Solubilization and Iterative Saturation Mutagenesis of α1,3‐fucosyltransferase from Helicobacter pylori to enhance its catalytic efficiency

α1,3‐Fucosyltransferase (α1,3‐FucT) is essential for the biosynthesis of biologically active α1,3‐fucosyloligosacchairdes (3‐FOs) from human milk oligosaccharides (HMO), particularly 3‐fucosyllactose (3‐FL) trisaccharide. α1,3‐FucT from Helicobacter pylori 26695 (FutA) accepts lactose and LacNAc as glycan acceptors and has a very low level of expression in Escherichia coli, and it shows a low catalytic activity for lactose in the large‐scale synthesis of 3‐FL. To overcome the poor solubility of FutA, codon optimization, and systematic truncation of the protein at the C‐terminus with only one heptad repeat remaining (Δ52 FutA) were conducted to yield 150–200 mg/L of soluble protein of FutA and resulting in more than an 18‐fold increase in the 3‐FL yield. To improve the low level of enzyme catalytic activity for lactose, focused directed evolution was attempted using a semi‐rational approach that combines structure‐guided computational analysis and subsequent iterative saturation mutagenesis (ISM). In order to select the functional residues in active site/substrate binding site, docking simulation was used together with HotSpot Wizard to target evolutionarily variable amino acid positions. A128 site was selected from the key residue located in the active site, and A128N mutant displayed a 3.4‐fold higher catalytic activity than wild‐type Δ52 FutA. Considering that the A128N mutation is located in the deep cleft of the lactose binding site, the residues within the substrate binding sites, especially on the two α‐helices for lactose and one α‐helix for GDP‐fucose, were subjected to structure‐guided ISM. The selected residues from each helix were clustered, and ISM was performed for each cluster in parallel. In particular, the mutant with triple mutations (A128N/H129E/Y132I) located on the α5 helix exhibited a 9.6‐fold improvement in specific activity when compared to wild‐type Δ52 FutA. When such clustered mutations on two α‐helices (α5 and α2/loop) were combined, mutants with triple (A128N/H129E/S46F) and quadruple mutations (A128N/H129E/Y132I/S46F) were generated, which showed the synergistic effects, that is 14.5‐ and 15.5‐fold improvement in specific activity relative to wild‐type Δ52 FutA, respectively. The mutations increased their binding affinity for lactose and kcat values for lactose and GDP‐fucose. The Δ52 FutA quadruple mutant (A128N/H129E/Y132I/S46F) was successfully applied to in vitro synthesis of 3‐FL with an improved yield and productivity (>96% yield based on 5 mM of GDP‐Fuc within 1 h). Biotechnol. Bioeng. 2016;113: 1666–1675. © 2016 Wiley Periodicals, Inc.