Ultrahigh‐Throughput Screening of an Artificial Metalloenzyme using Double Emulsions

Abstract 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.


Experimental Section
Fluorochem. Tris-(acetonitrile)-cyclopentadienylruthenium(II)-hexafluorophosphate was purchased from Sigma Aldrich. Water was 23 purified with a Milli-Q-system (Millipore). Antibiotics were purchased from Applichem GmbH. All enzymes, the Monarch plasmid 24 extraction kit, the Monarch PCR and DNA clean-up kit, and the Gibson assembly master mix, were purchased from New England 25 BioLabs. Magnetic beads used for DNA purification were AMPure XP purchased from Beckman-Coulter Life Sciences. Sodium dodecyl 26 sulfate (SDS) was bought from abcr. Poly(dimethylsiloxane) (PDMS, Sylgard 184) was purchased from Dow Corning. Hydrofluoroether 27 (HFE 7500) with 5% 008-FluoroSurfactant was purchased from RAN Biotechnologies, Inc. All supplies for Nanopore sequencing were 28 purchased from Oxford Nanopore Technologies.

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The bacterial strain Top10 DE3 was designed and generously provided by the Panke laboratory [1] . Chemically competent cells 30 (prepared according to the RbCl-method following the Hanahan protocol) and electrocompetent cells bearing the mNectarine plasmid 31 were prepared in the laboratory [2] .

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The pET30b vector ( Supplementary Fig. 4) was used for the generation of the mutant library. The pUA66 vector (Supplementary Fig.   33 5) was used for mNectarine [3] . Primers for NGS were designed individually and were synthesized at IDT technologies or Microsynth AG 34 (Supplementary Table 5   49 400-variants library: pET30b_Sav_library. A glycerol stock containing E. coli cells with the 400-mutant library at positions S112 and 50 K121 was grown overnight and the plasmid extracted. The purified plasmid (~100 ng) was transformed into electrocompetent 51 Top10(DE3) cells (50 µL), containing the plasmid pSC101 with mNectarine encoded. Electroshock was applied using the MicroPulser 52 electroporator by Bio-Rad Laboratories, Inc. Immediately after the electroshock, SOC-medium (450 µL) was added, the reaction 53 transferred to a sterile Eppendorf tube and incubated at 37 °C for 40-60 min. The transformation was split into four equal parts and 54 plated on 12 cm × 12 cm LB-agar plates supplemented with kanamycin and chloramphenicol and incubated overnight at 37 °C. All 55 plates were scraped by the addition of LB-medium (2 mL per plate) and the combined cell suspension was aliquoted. 100 µL cell 56 suspension was mixed with 100 µL of a glycerol stock solution (50%) to obtain 20 aliquots with a ~25 % final glycerol concentration.

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The glycerol stocks were immediately frozen in liquid N2 and finally stored at −80 °C. One whole such aliquot was used for the inoculation 58 of cultures for the screening assay.

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General protocol for Sav expression for the screening. A preculture of LB medium (5 mL) supplemented with chloramphenicol (32 72 mg/mL) and kanamycin (50 mg/mL) was inoculated with the previously prepared glycerol stock of the library of interest and incubated 73 for 8 h at 37 °C and 300 rpm. A culture of LB medium (25 mL) supplemented with chloramphenicol (32 mg/mL) and kanamycin (50 74 mg/mL) in a shaking flask (250 mL) was inoculated with the preculture to a starting OD600 = 0.05 and incubated for ~1-2 h at 37 °C and 75 300 rpm (until an OD600 = 0.5-0.8 was reached). Sav expression was induced by the addition of IPTG (50 µM final concentration) and 76 expression was performed overnight at 25 °C and 300 rpm. One sample (1 mL) of cell culture with an OD600 = 0.20 was prepared and 77 centrifuged (5 min; 17000 g). The supernatant was discarded and the pellet was resuspended in PBS (990 µL, pH 7.4). To this cell 78 suspension, the cofactor 1 stock solution (10 µL, 1 mM, 10 µM final concentration) was added to afford the cell-cofactor mixture for the 79 droplet production.

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Microfluidic platform fabrication. The devices were produced according to a protocol described previously [7] . Briefly, the microfluidic 82 chips were produced using a SU-8 master mold on which a mixture of PDMS and curing agent (ratio 10:1) was poured. The wafer was 83 cured at 80 °C for 3 h. After punching inlets and outlets with a biopsy puncher (diameter 0.5 mm), the chips were plasma bonded to 84 PDMS-coated glass slides. The device has four inlets for the outer aqueous phase (OA), the oil phase (OP) and the inner aqueous 85 phases (IA1 and IA2), and one outlet. To allow for DE formation, a 2.5 % poly(vinyl alcohol) (PVA) solution was used to coat the OA 86 and outlet channels with a hydrophilic layer, according to a protocol previously described by Deshpande et al [8] .

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Processing of NGS data: We used in-house bash and R scripts to analyze the NGS data. Fastq files containing the forward and 117 reverse reads were obtained following NGS. The reads were extracted and paired. The reads were filtered using the 24-bp fixed region 118 located between position 112 and position 121 ( Supplementary Fig. 4), allowing for a maximum of three mismatches. The target 119 fragments were attributed to each sample using their unique barcode (Supplementary Table 5