Whole-cell screening of oxidative enzymes using genetically encoded sensors

Abstract

Genetically encoded biosensors enable efficient high-throughput screening of oxidative enzyme libraries.

A Flow Cytometry‐Based Ultrahigh‐Throughput Screening Method for Directed Evolution of Oxidases

“…Another fluorescent reporter system encapsulates single cells carrying active oxidase within a fluorescent hydrogel [16] . However, the above systems either cannot be used in flow cytometry‐based sorting [15b] or require intricate chemosynthetic fluorescent labeling.…”

Enzyme directed evolution using genetically encodable biosensors

“…The best BM3 variant isolated exhibited an 11-fold increase in o-cresol formation over the parent enzyme. 78 Likewise, Siedler et al developed a redox-sensitive GEB using the E. coli transcription factor SoxR. 79 Unlike OxyR, SoxR senses cellular redox balance via oxidation of its iron-sulfur cluster and is thought to report on the intracellular ratio of NADPH/NADP + .…”

“…An example of this is DmpR, which was used to detect phenol, o-cresol, or 2-aminophenol to evolve three enzymes with distinctive mechanisms. 55,78,80 Thus, natural promiscuity can be employed in creative ways to obtain more mileage from a single GEB. Additionally, indirect screening strategies that detect by-product formation or cofactor utilization could allow a single GEB to be used to evolve multiple target enzymes, or even an enzyme class that catalyzes a common type of reaction.…”

“…Kardashliev et al used the E. coli oxidative stress response regulator OxyR to evolve P. chrysosporium alcohol oxidase (AOX) for improved glycerol oxidation to d -glyceraldehyde (Table 1 entry 16). 78 H 2 O 2 produced as a byproduct of this reaction can be directly sensed by OxyR to activate transcription from P oxyS . Notably, H 2 O 2 poses little threat of crosstalk because it readily reacts with cellular components and is actively degraded by intracellular catalases and peroxidases before it can diffuse out of the cell.…”

“…Encouraged by these results, the authors also applied this biosensor to B. megaterium P450 monooxygenase BM3 hydroxylation of toluene to o -cresol (Table 1 entry 17). 78 Although not directly formed from toluene oxidation, H 2 O 2 generated by P450 “uncoupling” could be used as a general indicator of P450 activity. Here, the OxyR GEB was used in concert with a second GEB that employed the o -cresol sensing activator DmpR to regulate gfp transcription from its cognate promoter, Po.…”