Development of a Microfluidics-Based Screening Assay for the High-Throughput Directed Evolution of Artificial Metalloenzymes

The present PhD thesis summarizes the scientific work conducted in the research group of Prof. Dr. Thomas R. Ward during the years 2016–2021. Research in the Ward group is focused on the development and optimization of artificial metalloenzymes with non-natural activities. These hybrid catalysts, resulting from an incorporation of a metal–containing cofactor within a protein or DNA scaffold, and can be optimized by either chemical or genetic means. The main part of this thesis deals with the genetic optimization of such systems and the development of higher throughput screening assays to facilitate the process. First attempts dealt with the development of a selection-based assay relying on the Carroll rearrangement (Chapter 2.6). Following, more high-throughput assays such as screening of cells relying on a fluorescent reporter protein (Chapter 3) or the screening of activity by an agar plate screening assay were pursued (Chapter 4.2). The main part of the thesis focuses on the method development of an ultrahigh-throughput screening platform for the in vivo directed evolution of artificial metalloenzymes using droplet microfluidics. The combination of ArMs and droplet microfluidics, can be a powerful tool for propelling directed evolution-based research forward. Systematic and high-throughput screening of ArMs in vivo using double emulsions could allow the screening of a much bigger sequence space, which is, to date, challenging. Identifying cooperative effects to improve catalysis or even remodelling whole enzymes to achieve new-to-nature reactivities are only two potential examples. Reactions based on ArMs could ultimately provide aqueous, environmentally friendly reaction pathways for industrial applications. Additionally, such big data sets could also be used as an input for machine learning applications, to further study active site plasticity, reaction pathways, or even protein-folding mechanisms. The developed method was then applied to libraries of different types and sizes, and recent findings of these screenings are highlighted in the fourth chapter. During the time in the research group of Prof. Dr. Ward, a deeper knowledge in molecular biology, especially library design, high-throughput screening using different approaches, microfluidic method development and fluorescence activated cell sorting (FACS), and the use of different sequencing techniques was garnered

Ultrahigh‐Throughput Screening of an Artificial Metalloenzyme using Double Emulsions

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols

Ultrahigh-Throughput Screening of an Artificial Metalloenzyme using Double Emulsions

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage throughout optimization, enabling reliable identification of improved mutants. The full potential of droplet microfluidics remains unexplored, however, as droplet sorting often relies on low-throughput, custom-made devices that typically only allow simultaneous analysis of two parameters. Here, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially-available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of allocprotected aminocoumarin. The most active variants, identified by next generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of enzymes and will significantly expand the accessibility of ultrahighthroughput DE screening protocols

An Enantioselective Artificial Suzukiase Based on the Biotin–Streptavidin Technology

Introduction of a biotinylated monophosphine palladium complex within streptavidin affords an enantioselective artificial Suzukiase. Site-directed mutagenesis allowed the optimization of the activity and the enantioselectivity of this artificial metalloenzyme. A variety of atropisomeric biaryls were produced in good yields and up to 90% ee. The hybrid catalyst described herein shows comparable TOF to the previous aqueous-asymmetric Suzuki catalysts, and excellent stability under the reaction conditions to realize higher TON through longer reaction time

E. coli surface display of streptavidin for directed evolution of an allylic deallylase

Artificial metalloenzymes (ArMs hereafter) combine attractive features of both homogeneous catalysts and enzymes and offer the potential to implement new-to-nature reactions in living organisms. Herein we present an E. coli surface display platform for streptavidin (Sav hereafter) relying on an Lpp-OmpA anchor. The system was used for the high throughput screening of a bioorthogonal CpRu-based artificial deallylase (ADAse) that uncages an allylcarbamate-protected aminocoumarin 1. Two rounds of directed evolution afforded the double mutant S112M-K121A that displayed a 36-fold increase in surface activity vs. cellular background and a 5.7-fold increased in vitro activity compared to the wild type enzyme. The crystal structure of the best ADAse reveals the importance of mutation S112M to stabilize the cofactor conformation inside the protein

Towards the Directed Evolution of Artificial Metalloenzymes

Artificial metalloenzymes (ArMs) are a class of enzymes holding great promise. In contrast to natural enzymes, the core of ArMs is a synthetic metallocofactor, with potential for bio-orthogonal reactivity, incorporated within a host protein. Next to chemical optimization of the metallocofactor, genetic optimization of the protein allows the further improvement of the ArM. Genetic optimization through directed evolution requires extensive screening of a large sequence-scape to enable the optimization of a desired phenotype. The process is however mostly limited by the throughput of the tools and methods available for screening. In recent years, versatile methods based on droplet microfluidics have been developed to address the need for higher throughput. This article aims to give an introduction into ArMs and the recent technological developments allowing high-throughput directed evolution of enzymes

Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey

Evolution is essential to the generation of complexity and ultimately life. It relies on the propagation of the properties, traits, and characteristics that allow an organism to survive in a challenging environment. It is evolution that shaped our world over about four billion years by slow and iterative adaptation. While natural evolution based on selection is slow and gradual, directed evolution allows the fast and streamlined optimization of a phenotype under selective conditions. The potential of directed evolution for the discovery and optimization of enzymes is mostly limited by the throughput of the tools and methods available for screening. Over the past twenty years, versatile tools based on droplet microfluidics have been developed to address the need for higher throughput. In this Review, we provide a chronological overview of the intertwined development of microfluidics droplet-based compartmentalization methods and in vivo directed evolution of enzymes.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

Droplet Microfluidics and Directed Evolution of Enzymes: an Intertwined Journey

Evolution is essential to the appearance of complexity and ultimately Life. It relies on the propagation of the properties, traits and characteristics that allow an organism to survive in a challenging environment. It is evolution that shaped our world over about four billion years by slow and iterative adaptation. While natural evolution based on selection is slow and gradual, directed evolution allows the fast and streamlined optimization of a phenotype under selective conditions. The potential of directed evolution for the discovery and optimization of enzymes is mostly limited by the throughput of the tools and methods available for screening. Over the past twenty years, versatile tools based on droplet microfluidics have been developed to address the need for higher throughput. In this review, we provide a chronological overview of the intertwined development of microfluidics droplet-based compartmentalization methods and in vivo directed evolution of enzymes

Directed Evolution of a Surface-Displayed Artificial Allylic Deallylase Relying on a GFP Reporter Protein

Artificial metalloenzymes (ArMs) combine characteristics of both homogeneous catalysts and enzymes. Merging abiotic and biotic features allows for the implementation of new-to-nature reactions in living organisms. Here, we present the directed evolution of an artificial metalloenzyme based on; Escherichia coli; surface-displayed streptavidin (Sav; SD; hereafter). Through the binding of a ruthenium-pianostool cofactor to Sav; SD; , an artificial allylic deallylase (ADAse hereafter) is assembled, which displays catalytic activity toward the deprotection of alloc-protected 3-hydroxyaniline. The uncaged aminophenol acts as a gene switch and triggers the overexpression of a fluorescent green fluorescent protein (GFP) reporter protein. This straightforward readout of ADAse activity allowed the simultaneous saturation mutagenesis of two amino acid residues in Sav near the ruthenium cofactor, expediting the screening of 2762 individual clones. A 1.7-fold increase of; in vivo; activity was observed for Sav; SD; S112T-K121G compared to the wild-type Sav; SD; (wt-Sav; SD; ). Finally, the best performing Sav isoforms were purified and tested; in vitro; (Sav; PP; hereafter). For Sav; PP; S112M-K121A, a total turnover number of 372 was achieved, corresponding to a 5.9-fold increase vs wt-Sav; PP; . To analyze the marked difference in activity observed between the surface-displayed and purified ArMs, the oligomeric state of Sav; SD; was determined. For this purpose, crosslinking experiments of; E. coli; cells overexpressing Sav; SD; were carried out, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The data suggest that Sav; SD; is most likely displayed as a monomer on the surface of; E. coli; . We hypothesize that the difference between the; in vivo; and; in vitro; screening results may reflect the difference in the oligomeric state of Sav; SD; vs soluble Sav; PP; (monomeric vs tetrameric). Accordingly, care should be applied when evolving oligomeric proteins using; E. coli; surface display

Single-Round Remodeling of the Active Site of an Artificial Metalloenzyme using an Ultrahigh-Throughput Double Emulsion Screening Assay

The potential of high-throughput compartmentalization renders droplet microfluidics an attractive tool for directed evolution of enzymes as it permits maintenance of the phenotype-genotype linkage throughout the entire optimization procedure. In particular, water-in-oil-in-water double emulsions droplets (DEs) produced by microfluidics enable the analysis of reaction compartments at ultra-high-throughput using commercially available fluorescence-activated cell sorting (FACS) devices. Here we report a streamlined method applicable for the ultrahigh-throughput screening of an artificial metalloenzyme (ArM), an artificial deallylase (ADAse), in double emulsions. The DE-protocol was validated by screening a four hundred member, double-mutant streptavidin library for the CpRu-catalyzed uncaging of aminocoumarin. The most active variants, identified by next generation sequencing of the sorted DE droplets with highest fluorescent intensity, are in good agreement with 96-well plate screening hits. These findings, thus, pave the way towards the systematic implementation of commercially available FACS for the directed evolution of metalloenzymes making ultrahigh-throughput screening more broadly accessible. The use of microfluidics for the formation of uniform compartments with precise control over reagents and cell encapsulation further facilitates the establishment of highly reliable quantitative assays