(624d) Mass Balance Based Semi-Quantification of Adipic Acid Producing Enzyme Cascade

Biological production of adipic acid is a promising alternative to the environmentally challenged petrochemical-based process currently used. Lignin is a promising feedstock for adipic acid production as it can be broken down into the aromatic intermediates that can be used to produce adipic acid. One common pathway for microbial production of adipic acid goes through a catechol intermediate to form muconic acid, which is then reduced to form adipic acid. To produce adipic acid in Escherichia coli, we utilize an enzyme cascade of catechol 1,2-dioxygenase (CatA) and enoate reductase (ER). Optimization of protein expression using synthetic biology allowed us to optimize adipic acid yield. Furthermore, we characterize our enzyme cascade by analyzing its metabolism of different substituted catechols available from lignin pyrolysis. We demonstrate production of substituted dicarboxylic acids that could be used in the production of nylon analogs with tunable properties. To quantify metabolism of different substituted catechols, we developed a semi-quantification method based on the conserved mass balance within our Escherichia coli system. This method allowed us to quantify compounds via liquid chromatography-mass spectroscopy that do not have commercially available standards, and compare conversion of substituted catechols by our adipic acid-producing enzyme cascade.