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Directed evolution of an α1,3-fucosyltransferase using a single-cell ultrahigh-throughput screening method

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Science Advances  09 Oct 2019:
Vol. 5, no. 10, eaaw8451
DOI: 10.1126/sciadv.aaw8451
  • Fig. 1 Fucosylation reactions catalyzed by fucosylation enzymes.

    (A) Structures of various disease-associated and human milk oligosaccharides. (B) Schematic depiction of fucosylation reactions catalyzed by FucTs and fucosidases. Typically, an FutA catalyzes the l-fucose transfer from a GDP-Fuc donor substrate to a LacNAc acceptor substrate via an α1,3-linkage, forming the Lex. α-l-Fucosidases can also transfer a fucose moiety from the para-nitrophenyl α-l-fucopyranoside (Fuc-α-pNP) to lactose, forming a 3-fucosyllactose.

  • Fig. 2 Scheme for the product entrapment strategy and the established cell-based fucosylation assay for FACS screening.

    (A) Two kinds of fluorescently labeled LacNAc derivatives (1 and 2) were designed and synthesized for cell entrapment analysis. (B) Fluorescently labeled acceptor substrates are transported into the cell via LacY; fucose enters into the cell via a fucosyl transporter (FucP) and was converted into GDP-fucose donor substrate by GDP-fucose synthase (FKP). After incubation and washing, E. coli cells expressing fucosylation enzymes accumulate fluorescent trisaccharide enzyme products, as the LacY transport rate for such products is significantly reduced compared to their disaccharide substrate form. Thus, the fluorescence intensity accumulation inside cells carries information about the catalytic activity of the fucosylation enzymes being assayed/screened. These cells with FutA activity can be further isolated using FACS. (C) Visualization of fluorescence entrapment within FutA(+) and FutA(−) cells under an ultraviolet light. (D) Flow cytometry profiles of FutA(+) and FutA(−) cell fluorescence after 30-min incubation with 1.5 mM fucose, 0.5 mM bodipy-LacNAc, and coumarin-LacNAc, followed by a washing step. Green and blue signals represent cells retaining bodipy and coumarin fluorescently labeled oligosaccharides, respectively.

  • Fig. 3 Analysis of FACS-based screening efficiency by competitive allele-specific TaqMan PCR.

    (A) The RBS-ATG spacing technique was used to create two populations of cells: (i) normal FutA(+) cells and (ii) FutA(+) cells with a weakened FutA activity resulting from reduced FutA expression [FutA(+)-RBS cells]. Cell mixtures of FutA(+) and FutA(+)-RBS were prepared and applied to one round of FACS sorting. The unsorted and sorted variant pools were quantified using competitive allele-specific TaqMan PCR, and then, enrichment factors were calculated according to FutA(+) cell ratios before and after sorting. (B) Flow cytometric screening of FutA(+) and FutA(+)-RBS cells. (C) Percentage of FutA(+) cells increased after sorting.

  • Fig. 4 Structural insight into the improved catalytic activity of the best M32 mutant.

    (A) Backbone diagram of the M32 mutant (PDB code 5ZOI) with mutations accumulations during directed evolution. Mutated residues are depicted in yellow sticks. Helix α5 having the triple mutations D127N/R128E/H131I located between the NTD and CTD is colored in green. (B) Enhanced interaction toward the LacNAc acceptor in the M32 mutant. The S45F mutation of M32 resulted in a new clamp-like structure with W33 and W34 at the bottom of the substrate-binding pocket. Key aromatic residues and S45 are shown in green sticks, and substituted residue F45 was represented in yellow stick. (C) Local electrostatic surface of M32 active pocket (red, electronegative; blue, electropositive; contoured from −8 to 8 kT/e). These D127N/R128E/H131I mutations showed a changed local electrostatic potential environment on the surface of hinge helix α5. (D) Root mean square fluctuation (RMSF) of wild-type FutA and M32 mutant residues from 122 to 148 region backbones in 100 ns constrained MD simulation. The segment of 122 to 148 residues are shown in cartoon.

  • Table 1 Kinetic parameters for wild-type FutA and selected beneficial mutants.

    The kinetic assays were performed in three independent replicates, and the fitting curves for the kinetic parameters are presented in fig. S7.

    LacNAc*GDP-FucLactose*GDP-Fuc
    Enzymekcat
    (s−1)
    KM
    (mM)
    kcat/KM
    (s−1 mM−1)
    kcat
    (s−1)
    KM
    (mM)
    kcat/KM
    (s−1 mM−1)
    kcat
    (s−1)
    KM
    (mM)
    kcat/KM
    (s−1 mM−1)
    kcat
    (s−1)
    KM
    (mM)
    kcat/KM
    (s−1 mM−1)
    Wild type7.91 ± 0.180.52 ± 0.0515.28 ± 1.1316.28 ± 0.620.09 ± 0.01180.89 ± 13.401.24 ± 0.0599.60 ± 0.080.01 ± 0.0050.05 ± 0.010.02 ± 0.012.53 ± 0.28
    M2630.60 ± 0.040.49 ± 0.0262.45 ± 1.7425.97 ± 0.570.08 ± 0.01324.63 ± 17.341.27 ± 0.0224.30 ± 1.000.05 ± 0.0010.22 ± 0.010.03 ± 0.017.35 ± 0.39
    M3234.27 ± 0.700.37 ± 0.0492.62 ± 8.2330.03 ± 0.040.08 ± 0.01375.38 ± 13.811.26 ± 0.039.20 ± 1.000.14 ± 0.010.31 ± 0.010.02 ± 0.0115.59 ± 1.31

    *Kinetic measurements at a non-limiting concentration of the fucosyl donor substrate GDP-Fuc (0.4 mM) but variable concentrations of the acceptors LacNAc (0.02 to 8 mM) or lactose (4 to 20 mM).

    †Kinetic measurements at non-limiting concentrations of the acceptors LacNAc or lactose (both 0.4 mM) but a variable concentration of the fucosyl donor substrate GDP-Fuc (0.02 to 8 mM).

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaaw8451/DC1

      Fig. S1. Scheme for the fluorescent product entrapment strategy and the cell-based FutC and FucD fucosylation assays using FACS.

      Fig. S2. Analysis of fluorescence retention in various cells.

      Fig. S3. Optimization of the FACS-based system.

      Fig. S4. Site-directed mutagenesis and ordered recombination of the mutation site.

      Fig. S5. Rational selection of candidates for “best hit” from Cα of catalytic key residue and clustering of α helices on substrate-binding sites for CAST and SSM.

      Fig. S6. LC-MS analyses of the LeX from LacNAc catalyzed by FutA variants.

      Fig. S7. Steady-state kinetics of wild type, M26, and M32 measured using various substrates.

      Fig. S8. Structural insight into the improved activity of the best M32 mutant.

      Table S1. Model screening of FutA(+) cells.

      Table S2. Specific activities of FutA and selected mutants using LacNAc and lactose as acceptors.

      Table S3. Activity comparison between the best mutant in the present study and previously reported FutA enzymes.

      Table S4. Primers used in this study.

      Table S5. Data collection and refinement statistics.

      References (44, 45)

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Scheme for the fluorescent product entrapment strategy and the cell-based FutC and FucD fucosylation assays using FACS.
      • Fig. S2. Analysis of fluorescence retention in various cells.
      • Fig. S3. Optimization of the FACS-based system.
      • Fig. S4. Site-directed mutagenesis and ordered recombination of the mutation site.
      • Fig. S5. Rational selection of candidates for “best hit” from Cα of catalytic key residue and clustering of α helices on substrate-binding sites for CAST and SSM.
      • Fig. S6. LC-MS analyses of the LeX from LacNAc catalyzed by FutA variants.
      • Fig. S7. Steady-state kinetics of wild type, M26, and M32 measured using various substrates.
      • Fig. S8. Structural insight into the improved activity of the best M32 mutant.
      • Table S1. Model screening of FutA(+) cells.
      • Table S2. Specific activities of FutA and selected mutants using LacNAc and lactose as acceptors.
      • Table S3. Activity comparison between the best mutant in the present study and previously reported FutA enzymes.
      • Table S4. Primers used in this study.
      • Table S5. Data collection and refinement statistics.
      • References (44, 45)

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