A structural and data-driven approach to engineering a plant cytochrome P450 enzyme

Area of Science:

• Biochemistry
• Metabolic Engineering
• Plant Natural Products

Background:

• Cytochrome P450s (P450s) are crucial enzymes in plant natural product biosynthesis.
• Engineering P450s offers a route to manipulate cucurbitacin and mogroside production.
• Current P450 engineering methods are often slow and lack high-throughput screening.

Purpose of the Study:

• To develop an efficient method for altering P450 substrate specificity.
• To engineer a multifunctional P450 (CYP87D20) for targeted hydroxylation.
• To demonstrate a novel integrated approach for P450 functional redesign.

Main Methods:

• Integrated computational protein design, evolutionary information, and experimental optimization.
• Iterative design and evaluation of 96 protein variants of CYP87D20.
• Focus on altering substrate specificity for specific hydroxylation reactions.

Main Results:

• Successfully transformed multifunctional CYP87D20 into a P450 mono-oxygenase with altered specificity.
• Achieved specific hydroxylation at the C11 position of cucurbitadienol.
• Demonstrated the efficacy of the integrated P450 engineering approach.

Conclusions:

• The integrated P450 engineering strategy is effective for precise enzyme functional redesign.
• This approach can be applied to create new biosynthetic pathways, such as for mogrol production.
• Enables enhanced structural diversity of plant triterpenoids through P450 manipulation.