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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Tran, Tuan M. Romero, Philip A. Abate, Adam R. |
| Description | Author Affiliation: Romero PA ( Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158.); Tran TM ( Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158.); Abate AR ( Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158 adam.abate@ucsf.edu.); |
| Abstract | Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space. |
| ISSN | 00278424 |
| e-ISSN | 10916490 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Issue Number | 23 |
| Volume Number | 112 |
| Language | English |
| Publisher | National Academy of Sciences |
| Publisher Date | 2015-06-01 |
| Publisher Place | United States |
| Access Restriction | Open |
| Subject Keyword | Glycoside Hydrolases Metabolism Microfluidic Analytical Techniques Mutation Genetics High-Throughput Nucleotide Sequencing Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Multidisciplinary |
| Content Type | Text |
| Resource Type | Article |
| Subject | Multidisciplinary |
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