Efficient Biosynthesis of 2'-Fucosyllactose Using an In Vitro Multienzyme Cascade

2'-Fucosyllactose (2-FL) is a fucose-containing oligosaccharide that is found in humans and is believed to have potential nutraceutical and pharmaceutical uses. Here, a promising in vitro multienzyme cascade catalysis system (MECCS) was designed to convert L-fucose and lactose to 2-FL. The cascade comprises L-fucokinase/GDP-L-fucose phosphorylase (FKP), α-1,2-fucosyltransferase (FucT), and pyruvate kinase (PK). This MECCS was able to efficiently regenerate ATP or GTP with 5.67-fold improvement of GDP-L-fucose. To address the rate-limiting step in the MECCS, various FucT orthologues were screened, and HpFucT from Helicobacter pylori showed the highest catalytic efficiency, with a (k_cat/K_M) of 39.28 min^-1 mM^-1, while TeFucT from Thermosynechococcus elongatus showed the highest thermostability, with a melting temperature (T_m) of 48 °C. The dissociation constant (K_D) of TeFucT (1.34 ± 0.41 μM) was 15-fold lower than that of HpFucT (20.24 ± 1.81 μM), suggesting that TeFucT had much higher affinity for GDP. Structural analysis of HpFucT indicated that Arg169 is part of a unique substrate-binding site that interacts with two oxygen atoms from the phosphate group of GDP-L-fucose. The 2-FL productivities of the MECCS in fed-batch reached 0.67 and 0.73 g/L/h with TeFucT and HpFucT, respectively. This research provides an alternative pathway for efficient production of 2-FL.