A High Throughput Platform for Enzyme Evolution

R. Jha
Los Alamos National Laboratory,
United States

Keywords: biofuel, enzyme


Evolution process for industrial enzymes and strains is often constrained by the limit in screening throughput. To this end, we engineered ‘smart’ microbial cells (SMCs) that can ‘catalyze’ and ‘sense’ a desired conversion reaction where the fluorescence output is linked to the conversion efficiency. Based on the cell fluorescence, SMCs can be screened using flow cytometry or simply on a plate and visualized under an illuminator. The key requirement for SMCs is availability of a sensor which shows high specificity and sensitivity for desired product. Here, I will describe engineering of transcription factors (TFs) for two very similar aromatic molecules, 4-hydroxybenzoate (4HB) and p-nitrophenol (pNP). 4HB is an industrial intermediate for a number of value-added chemicals while pNP is a leaving group in a wide range of hydrolysis reactions that utilizes p-nitrophenyl based laboratory substrates: esterases, lipases, xylanases and glucanases to name a few. Using an Acinetobacter TF, PobR as a scaffold, we created a variant, PobR-DM with enhanced sensitivity and dynamic range for 4HB sensing. Further, homology modeling assisted diversified TF library and selection for pNP sensing, fetched a variant, pNPmut, that responded to pNP in a dose-dependent manner. PobR-DM and pNPmut were specific and did not respond to pNP and 4HB respectively, confirming a specificity switch. Each sensor was applied for enzyme evolution. In one case, pNP sensor was used to enhance the catalytic efficiency of a phosphotriesterase (PON1) with therapeutic and prophylactic application. In another case, 4HB sensor was applied for relieving the product inhibition in an industrial enzyme. The evolved enzyme when used in a production strain for cis,cis muconic acid, resulted in more than twofold increase in the yield. Our technology is generalizable for rapid evolution of other industrial enzymes and strains utilizing a natural or designed TF as a sensor.