(281b) Deploying Orthogonal Optogenetic Circuits for Improving Microbial Chemical Production

Metabolic engineering aims to maximize production of valuable compounds by using microbes as biological catalysts. Nevertheless, significant progress is still necessary to drive titers and yields of chemical products towards commercially viable levels. A major hurdle faced in metabolic engineering is that overburdening an organism’s native metabolism with exogenous pathways often leads to growth defects and loss of productivity. These drawbacks may occur due to toxicity of products or pathway intermediates, cofactor imbalance, or siphoning of cellular resources away from essential metabolites. This issue has been mediated through various forms of dynamic control: decoupling growth and production via two-phase fermentations. Current methods of dynamically regulating cellular metabolism typically use chemicals, nutrients, or process conditions like temperature to induce or repress gene expression. Nevertheless, such inducers can have various drawbacks, such as cost, toxicity, and irreversibility.

Our group has explored new modes of dynamic control that allow for rapid and robust regulation of microbial metabolism using optogenetics: light-mediated control of gene expression. Several advantages of optogenetic systems are that light is inexpensive, easily applied, and rapidly transmitted, with minimal toxicity and off-target effects. Reversible and dose-dependent gene expression can also be achieved by varying the exposure time to light, allowing for tremendous temporal control. We have developed several optogenetic circuits that allow for flexible switching between cellular growth and chemical production. By optogenetically regulating the abundances of transcriptional activators and repressors, these circuits are able to induce gene expression in either blue light or darkness. Moreover, by implementing protein pairs that do not interfere with each other’s activities, we have allowed for simultaneous signal amplification and inversion. Our circuits display high expression levels, rapid activation kinetics, and minimal cross-talk. Through co-utilization of these optogenetic amplifier and inverter circuits, we have demonstrated improvements in 2,3-butanediol production over using each circuit individually. We believe that our results vindicate the potential for applying optogenetics towards sustainable and economically feasible production of valuable chemicals.