(390f) Layered Regulation Strategies to Improve D-Glucaric Acid Production

Microbial synthesis can be a sustainable method of chemical and material production. In order for microbial production to become economically viable, the titer, yield, and selectivity frequently must be improved through strain engineering. One strain engineering technique for pathway improvement is dynamic regulation, where cells are designed to sense extracellular or intracellular changes and alter their behavior accordingly. Dynamic regulation has been used to redirect flux from byproducts to products, balance the usage of key intermediates, and minimize production of toxic intermediates. We applied dynamic regulation to the production of D-glucaric acid, a precursor to nylons and detergents. Two orthogonal, autonomous, and tunable dynamic regulation systems were engineered into the D-glucaric acid pathway, resulting in the highest glucaric acid titer reported thus far in the Escherichia coli K-12 strains. The first regulation scheme is a pathway-independent quorum sensing system that knocks down the glycolytic enzyme PfkA at a critical cell density, switching the culture from growth to production mode. The second regulation system controls expression of a downstream enzyme on an as-needed basis – if sufficient levels of a key intermediate are detected via a biosensor, the enzyme is transcribed. While the two regulation strategies independently increase titers 2-to-3-fold, the combination of the two systems achieves a 10-fold increase in titer over the un-regulated control. This work demonstrates the use of synthetic biology tools to implement layered dynamic regulation, a promising strain engineering strategy for improving microbial production.