Femmer C Bechtold M Held M Panke S 2020 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. https://sonar.ch/global/documents/185799 eng info:eu-repo/semantics/altIdentifier/doi/10.1016/j.ymben.2020.01.003 info:eu-repo/semantics/altIdentifier/pmid/31926305 Metabolic engineering. - 2020 Amino acid racemase Antimetabolite Directed evolution High-throughput determination of growth Protein engineering In vivo directed enzyme evolution in nanoliter reactors with antimetabolite selection. http://purl.org/coar/resource_type/c_6501