Activity-based directed evolution of a membrane editor in mammalian cells

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Cellular membranes comprise diverse lipids, but in situ manipulation for functional studies remains challenging.
• Phospholipids are key membrane components, and understanding their roles requires methods for controlled compositional changes.

Purpose of the Study:

• To develop a novel strategy for editing phospholipids within biological membranes.
• To engineer enhanced phospholipase D (PLD) enzymes for improved intracellular activity and specificity.

Main Methods:

• Utilized a bacterial phospholipase D (PLD) as the basis for a membrane editing system.
• Employed activity-dependent directed enzyme evolution in mammalian cells to generate 'superPLDs'.
• Characterized the structure and function of engineered superPLDs.

Main Results:

• Developed superPLDs with up to 100-fold enhanced intracellular activity compared to wild-type PLD.
• Demonstrated optogenetics-enabled phospholipid editing in specific organelle membranes of live cells.
• Showcased biocatalytic synthesis of natural and unnatural phospholipids in vitro.

Conclusions:

• Engineered superPLDs provide a powerful tool for precise manipulation of membrane phospholipid composition.
• The developed approach facilitates both in vivo cellular studies and in vitro lipid synthesis.
• Activity-based directed enzyme evolution in mammalian cells is a versatile platform for engineering chemoenzymatic biomolecule editors.