pH-modulated soluble expression of alcohol dehydrogenases in Escherichia coli using adaptive laboratory evolution

Industrial production of alcohol dehydrogenases (ADHs) is severely hampered by their propensity to form insoluble inclusion bodies during recombinant expression. This work achieves soluble expression of such historically aggregate-prone enzymes in Escherichia coli by addressing the fundamental physicochemical incompatibilities between recombinant proteins and their host environment. Elevating the expression medium to pH 9 and engineering E. coli to adapt to high- pH improved catalytically active soluble yields for industrially significant Rhodococcus ADH-A and ADH-G by 18.55-fold and 26.59-fold, respectively. The evolved E. coli overcame alkaline stress by optimizing energy-intensive metabolic pathways for cytoplasmic pH homeostasis rather than by modulating ribosomal machinery and translation. Seven key mutations acquired during adaptive evolution conferred strategic modifications to cell envelope stress responses, metabolism, and genomic regulation, collectively enhancing fitness. This robust framework proposed for matching the biochemical requirements of heterologous enzymes with host physiology offers a powerful solution for the high-yield production of challenging industrial biocatalysts.