Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases

Area of Science:

• Biochemistry
• Enzyme Engineering
• Synthetic Biology

Background:

• Plant thiamin (vitamin B1) synthesis relies on cysteine-dependent THI4 enzymes with high turnover rates, leading to significant energy expenditure.
• Sulfide-dependent THI4 enzymes offer a potential alternative but are typically anaerobic and inefficient under plant aerobic conditions.

Purpose of the Study:

• To enhance the aerotolerance and catalytic activity of sulfide-dependent bacterial THI4 enzymes for potential application in plant thiamin biosynthesis.
• To demonstrate the feasibility of using directed evolution in a yeast system to adapt non-plant enzymes for improved function in aerobic environments.

Main Methods:

• Continuous directed evolution of two sulfide-dependent bacterial THI4 enzymes was performed under aerobic conditions using the yeast OrthoRep system.
• Beneficial mutations were identified through selection and characterized, with structural modeling used to predict active-site cleft locations.

Main Results:

• Seven beneficial single mutations were identified that significantly improved enzyme performance.
• Five mutations were located within the predicted active-site cleft, and two mutations mimicked features of naturally aerotolerant THI4 enzymes.
• The study demonstrated substantial performance gains from single mutations, suggesting further improvements are possible by combining mutations.

Conclusions:

• Sulfide-dependent THI4 enzyme performance in aerobic conditions is evolvable, offering a pathway to engineer more efficient thiamin synthesis in plants.
• The yeast OrthoRep system serves as an effective platform for adapting non-plant enzymes to aerobic conditions, acting as a plant-like bridge for enzyme improvement.