Optovolution enables continuous directed evolution of dynamic, multi-state, and computational protein functionalities by coupling protein output to engineered cell cycle regulators that must oscillate for proliferation, yielding El222 variants with 16-fold enhanced light sensitivity and unprecedented green-light responsiveness, revealing that YOR1 deletion eliminates chromophore supplementation requirements for PhyB-Pif3 systems, and improving non-optogenetic AND gate performance.

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.