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

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Carbohydrate Chemistry

Background:

• 2 -Fucosyllactose (2-FL) is a human milk oligosaccharide with potential nutraceutical and pharmaceutical applications.
• Efficient and scalable production of 2-FL remains a challenge.
• Enzyme cascade systems offer a promising approach for complex carbohydrate synthesis.

Purpose of the Study:

• To design and optimize an in vitro multienzyme cascade catalysis system (MECCS) for the efficient synthesis of 2-FL.
• To identify and characterize optimal fucosyltransferase (FucT) orthologues for enhanced 2-FL production.
• To investigate the kinetic and structural properties of selected FucT enzymes.

Main Methods:

• Development of a three-enzyme cascade involving L-fucokinase/GDP-L-fucose phosphorylase (FKP), α-1,2-fucosyltransferase (FucT), and pyruvate kinase (PK).
• Screening of various FucT orthologues to identify enzymes with high catalytic efficiency and thermostability.
• Kinetic analysis (kcat/KM, KD) and structural insights into FucT-GDP interactions.
• Fed-batch cultivation of the MECCS to determine 2-FL productivity.

Main Results:

• The MECCS efficiently regenerated ATP/GTP, improving GDP-L-fucose availability by 5.67-fold.
• HpFucT from *Helicobacter pylori* exhibited the highest catalytic efficiency (kcat/KM = 39.28 min⁻¹ mM⁻¹).
• TeFucT from *Thermosynechococcus elongatus* demonstrated superior thermostability (Tm = 48 °C) and GDP affinity (KD = 1.34 μM).
• 2-FL productivities reached 0.67 g/L/h (TeFucT) and 0.73 g/L/h (HpFucT) in fed-batch mode.

Conclusions:

• A novel and efficient in vitro multienzyme cascade catalysis system (MECCS) was successfully established for 2-FL synthesis.
• Enzyme engineering and selection of optimal FucT orthologues are crucial for enhancing 2-FL production.
• This study provides a viable alternative pathway for the cost-effective and scalable production of 2-FL.