TOWARD A MICROFLUIDIC PLATFORM FOR CELL FREE SYNTHESIS OF MORPHINE DEGRADATION PATHWAY ENZYMES

Abstract

Opioids are a class of drugs highly valued for their potent analgesic properties; however, they are also highly addictive and cause severe side effects. Alternative manufacturing methods may be the isruption needed to overcome the cost and availability issues associated with naloxone, the opioid overdose antidote. Toward this need, we propose the development of a biosynthetic route of naloxone production with morphine as the precursor. In this work, we produced morphine dehydrogenase - an enzyme that catalyzes the oxidation of morphine to morphinone - by cell free protein synthesis (CFPS), taking advantage of the speed of CFPS compared to cell-based culture. To supplement our constraint-based metabolic modeling and detect compounds of interest with rigor, we developed a method for absolute quantification of metabolites and nucleotide sugars by reversed-phase liquid chromatography-mass spectrometry. Finally, we have characterized and begun modeling a microreactor with an eye toward its future use as our main bioprocessing platform. Taken together, we have developed a rapid protocol for expression of what could serve as the first enzyme in a novel biosynthetic pathway and laid the groundwork for reactor optimization.