Lighting the Path to Neofunctionalization: Tuning Yeast Evolution with OptoRep

The pursuit of rapid, targeted protein evolution has been significantly advanced by systems like OrthoRep, which allows for high-rate in vivo hypermutation of specific genes in Saccharomyces cerevisiae without compromising genomic integrity. Despite its utility, OrthoRep typically relies on activity-dependent growth selection, which can be a bottleneck when attempting to evolve entirely new functions in genes that lack even basal levels of the desired activity. To overcome this limitation, researchers in José Avalos’ lab at Princeton recently developed OptoRep, a strategy that integrates optogenetics with continuous evolution to allow for the fine-tuning of selection pressure using light.

The challenge of neofunctionalization is particularly evident in mevalonate metabolism. While mevalonate is a critical precursor for isoprenoids, including biofuels like farnesene, S. cerevisiae possesses no known native mevalonate importer. Traditional OrthoRep struggles here because there is no initial transport activity to support growth under conditions of diminishing nutrients. By implementing the OptoRep system, the researchers created a light-sensitive mevalonate auxotrophic strain termed OptoMEV. In this strain, the expression of hydroxymethylglutaryl-CoA (HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA to mevalonate, is placed under the control of the blue light-activated EL222 transcription factor, a bacterial Light-Oxygen-Voltage protein that binds DNA when illuminated with blue light. This allows researchers to use light pulses to maintain semi-permissive growth conditions, providing enough mevalonate for cellular viability while maintaining a selection pressure that favors the evolution of a de novo importer.

Using this setup, the team targeted JEN1, a known monocarboxylate transporter. They utilized a glucose-insensitive truncated form, JEN1t, to avoid native regulatory mechanisms that would otherwise degrade the protein in the presence of glucose. Remarkably, after only three passages in semi-permissive light conditions, three independent lineages evolved the same A539G gene transition mutation, resulting in a protein Y180C substitution. This single missense mutation was sufficient to neofunctionalize JEN1t into an effective mevalonate importer, enabling growth in mevalonate-supplemented media even under nonpermissive dark conditions or in HMG-CoA reductase-null backgrounds.

Further refinement of the transporter through site-saturation mutagenesis at the Y180 residue identified even more efficient variants, such as JEN1tY180G. Structural modeling via AlphaFold and molecular docking simulations provided insight into why this residue is so critical. It appears that substituting the bulky tyrosine at position 180 with smaller residues like cysteine or glycine enlarges the substrate-binding pocket and facilitates better interaction between mevalonate and the R95 residue, which is essential for the translocation process. These findings suggest that Y180 is a key gatekeeper for substrate recognition in Jen1p.

The biotechnological utility of these evolved transporters was demonstrated through the production of farnesene, a renewable aviation biofuel. Strains expressing JEN1tY180G showed enhanced farnesene production when fed exogenous mevalonate, even at low concentrations. Perhaps most impressively, the evolved importer enabled the creation of a functional yeast-yeast microbial consortium. In this system, an upstream strain was engineered to overproduce and secrete mevalonate, while a downstream strain expressing JEN1tY180G imported that mevalonate to convert it into farnesene. This consortium achieved a 32% increase in farnesene production compared to the individual strains, demonstrating a way to recover carbon that would otherwise be lost as a byproduct.

The development of OptoRep represents a significant step forward for metabolic engineering and synthetic biology. By providing a highly flexible, user-defined selection setpoint through light, OptoRep allows for the evolution of gain-of-function mutations that were previously inaccessible. This platform could potentially be used to evolve a variety of other transmembrane transporters or optimize intracellular pathways by linking metabolic flux to cellular fitness through light-conditional auxotrophy. As microbiologists look toward more complex designs for chemical production and cellular biosensors, the ability to "tune" evolution with light offers a powerful new tool for the laboratory.

Wegner SA, Jiang V, Cortez JD, Avalos JL. Orthogonal replication with optogenetic selection evolves yeast JEN1 into a mevalonate transporter. Mol Syst Biol. 2025 Sep;21(9):1190-1213. doi: 10.1038/s44320-025-00113-5. Epub 2025 Jun 11. PMID: 40500463; PMCID: PMC12405511. https://link.springer.com/article/10.1038/s44320-025-00113-5