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

Femmer C; Bechtold M; Held M; Panke S

doi:10.1016/j.ymben.2020.01.003

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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

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Metabolic engineering. - 2020

High-throughput determination of growth

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.

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