Semi-continuous biomanufacturing for maximizing the production of complex chemicals and fuels: a case study of amorpha-4,11-diene.

Biomanufacturing is emerging as a key technology for the sustainable production of chemicals, materials, and food ingredients using engineered microbes. However, despite billions of dollars of investment, few processes have been successfully commercialized due to a lack of attention on industrial-scale bioprocess design and innovation. In this study, we address this challenge through the development of a novel semi-continuous bioprocess for the production of the terpene amorpha-4,11-diene (AMD4,11) using engineered Escherichia coli. Using a hydrophilic membrane for product and biomass retention, we successfully decoupled production at low growth rates (~0.01 1/h) and improved reactor productivity up to 166 mg/lReactor h, threefold compared with traditional fed-batch fermentations. When cell recycling was implemented, we showed sustained production at the highest conversion yield and production rate for up to three cycles, demonstrating the robustness of both the strain and the process and highlighting the potential for new bioprocess strategies to improve the economic viability of industrial biomanufacturing.

Enabling commercial success of industrial biotechnology.

Commercial-scale research translation has been muted.

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.