Expression, Purification, and Expression of Ring-Cleaving Dioxygenases for Synthesis and Bioremediation

Abstract

The development of sustainable routes to organic building blocks is a critical endeavor for reducing the environmental impact of traditional organic chemical synthesis. Biocatalysts offer an alternative method to facilitate sustainable synthesis, as they perform highly selective reactions at an increased rate. Ring-cleaving dioxygenases (RCDs) are a class of enzymes responsible for selectively breaking open the ring of benzene derivatives to provide a carbon source for microorganisms in bioremediation. To access the biocatalysts, many microbiology methods were utilized. The E. coli cells were transformed to contain the desired gene, the cells were then grown until there optical density was at the ideal value for cell viability (0.6-0.8) and induced with IPTG to facilitate protein expression. After heterologous expression, the enzyme was purified to homogeneity by immobilized metal affinity chromatography. We continue to analyze RCD types (type I, II, and III) through endpoint screening and product isolation using various substituted catechols. We envision that this approach to muconic acid synthesis will contribute to ongoing efforts to streamline synthesis of these important organic building blocks and reduce the usage of fossil fuels for organic synthesis.