Acetate formation during recombinant protein production in Escherichia coli K-12 with an elevated NAD(H) pool

Acetate formation is a disadvantage in the use of Escherichia coli for recombinant protein production, and many studies have focused on optimizing fermentation processes or altering metabolism to eliminate acetate accumulation. In this study, E. coli MEC697 (MG1655 nadR nudC mazG) maintained a larger pool of NAD(H) compared to the wild-type control, and also accumulated lower concentrations of acetate when grown in batch culture on glucose. In steady-state cultures, the elevated total NAD(H) found in MEC697 delayed the threshold dilution rate for acetate formation to a growth rate of 0.27 h^-1. Batch and fed-batch processes using MEC697 were examined for the production of β-galactosidase as a model recombinant protein. Fed-batch culture of MEC697/pTrc99A-lacZ compared to MG1655/pTrc99A-lacZ at a growth rate of 0.22 h^-1 showed only a modest increase of protein formation. However, 1 L batch growth of MEC697/pTrc99A-lacZ resulted in 50% lower acetate formation compared to MG1655/pTrc99A-lacZ and a two-fold increase in recombinant protein production.