(75b) Optogenetic Control of Gene Expression for Metabolic Engineering

Metabolic engineering to produce chemicals faces the following challenges: (1) biosynthetic pathways of interest frequently compete with essential metabolic pathways, (2) desired compounds are often toxic to the cells engineered to produce them, and (3) imbalances in enzyme levels can greatly affect the performance of engineered metabolic pathways. To address these challenges, we introduce a new technological platform that utilizes light-sensitive proteins to control gene expression, and achieve unprecedented control over engineered metabolic pathways. This platform includes optogenetic circuits to control metabolic valves for essential pathways that compete with pathways of interest, as well as biosynthetic pathways of toxic compounds. To avoid light shading effects of high cell density fermentations that would limit the applicability of optogenetics to metabolic engineering, we have developed systems that respond to very low light levels, as well as inverter circuits that repress toxic pathways in the light, during the growth phase (when cell densities are low), and induce them in the dark during the production phase (when cell densities are high). There are many advantages of using light to control gene expression for metabolic engineering, compared to established methods that use chemical inducers. Light is inexpensive relative to many chemical inducers, non-toxic, and less likely to produce off-target effects. Also, our optogenetic circuits are tunable, and reversible, which allows for real-time corrections that are not available to chemical inducers. Furthermore, this technology enables a periodic operation of fermentations, using light pulses to maximize product formation. With this new platform, we have surpassed the maximum yields, titers, and productivities of desired products reported in the literature. Our approach significantly departs from existing strategies to control gene expression for metabolic engineering, and raises the possibility of optimizing engineered strains by simultaneously balancing biosynthetic pathways for products of interest, while keeping in check essential pathways that compete with product formation, and rates of toxic product formation.