Design and optimizing a new CDP-choline in vitro multienzyme producing process starts from d-ribose

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Metabolic Engineering

Background:

• CDP-choline is a vital intermediate for phospholipid synthesis.
• Efficient enzymatic production of CDP-choline is challenging due to complex reaction pathways.
• Existing methods often suffer from low yields and high costs.

Purpose of the Study:

• To design and optimize an in vitro multienzyme system for CDP-choline production from d-ribose.
• To develop an efficient one-pot catalytic system integrating multiple enzymes.
• To enhance the industrial feasibility of CDP-choline biosynthesis.

Main Methods:

• Integration of a 10-enzyme cascade system.
• Implementation of an acetate kinase/acetyl phosphate-based ATP regeneration module.
• Optimization of enzyme building blocks and expression parameters.

Main Results:

• Achieved a CDP-choline titer of 12.2 mM (6 g/L), a significant improvement from 0.2 g/L.
• Demonstrated a robust one-pot multienzyme system for energy-rich compound production.
• Successfully optimized enzyme selection and expression for higher yields.

Conclusions:

• The developed one-pot multienzyme system offers a promising route for industrial CDP-choline production.
• The optimization strategy provides a valuable framework for improving other multienzyme systems.
• This work advances biocatalytic approaches for synthesizing valuable biochemicals.