1. A paper-based whole-cell screening assay for directed evolution-driven enzyme engineering
- Author
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Xian Yang, Tadesse Fantaye Bogale, Ijaz Gul, Yong Chen, Ruiqin Fang, Hui Gao, Lixia Tang, and Juan Feng
- Subjects
Paper ,Hydrolases ,Computer science ,Halohydrin dehalogenase ,Applied Microbiology and Biotechnology ,Catalysis ,03 medical and health sciences ,Digital image ,Software ,Escherichia coli ,Instrumentation (computer programming) ,Gene Library ,030304 developmental biology ,0303 health sciences ,030306 microbiology ,business.industry ,Reproducibility of Results ,Screening assay ,General Medicine ,Protein engineering ,Paper based ,Directed evolution ,High-Throughput Screening Assays ,Mutagenesis ,Mutation ,Colorimetry ,Smartphone ,Biochemical engineering ,Directed Molecular Evolution ,business ,Biotechnology - Abstract
Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 μl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes.Key Points• A frugal method was developed for directed enzyme evolution.• Mutagenesis libraries were successfully screened on a paper platform.• Smartphone imaging was efficiently used to measure enzyme activities.
- Published
- 2020