Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Enzymatic Cascades and Directed Evolution

Liang, Alexandria Deliz and Serrano-Plana, Joan and Peterson, Ryan L. and Ward, Thomas R.. (2019) Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Enzymatic Cascades and Directed Evolution. Accounts of Chemical Research, 52 (3). pp. 585-595.

PDF - Published Version

Official URL: https://edoc.unibas.ch/70592/

Downloads: Statistics Overview


Artificial metalloenzymes (ArMs) result from anchoring a metal-containing moiety within a macro molecular scaffold (protein or oligonucleotide). The resulting hybrid catalyst combines attractive features of both homogeneous catalysts and enzymes. This strategy includes the possibility of optimizing the reaction by both chemical (catalyst design) and genetic means leading to achievement of a novel degree of (enantio)selectivity, broadening of the substrate scope, or increased activity, among others. In the past 20 years, the Ward group has exploited, among others, the biotin (strept)avidin technology to localize a catalytic moiety within a well-defined protein environment. Streptavidin has proven versatile for the implementation of ArMs as it offers the following features: (i) it is an extremely robust protein scaffold, amenable to extensive genetic manipulation and mishandling, (ii) it can be expressed in E. coli to very high titers (up to >8 g.L-1 in fed-batch cultures), and (iii) the cavity surrounding the biotinylated cofactor is commensurate with the size of a typical metal-catalyzed transition state. Relying on a chemogenetic optimization strategy, varying the orientation and the nature of the biotinylated cofactor within genetically engineered streptavidin, 12 reactions have been reported by the Ward group thus far. Recent efforts within our group have focused on extending the ArM technology to create complex systems for integration into biological cascade reactions and in vivo. With the long-term goal of complementing in vivo natural enzymes with ArMs, we summarize herein three complementary research lines: (i)With the aim of mimicking complex cross-regulation mechanisms prevalent in metabolism, we have engineered enzyme cascades, including cross-regulated reactions, that rely on ArMs. These efforts highlight the remarkable (bio)compatibility and complementarity of ArMs with natural enzymes. (ii) Additionally, multiple-turnover catalysis in the cytoplasm of aerobic organisms was achieved with ArMs that are compatible with a glutathione-rich environment. This feat is demonstrated in HEK-293T cells that are engineered with a gene switch that is upregulated by an ArM equipped with a cell penetrating module. (iii) Finally, ArMs offer the fascinating prospect of "endowing organometallic chemistry with a genetic memory." With this goal in mind, we have identified E. coli's periplasmic space and surface display to compartmentalize an ArM, while maintaining the critical phenotype genotype linkage. This strategy offers a straightforward means to optimize by directed evolution the catalytic performance of ArMs. Five reactions have been optimized following these compartmentalization strategies: ruthenium-catalyzed olefin metathesis, ruthenium-catalyzed deallylation, iridium-catalyzed transfer hydrogenation, dirhodium-catalyzed cyclopropanation and carbene insertion in C H bonds. Importantly, >100 turnovers were achieved with ArMs in E. coli whole cells, highlighting the multiple turnover catalytic nature of these systems.
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Bioanorganische Chemie (Ward)
UniBasel Contributors:Ward, Thomas R. R. and Liang, Alexandria Deliz and Serrano Plana, Joan and Peterson, Ryan
Item Type:Article, refereed
Article Subtype:Further Journal Contribution
Publisher:American Chemical Society
Note:Publication type according to Uni Basel Research Database: Journal item
Related URLs:
Identification Number:
edoc DOI:
Last Modified:07 Apr 2021 13:19
Deposited On:23 May 2019 13:13

Repository Staff Only: item control page