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In vivo directed enzyme evolution in nanoliter reactors with antimetabolite selection.
Journal article

In vivo directed enzyme evolution in nanoliter reactors with antimetabolite selection.

  • Femmer C ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058, Basel, Switzerland.
  • Bechtold M ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058, Basel, Switzerland.
  • Held M ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058, Basel, Switzerland.
  • Panke S ETH Zurich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058, Basel, Switzerland. Electronic address: sven.panke@bsse.ethz.ch.
  • 2020-01-12
Published in:
  • Metabolic engineering. - 2020
Amino acid racemase
Antimetabolite
Directed evolution
High-throughput determination of growth
Protein engineering
English Scoring changes in enzyme or pathway performance by their effect on growth behavior is a widely applied strategy for identifying improved biocatalysts. While in directed evolution this strategy is powerful in removing non-functional catalysts in selections, measuring subtle differences in growth behavior remains difficult at high throughput, as it is difficult to focus metabolic control on only one or a few enzymatic steps over the entire process of growth-based discrimination. Here, we demonstrate successful miniaturization of a growth-based directed enzyme evolution process. For cultivation of library clones we employed optically clear gel-like microcarriers of nanoliter volume (NLRs) as reaction vessels and used fluorescence-assisted particle sorting to estimate the growth behavior of each of the gel-embedded clones in a highly parallelized fashion. We demonstrate that the growth behavior correlates with the desired improvements in enzyme performance and that we can fine-tune selection stringency by including an antimetabolite in the assay. As a model enzyme reaction, we improve the racemization of ornithine, a possible starting block for the large-scale synthesis of sulphostin, by a broad-spectrum amino acid racemase and confirm the discriminatory power by showing that even moderately improved enzyme variants can be readily identified.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://sonar.ch/global/documents/185799
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