P118: Engineering E. coli for the production of organic acids from glycerol under microaerobic conditions

Glycerol is an attractive feedstock for the microbial synthesis of biochemicals because of its high degree of reduction, abundance, and plummeting prices due to biodiesel production. We previously delineated the pathways for the fermentative use of glycerol by Escherichia coli in minimal salts media under microaerobic conditions. Here, we capitalized on such results to engineer E. coli to produce value-added organic acids (succinate/lactate) that can act as platform chemicals to produce a wide spectrum of products.

Through rational design, succinate production was elevated by 1) blocking pathways for the competing production of lactate (ΔldhA), ethanol (ΔadhE), and acetate (Δpta), and 2) expressing Lactococcus lactis pyruvate carboxylase in E. coli to sequester CO₂ and drive the generation of succinate from pyruvate, circumventing low phosphoenolpyruvate levels. This metabolic engineering strategy coupled cell growth to succinate production because the creation of succinate remained as the primary route of NAD+ regeneration

E. coli was also engineered for the efficient conversion of glycerol to D-lactic acid (D-lactate) (3C), a negligible product in wild-type strains. Engineering a homofermentative D-lactate route was based on 1) overexpressing pathways involved in the conversion of glycerol to glycolytic intermediates and subsequently D-lactate (i.e. GlpK-GlpD and D-lactate dehydrogenase, respectively), 2) disrupting pathways leading to the synthesis of competing products ethanol, acetate, and succinate (ΔpflB, ΔpoxB, ΔfrdA) and 3) blocking the aerobic utilization of D-lactate (Δdld). Further intensive engineering is currently underway for the homofermentative production of L-lactate from glycerol as pure enantiomers are important for downstream applications such as poly(lactic acid).