Engineering analysis of multienzyme cascade reactions for 3'-sialyllactose synthesis

Area of Science:

• Biotechnology
• Carbohydrate Chemistry
• Enzyme Engineering

Background:

• Sialo-oligosaccharides are valuable nutritional ingredients produced via biotechnology.
• Enzyme cascade catalysis is key for sialo-oligosaccharide synthesis, but optimization strategies are underdeveloped.
• Mathematical modeling offers a systematic approach to optimize multienzyme transformations.

Purpose of the Study:

• To develop and apply a mathematical modeling-guided approach for optimizing 3'-sialyllactose (3SL) synthesis.
• To compare the efficiency of different N-acetyl-d-neuraminic acid (Neu5Ac) synthesis pathways (lyase vs. synthase).
• To optimize enzyme loading and activity ratios for efficient 3SL production.

Main Methods:

• Mathematical modeling and simulation of enzyme cascade reactions.
• In situ synthesis of Neu5Ac using Neu5Ac lyase or Neu5Ac synthase.
• Enzymatic synthesis of 3SL from Neu5Ac and lactose using cytidine 5'-triphosphate.
• Comprehensive time-course studies and experimental validation.
• Model-based optimization of protein loading and enzyme activity ratios.

Main Results:

• The Neu5Ac synthase pathway significantly increased 3SL yield (up to 75%) and doubled initial productivity compared to the lyase pathway.
• Mathematical modeling guided optimization reduced total protein loading by up to 43% while maintaining target 3SL yields (61-75%).
• Achieved high 3SL yields (10.4 g/L) and productivity (15 g/L/h) through optimized enzymatic synthesis.

Conclusions:

• Kinetic advantages of the Neu5Ac synthase reaction are critical for high 3SL yield and productivity.
• Engineering analysis and mathematical modeling are essential for optimizing multienzyme systems in oligosaccharide production.
• This approach enables efficient and scalable production of sialo-oligosaccharides for biotechnological applications.