(317d) Alleviation of Enzyme Product Inhibition By Genetic Biosensor-Based Evolution and Its Application in Enhanced Cis,Cis-Muconic Acid Production in Pseudomonas Putida

Product inhibition of enzymes is a major bottleneck in functionalizing a biosynthetic pathway resulting in low product titers. Alleviation of such a limitation by site directed mutagenesis or screening for one from a library of mutants by traditional methods are often cumbersome. In this study we have developed a genetically encoded biosensor in Pseudomonas putida for 4-hydroxybenzoate – a key intermediate in the shikimate pathway for the production of important industrial precursors. With a surrogate molecule that inhibits the activity of the enzyme but does not affect the biosensor, we have created a library of variants of the enzyme. Using whole cell biosensing and catalysis, we arrived at an evolved variant with reduced product inhibition and improved kinetic parameters. When this evolved enzyme was expressed in the biosynthetic pathway, we observed >140 % improvement in the yields of cis,cis-muconic acid, a bioplastic precursor.