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Updated: Jun 13, 2025

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications
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Orthogonal replication with optogenetic selection evolves yeast JEN1 into a mevalonate transporter.

Scott A Wegner1, Virginia Jiang2, Jeremy D Cortez1

  • 1Department of Molecular Biology, Princeton University, Princeton, NJ, 08544, USA.

Molecular Systems Biology
|June 11, 2025
PubMed
Summary

OptoRep, a novel system combining orthogonal replication (OrthoRep) and optogenetics, enables precise control over gene evolution in yeast. This system successfully engineered a mevalonate importer for producing the biofuel farnesene.

Keywords:
JEN1Continuous EvolutionMevalonate Transporter EvolutionOptogeneticsOrthoRep

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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 (JEN1tY180C/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.