reduced acetate accumulation, indicating improved metabolic balance. Proteomic analysis revealed notable changes in protein abundance during the early log and post-induction mid-log phases, with supplemented cultures showing enhanced metabolic capacity and reduced stress levels. Gene expression analysis demonstrated that amino acid availability downregulated stress response genes and upregulated genes associated with nutrient uptake and metabolism, boosting growth and productivity. Specifically, genes lldR, ydgR, tppB, and yhhN were upregulated, while adiA and ydeI were downregulated, reflecting better metabolic conditions. Stringent response analysis showed elevated (p)ppGpp levels and increased activities of SpoT and RelA without supplementation, indicative of nutrient stress. Supplementation lowered these stress markers, fostering a favourable metabolic state. The study underscores the importance of amino acid supplementation for optimizing biomass production and recombinant protein yields, offering insights into E. coli physiology and strategies for metabolic engineering.