Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Directed evolution is a powerful method in biological engineering. Current approaches draw on time-invariant selection mechanisms, ideal for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to continuously evolve dynamic, multi-state, or computational functionalities, e.g., on-off kinetics, state-specific activity, stimulus-responsiveness, or switching and logic capabilities. These require selection pressure on all of the states of a protein of interest (POI) and the transitions between them. We realized that optogenetics and cell cycle oscillations could be leveraged for a novel directed evolution paradigm (‘optovolution’) that is germane for this need: We designed a signaling cascade in budding yeast where optogenetic input switches the POI between off (0) and on (1) states. In turn, the POI controls a Cdk1 cyclin, which in the re-engineered cell cycle system is essential for one cell cycle stage but poisonous for another. Thus, the cyclin must oscillate (1-0-1-0…) for cell proliferation. In this system, evolution can act efficiently on the POI’s different states, input-output relations, and dynamics on the timescale of minutes in every cell cycle. Further, controlling the pacemaker, light, directs and tunes selection pressures. Optovolution is in vivo , continuous, self-selecting, and efficient. We first evolved two optogenetic systems, which relay 0/1 input to 0/1 output: We obtained 19 new variants of the LOV transcription factor El222 that were stronger, less leaky, or green light responsive in vivo . We demonstrate the utility of the latter mutations for orthogonal color-multiplexing with only LOV domains for the first time. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss of YOR1 makes supplementing the chromophore phycocyanobilin (PCB) unnecessary. Finally, we demonstrate the generality of the method by evolving a destabilized rtTA transcription factor, which performs an AND operation between transcriptional and doxycycline input. Optovolution makes coveted, difficult-to-change protein functionalities continuously evolvable.