Light-directed evolution of dynamic, multi-state, and computational protein functionalities
TL;DR
This technique could revolutionize biotechnology by making it easier to create proteins that act like biological switches, sensors, or even computers inside living cells. Such programmable proteins could lead to better medical treatments, more efficient biomanufacturing, or new types of biological devices that respond to environmental changes in real-time.
Evolving dynamic, multi-state, and computational protein functionalities is challenging because it requires selection pressure on all the states of a protein of interest (POI) and the transitions between them. To create a continuous directed evolution paradigm for such properties, we genetically engineered budding yeast for optogenetic input to switch a POI "on" and "off," which, in turn, controls a Cdk1 cyclin that is essential for one cell-cycle stage but detrimental for another. The method, "optovolution," generates dynamic selection pressure on POI cycling at the timescale of tens of minutes. We used it to evolve 19 new variants of the LOV transcription factor El222, including in vivo green-light-responsive variants allowing LOV color-multiplexing. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss of YOR1 makes supplementing phycocyanobilin (PCB) unnecessary. Finally, we demonstrated the generality of the method by evolving a non-light-responsive AND gate (PEST-rtTA). Optovolution makes difficult-to-engineer protein functionalities continuously evolvable.
- 1Developed 'optovolution' - a method that uses light to create continuous evolutionary pressure on proteins that need to switch between different states
- 2Successfully evolved 19 new variants of the LOV light-sensing protein, including versions that respond to green light for better color control
- 3Discovered that removing the YOR1 gene eliminates the need for an expensive supplement (PCB) when evolving light-responsive proteins
- 4Proved the method works beyond light-responsive proteins by evolving a non-light AND gate protein
- 5Created selection pressure that operates on a timescale of tens of minutes, much faster than traditional evolution methods
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