UC Berkeley

The field of green chemistry has been critical in helping reduce hazardous waste production in the chemical industry. An increasingly utilized approach in reducing waste has been the use of biocatalysts, enzymes, in industrial chemical processes. In the majority of applications of biocatalysts, single enzymes are used, however there remains the potential for further improving these syntheses using metabolically engineered organisms to synthesize small molecules in consolidated processes from renewable sugars. Here we present two strategies for improving the implementation of genetically engineered microorganisms for these green chemistry solutions. In the first strategy, we use combinatorial expression optimization to simultaneously vary expression of eight enzymes involved in the fungal xylose utilization pathway in Saccharomyces cerevisiae. We demonstrate how this tool can be used to map optimal expression space, which we apply to understanding how optimal expression changes with varying conditions. We also use this expression mapping to identify rate-controlling activities for subsequent engineering. In the second strategy, we describe the use of ester biochemical protecting groups as a means of controlling product reactivity. We propose protecting the indigo precursor, indoxyl, with either the base-labile acetyl or malonyl protecting groups for use in a green alternative to the current indigo dyeing process. Use of biochemical protecting groups to tailor chemical properties is a promising avenue to developing novel green chemistry applications for current bioproduction challenges.