Fucosyltransferases (FucTs) are involved in the natural fucosylation reactions, which catalyze the transfer of L-fucose from the donor GDP-L-fucose to various sugar acceptors, including oligosaccharides, glycoproteins, and glycolipids [1]. Fucosylation serves as the concluding stage in the biosynthesis of oligosaccharides and glycoconjugates. It holds a significant and indispensable role in numerous intricate physiological and pathological processes [2,3].

α-1,2-Fucosyltransferase (α-1,2-FucT, EC 2.4.1.69) belongs to the GT 11 family, which not only catalyzes the substrate N-acetyl-D-lactosamine (LacNAc) form LewisX antigen for use in anti-inflammatory drugs and anti-tumor vaccines but also catalyzes lactose to generate 2’-FL, an essential prebiotic in human milk oligosaccharides (HMOs) [4,5]. 2’-FL, which accounts for about 30 % of total HMOs, has been approved by the U.S. Food and Drug Administration (FDA) as Generally Recognized as Safe (GRAS) food [6]. The beneficial properties of 2’-FL (e.g. maintenance of intestinal ecological balance, resistance to adhesion of pathogenic bacteria, immunomodulation, and promotion of neurological development and repair) have attracted considerable attention for its potential applications in nutritional and pharmaceutical applications [7,8].

To date, five bacterial-derived α-1,2-FucT with known sequences have been identified, including WsFucT from Escherichia coli O128:B12 [9], WbiQ from Escherichia coli O127:K63(B8) [10], WbgL from Escherichia coli O126 [11], HpFucT from Helicobacter pylori NCTC11639 [12], and TeFucT from Thermosynechococcus elongatus BP-1[13]. However, no crystal structures have been reported for microbial α-1,2-FucT. Most of the excavated α-1,2-FucT exhibited low catalytic activity and poor soluble expression in microbial expression systems [14,15,16]. Furthermore, the in vitro screening process of this enzyme was time-consuming and the cost of the donor GDP-fucose was expensive [17]. Therefore, α-1,2-FucT becomes the rate-limiting enzyme in 2’-FL biosynthesis (including de novo and salvage pathways) [18,19,20]. Several effective strategies such as ribosome binding site (RBS), fusion peptides, and gene copy number screening were applied to improve the expression level of α-1,2-FucT and thus enhance 2’-FL production [4,21]. Recently, site-directed saturation mutations based on structure and sequence alignment have been widely used to improve enzyme performance [13]. Yao et al. used multiple sequence alignment to predict mutants with improved activity and applied the protein structure predicted by AlphaFold 2 to mutate 21 amino acids near the catalytic triplet, ultimately increasing urethanase activity by 3.1-fold [22]. Liu et al. adopted model-guided targeted mutations and combinations to improve the recognition of α-1,2-FucT for lactose, and the high synthetic efficiency of the mutant was verified by in vitro biotransformation of 2’-FL [16]. Interestingly, Shin et al. established biosensors linking proteolysis to antimicrobial resistance, improved soluble α-1,2-FucT expression and enhanced 2’-FL titer and yield by 1.72-fold and 1.51-fold via directed evolution [23]. Nevertheless, enzyme engineering studies on α-1,2-FucT are still rarely reported. Molecular modifications to improve enzyme activity, soluble expression and thermostability are of great importance for solving the technical bottleneck of α-1,2-FucT and for the application of 2’-FL.

Helicobacter pylori α-1,2-FucT (HpfutC) was selected for this study as it is the optimal enzyme source previously reported for 2’-FL production [4]. Multiple sequence alignment of α-1,2-FucT was performed to explore the sequence-evolution-function relationship and to guide the rational design of HpfutC with higher activity. Site-specific saturation mutation was performed on 17 conserved but different (CbD) residue sites. Six single-point mutants and six combination mutants were obtained by in vivo screening of the mutant library and in vitro enzymatic activity assays. Additionally, the enzymatic properties of wild-type HpfutC and its variants were evaluated. The final three-dimensional (3D) structure of HpfutC predicted by AlphaFold 2 [24] was applied to analyze and investigate the mechanism of enzymatic activity enhancement of the mutants. The enzyme engineering strategies adopted in this study may be valuable for the engineering design of other microbial enzymes with industrial applications.