Balancing glucose and oxygen uptake rates to enable high amorpha‐4,11‐diene production in Escherichia coli via the methylerythritol phosphate pathway

Amorpha‐4,11‐diene (AMD4,11) is a precursor to artemisinin, a potent antimalarial drug that is traditionally extracted from the shrubs of Artemisia annua. Despite significant prior efforts to produce artemisinin and its precursors through biotechnology, there remains a dire need for more efficient biosynthetic routes for its production. Here, we describe the optimization of key process conditions for an Escherichia coli strain producing AMD4,11 via the native methylerythritol phosphate (MEP) pathway. By studying the interplay between glucose uptake rates and oxygen demand, we were able to identify optimal conditions for increasing carbon flux through the MEP pathway by manipulating the availability of NADPH required for terpenoid production. Installation of an optimal qO2/qglucose led to a 6.7‐fold increase in product titers and a 6.5‐fold increase in carbon yield. We have established that increase in q02/qGluocse negatively impacts MEP pathway flux and hence amorpha‐4,11‐diene (AMD4,11) productivities. Increase in glucose uptake rates leads corresponding rise in oxygen uptake rates, while increasing flux through PDH into the TCA cycle. This shift in carbon flux had the effect of reducing the amount of pyruvate for the MEP pathway. Simultaneously, higher oxygen uptake rates led to a higher utilization of NADH which was partially provided by the transhydrogenase‐mediated conversion of NADPH to NADH. Lower overall availability of NADPH also had the effect of reducing the flux through the MEP pathway and towards amorpha‐4,11‐diene production.