METABOLIC ENGINEERING OF ESCHERICHIA-COLI TO ENHANCE RECOMBINANT PROTEIN-PRODUCTION THROUGH ACETATE REDUCTION

Genetic and metabolic engineering provide powerful and effective tools for the systematic manipulation and fine tuning of cellular metabolic activities. In this study, successful application of such techniques to enhance recombinant protein production by reducing acetate accumula tion in Escherichia coli is presented. The alsS gene from Bacillus sub tilis encoding the enzyme acetolactate synthase was introduced into E. coli cells using a multicopy plasmid. This newly introduced heterolog ous enzyme modifies the glycolytic fluxes by redirecting excess pyruva te away from acetate to acetolactate. Acetolactate is then converted t o a nonacidic and less harmful byproduct acetoin, which appears in the broth. Furthermore, comparative fermentation studies show that the re duction in acetate accumulation leads to a significant improvement of recombinant protein production. The expression of a model recombinant CadA/beta-galactosidase fusion protein, under the control of a strong pH-regulated promoter, was found to increase by about 60% for the spec ific protein activity (to a level of 30% of total cellular protein) an d 50% in terms of the volumetric activity in a batch fermenter. In fed -batch cultivation, the engineered strain achieved a volumetric recomb inant protein yield of 1.6 million units/mL (about 1.1 g/L of beta-gal actosidase), which represented a 220% enhancement over the control str ain. In the meantime, acetate excretion was maintained below 20 mM com pared with 80 mM for the control, and the final cell density was impro ved by 35%.