Kinetic Modeling and Isotopic Investigation of Isobutanol Fermentation by Two Engineered Escherichia coli Strains

Abstract

We constructed an Escherichia coli BL21 strain with the Ehrlich pathway (the low-performance strain for isobutanol production). We also obtained a high isobutanol-producing E. coli strain JCL260 from the James Liao group (University of California). To compare the fermentation performances of the two engineered strains, we employed a general Monod-based model coupled with mixed-growth-associated isobutanol formation kinetics to simulate glucose consumption, biomass growth, and product secretion/loss under different cultivation conditions. On the basis of both kinetic data and additional 13C-isotopic investigation, we found that the low-performance strain demonstrated robust biomass growth in the minimal growth medium (20 g/L glucose), achieving isobutanol production (up to 0.95 g/L). It utilized significant amounts of yeast extract to synthesize isobutanol when it grew in the rich medium. The rich medium also enhanced waste product secretion, and thus reduced the glucose-based isobutanol yield. In contrast, JCL260 had poor biomass growth in the minimal medium due to an inflated Monod constant (KS), while the rich medium greatly promoted both biomass growth and isobutanol productivity (∼60% of the theoretical isobutanol yield). With the optimized keto-acid pathway, JCL260 synthesized isobutanol mostly from glucose even in the presence of sufficient yeast extract. This study not only provided a kinetic model for scaled-up isobutanol fermentation but also offered metabolic insights into the performance trade-off between the two engineered E. coli strains.