Directed Evolution to Enhance Phytoene Synthase Activity for Metabolic Engineering

Additional Authors

My committee members Dr. Dhirendra Kumar, Biological Sciences Dr. Anoop Arunagiri, Biological Sciences

Abstract

Imagine a tomato that not only looks vibrant but also packs a powerful nutritional punch. Tomatoes contain carotenoids that are crucial for plant growth and human nutrition. Metabolic engineering efforts have focused on increasing carotenoid production in crops, particularly targeting provitamin A biosynthesis. Phytoene synthase (PSY), the first committed enzyme in the carotenoid pathway, is a major rate-limiting step. Therefore, it is the main target for provitamin A biofortification in many crops. Tomato has multiple copies of PSY, while PSY1 is dominant in fruits, it is less efficient in promoting carotenoid biosynthesis compared to PSY2. This provides us a target for further improvement not only in tomato but also in other crops since PSY sequences have high homology in plants. In this study, we took advantage of a bacteria system, which can accumulate different level of colored carotenoid products based on the activity of PSY introduced. Structure-guided site-directed and random mutagenesis are introduced in PSY2 gene. Therefore, the mutants of the highest enzymatic activity can be easily screened out by the color accumulated in bacteria culture, which will provide novel targets for carotenoid metabolic engineering. By applying random mutation and directed evolution, we aim to identify key amino acid residues which can increase PSY activity significantly. Ultimately, those sites can be used for prime editing to improve provitamin A carotenoid production in crops.

Start Time

16-4-2025 2:30 PM

End Time

16-4-2025 3:30 PM

Room Number

303

Presentation Type

Oral Presentation

Presentation Subtype

Grad/Comp Orals

Presentation Category

Science, Technology and Engineering

Faculty Mentor

Tianhu Sun

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Apr 16th, 2:30 PM Apr 16th, 3:30 PM

Directed Evolution to Enhance Phytoene Synthase Activity for Metabolic Engineering

303

Imagine a tomato that not only looks vibrant but also packs a powerful nutritional punch. Tomatoes contain carotenoids that are crucial for plant growth and human nutrition. Metabolic engineering efforts have focused on increasing carotenoid production in crops, particularly targeting provitamin A biosynthesis. Phytoene synthase (PSY), the first committed enzyme in the carotenoid pathway, is a major rate-limiting step. Therefore, it is the main target for provitamin A biofortification in many crops. Tomato has multiple copies of PSY, while PSY1 is dominant in fruits, it is less efficient in promoting carotenoid biosynthesis compared to PSY2. This provides us a target for further improvement not only in tomato but also in other crops since PSY sequences have high homology in plants. In this study, we took advantage of a bacteria system, which can accumulate different level of colored carotenoid products based on the activity of PSY introduced. Structure-guided site-directed and random mutagenesis are introduced in PSY2 gene. Therefore, the mutants of the highest enzymatic activity can be easily screened out by the color accumulated in bacteria culture, which will provide novel targets for carotenoid metabolic engineering. By applying random mutation and directed evolution, we aim to identify key amino acid residues which can increase PSY activity significantly. Ultimately, those sites can be used for prime editing to improve provitamin A carotenoid production in crops.