Flow cytometric screening of FutA activity was carried out essentially as described before (15, 17), except that the two fluorescent substrates were used. Briefly, we transformed E. coli JM107 LacZ (12, 14) cells with pACKC18-FKP recombinant plasmid encoding for GDP-fucose synthase and then named it as JM107*. Plasmid DNA (pUC18-FutA) encoding the FutA libraries was transformed into JM107* competent cells and used to directly inoculate LB media supplemented with ampicillin and chloramphenicol (100 mg/ml) and grown overnight at 37°C. The cells were then diluted 1:50 in M9 mineral cultured media and grown at 37°C with vigorous shaking. When the OD600 (optical density at 600 nm) reached 0.5 to 0.7, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the cultures were transferred to 20°C and grew overnight. Cells were spun down (2 ml) and resuspended in 50 μl of M9 media supplemented with 1.5 mM fucose (RayGood Biotech, Shanghai, China), 0.5 mM concentrations of fluorescently labeled substrates. Following 30 min of incubation, cells were spun down, and the excess acceptor sugars were removed by washing the cells with LB media and PBS (pH 7.4).

The cells were diluted with PBS to obtain a flow cytometric event rate of ~6000/s in a FACSAria II flow cytometer (Becton Dickinson) using PBS as the sheath fluid. The threshold for event detection was set to forward and side scattering. The average sort rate was ~6000 events/s, using a 85-μm nozzle, exciting argon ion (508 nm) and 405-nm lasers, and measuring emissions passing the 515-nm (fluorescein isothiocyanate) band-pass filter for the bodipy emission and the 450-nm (violet 1) filter for the coumarin emission. The detection threshold was set at forward scatter (FSC) > 10,000 and side scatter (SSC) > 10,000. Cells were sorted into 1.5-ml tubes and centrifuged at 14,000g for 30 min. Genes from positive cells were amplified using the FutA-F and FutA-R primers (table S4) with Proofast Super-Fidelity DNA polymerase (ATG Biotech). The 50 μl of reaction mixtures contained ~11 μl of water, 25 μl of Proofast buffer (2×), 1 μl of 10 mM dNTPs (deoxynucleoside triphosphates), 10 μl of sorted cells, 20 μM primers mix (1 μl each), and 1 μl of Proofast Super-Fidelity DNA polymerase using the following PCR thermocycling profile: 95°C for 3 min; 40 cycles of 95°C for 15 s, 55°C for 15 s, 72°C for 90 s, and 72°C for 10 min. The PCR product was digested and ligated into the pUC18 vector using HindIII and EcoRI restriction sites, and the ligation mixture was electroporated into E. coli 10G elite cells (Lucigen) according to the manufacturer’s protocol. Extracted plasmids were then electroporated into E. coli JM107* cells for the next round of FACS enrichment. FACS data were processed using FlowJo software 10 (TreeStar).

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