Conversion of glycerol to pyruvate by Escherichia coli using acetate- and acetate/glucose-limited fed-batch processes

We report the conversion of glycerol to pyruvate by E. coli ALS929 containing knockouts in the genes encoding for phosphoenolpyruvate synthase, lactate dehydrogenase, pyruvate formate lyase, the pyruvate dehydrogenase complex, and pyruvate oxidase. As a result of these knockouts, ALS929 has a growth requirement of acetate for the generation of acetyl CoA. In steady-state chemostat experiments using excess glycerol and limited by acetate, lower growth rates favored the formation of pyruvate from glycerol (0.60 g/g at 0.10 h(-1) versus 0.44 g/g at 0.25 h(-1)), while higher growth rates resulted in the maximum specific glycerol consumption rate (0.85 g/g h at 0.25 h(-1) versus 0.59 g/g h at 0.10 h(-1)). The presence of glucose significantly improved pyruvate productivity and yield from glycerol (0.72 g/g at 0.10 h(-1)). In fed-batch studies using exponential acetate/glucose-limited feeding at a constant growth rate of 0.10 h(-1), the final pyruvate concentration achieved was about 40 g/L in 36 h. A derivative of ALS929 which additionally knocked out methylglyoxal synthase did not further increase pyruvate productivity or yield, indicating that pyruvate formation was not limited by accumulation of methylglyoxal.