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Proc Natl Acad Sci U S A. 2015 Jun 9;112(23):7159-64. doi: 10.1073/pnas.1422285112. Epub 2015 May 26.

Dissecting enzyme function with microfluidic-based deep mutational scanning.

Author information

1
Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158.
2
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.

KEYWORDS:

droplet-based microfluidics; high-throughput DNA sequencing; protein engineering

PMID:
26040002
PMCID:
PMC4466731
DOI:
10.1073/pnas.1422285112
[Indexed for MEDLINE]
Free PMC Article

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