Los Alamos National Laboratory,
Keywords: biofuel, enzyme
Summary: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.