Computationally Guided Engineering of Multi-Enzyme Cascades Enables Efficient Trehalose Biosynthesis

Area of Science:

• Biotechnology
• Enzyme Engineering
• Metabolic Engineering

Background:

• Trehalose is a disaccharide valued for its biomolecule-stabilizing properties.
• Current multienzyme cascade production methods face challenges with low enzyme expression and suboptimal catalytic efficiency.
• Efficient and scalable production of trehalose is crucial for its widespread application.

Purpose of the Study:

• To develop an improved multienzyme cascade for efficient trehalose biosynthesis.
• To enhance enzyme performance through protein engineering and computational screening.
• To establish a scalable Bacillus subtilis platform for high-titer trehalose production.

Main Methods:

• Intracellular expression of thermostable maltooligosyltrehalose synthase (TreY) and trehalohydrolase (TreZ) in Bacillus subtilis.
• Computational screening and semirational engineering of 4-α-glucanotransferase (MalQ-3) to generate MalQ-3-M2 with improved activity and stability.
• Integration of engineered enzymes into a crude-lysate cascade for trehalose synthesis.

Main Results:

• Initial cascade achieved 281.4 g/L trehalose with a yield of 0.7 g/g maltodextrin.
• Engineered MalQ-3-M2 exhibited enhanced activity and stability, improving substrate processing.
• The optimized cascade incorporating MalQ-3-M2 increased trehalose titer to 338 g/L and yield to 0.85 g/g maltodextrin.

Conclusions:

• A scalable Bacillus subtilis platform for efficient trehalose production was successfully established.
• Enzyme engineering and cascade optimization significantly improved trehalose biosynthesis efficiency.
• The developed system offers a promising approach for industrial-scale trehalose manufacturing.