Artificial metalloenzymes (ArMs) catalyzing new-to-nature reactions under mild conditions could play an important role in the transition to a sustainable, circular economy. While ArMs have been created for a variety of bioorthogonal transformations, attempts at optimizing their performance by enzyme engineering have been case-specific and resulted only in modest improvements. To realize the full potential of ArMs, methods that enable the rapid discovery of highly active ArM variants for any reaction of interest are required. Here, we introduce a broadly applicable, automation-compatible ArM engineering platform, which relies on periplasmic compartmentalization in Escherichia coli to rapidly and reliably identify improved ArM variants based on the biotin-streptavidin technology. We systematically assess 400 ArM mutants for five bioorthogonal transformations involving different metal cofactors, reaction mechanisms and substrate-product pairs, including novel ArMs for gold-catalyzed hydroamination and hydroarylation. The achieved activity enhancements of up to fifteen-fold over wild type highlight the potential of the systematic approach to ArM engineering. We further capitalize on the sequence-activity data to suggest and validate smart strategies for future screening campaigns. This systematic, multi-reaction study has important implications for the development of highly active ArMs for novel applications in biocatalysis and synthetic biology. ### Competing Interest Statement The authors have declared no competing interest.
- Downloaded 861 times
- Download rankings, all-time:
- Site-wide: 29,482
- In synthetic biology: 377
- Year to date:
- Site-wide: 42,347
- Since beginning of last month:
- Site-wide: 48,949
Downloads over time
Distribution of downloads per paper, site-wide
- 27 Nov 2020: The website and API now include results pulled from medRxiv as well as bioRxiv.
- 18 Dec 2019: We're pleased to announce PanLingua, a new tool that enables you to search for machine-translated bioRxiv preprints using more than 100 different languages.
- 21 May 2019: PLOS Biology has published a community page about Rxivist.org and its design.
- 10 May 2019: The paper analyzing the Rxivist dataset has been published at eLife.
- 1 Mar 2019: We now have summary statistics about bioRxiv downloads and submissions.
- 8 Feb 2019: Data from Altmetric is now available on the Rxivist details page for every preprint. Look for the "donut" under the download metrics.
- 30 Jan 2019: preLights has featured the Rxivist preprint and written about our findings.
- 22 Jan 2019: Nature just published an article about Rxivist and our data.
- 13 Jan 2019: The Rxivist preprint is live!