Concentration Recognition-Based Auto-Dynamic Regulation System (CRUISE) Enabling Efficient Production of Higher Alcohols

Microbial factories lacking the ability of dynamically regulating the pathway enzymes overexpression, according to in situ metabolite concentrations, are suboptimal, especially when the metabolic intermediates are competed by growth and chemical production. The production of higher alcohols (HAs), which hijacks the amino acids (AAs) from protein biosynthesis, minimizes the intracellular concentration of AAs and thus inhibits the host growth. To balance the resource allocation and maintain stable AA flux, this work utilizes AA-responsive transcriptional attenuator ivbL and HA-responsive transcriptional activator BmoR to establish a concentration recognition-based auto-dynamic regulation system (CRUISE). This system ultimately maintains the intracellular homeostasis of AA and maximizes the production of HA. It is demonstrated that ivbL-driven enzymes overexpression can dynamically regulate the AA-to-HA conversion while BmoR-driven enzymes overexpression can accelerate the AA biosynthesis during the HA production in a feedback activation mode. The AA flux in biosynthesis and conversion pathways is balanced via the intracellular AA concentration, which is vice versa stabilized by the competition between AA biosynthesis and conversion. The CRUISE, further aided by scaffold-based self-assembly, enables 40.4 g L⁻¹ of isobutanol production in a bioreactor. Taken together, CRUISE realizes robust HA production and sheds new light on the dynamic flux control during the process of chemical production.