Orthogonal replication with optogenetic selection evolves yeast JEN1 into a mevalonate transporter.

Area of Science:

• Synthetic Biology
• Biotechnology
• Metabolic Engineering

Background:

• The OrthoRep system facilitates rapid gene evolution in Saccharomyces cerevisiae but struggles with neofunctionalization due to selection pressure limitations.
• Optogenetics offers precise control over biological processes using light, presenting an opportunity to refine selection strategies.

Purpose of the Study:

• To develop a refined gene evolution system by integrating OrthoRep with optogenetics, termed OptoRep.
• To engineer a de novo mevalonate importer for enhanced biofuel production.

Main Methods:

• Combined OrthoRep with optogenetics to create the OptoRep system for light-controlled selection pressure.
• Evolved a truncated monocarboxylate transporter (JEN1t) using OptoRep.
• Demonstrated the functionality of the evolved transporter in farnesene production.

Main Results:

• Successfully evolved JEN1t into a functional de novo mevalonate importer (JEN1t^Y180C/G).
• Utilized the engineered JEN1t for the production of farnesene, a renewable aviation biofuel.
• Showcased the ability to fine-tune selection pressure for gene evolution.

Conclusions:

• OptoRep enhances the evolution of novel gene functions and allows continuous evolution of genes with narrow selection windows.
• The engineered mevalonate importer is effective for farnesene production, highlighting the potential of OptoRep in synthetic biology and biofuel development.