New engineered phenolic biosensors based on the AraC regulatory protein.

Customized transcription factors that control gene expression in response to small molecules can act as endogenous molecular biosensors and are valuable tools for synthetic biology. We previously engineered the Escherichia coli regulatory protein AraC to respond to non-native inducers such as D-arabinose and triacetic acid lactone. Those prior studies involved the construction and screening of individual 4- or 5-site saturation mutagenesis libraries, followed by iterative rounds of positive- and negative fluorescence-activated cell sorting (FACS). Here we describe an improved screening platform and the isolation of several new and potentially useful AraC variants that respond to vanillin and salicylic acid. To increase throughput and reduce total screening time, selection steps were added to the sorting workflow. Two different site-saturation libraries and a random mutagenesis library were pooled together and >108 variants were subjected to iterative FACS and selection in search of variants responding to a panel of compounds. The new phenolic-sensing variants show responses >100-fold over background and are highly specific towards their target compound. The isolation of these variants further demonstrates the potential for engineering the AraC transcriptional regulatory protein for molecular sensing and reporting, and our improved screening system should prove effective in designing similar biosensors.

Cost-effective whole-cell assay for laboratory evolution of hydroxylases in Escherichia coli.

Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and longchain fatty acids at the omega-1, omega-2, and omega-3 positions. A continuous spectrophotometric assay for P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycarboxylic acids and the chromophore p-nitrophenolate was reported recently. However, this pNCA assay procedure contained steps that limited its application in high throughput screening, including expression of P450 BM-3 variant F87A in 4-ml cultures, centrifugation, resuspension of the cell pellet, and cell lysis. We have shown that permeabilization of the outer membrane of Escherichia coli DH5alpha with polymyxin B sulfate, EDTA, polyethylenimine, or sodium hexametaphosphate results in rapid conversion of 12-pNCA. A NADPH-generating system consisting of NADP(+), D/L-isocitric acid, and the D/L-isocitrate dehydrogenase of E. coli DH5alpha reduced the cofactor expense more than 10-fold. By avoiding cell lysis, resuspension, and centrifugation, the high throughput protocol allows screening of thousands of samples per day.