Tobacco etch virus protease (TEV) is one of the most widely-used proteases in biotechnology because of its exquisite sequence-specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV's cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our S153N mutant (uTEV1Δ), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-second temporal resolution.Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed evolution platform used to generate them, could be beneficial across a wide range of applications. Main textProteases are ubiquitous in biology, frequently initiating or terminating endogenous signaling cascades. Their peptide bond cleavage activities have been harnessed for a wide range of biotechnological applications, including bottom-up mass spectrometry (MS)-based proteomics, affinity purification, neuronal silencing (e.g., botulinum protease [1]), light-regulated apoptosis [2], tagging of newly synthesized proteins [3], assembly of protein droplets [4], protease-based synthetic circuits [5,6], regulation of TALENs [7], transcriptional readout of calcium [8,9] and protein-protein interactions [10,11,12].One of the most frequently-used proteases in biotechnology is TEV, the 27 kD cysteine protease from tobacco etch virus. TEV is appealing because it is active in the mammalian cytosol, has no required cofactors, recognizes a 7-amino acid consensus peptide substrate, and, most importantly, is highly sequence-specific, exhibiting negligible activity towards endogenous mammalian proteomes.Consequently, TEV has been harnessed for sequence-specific transcription factor release in response to calcium and light in FLARE [8], GPCR activation in TANGO [10], and GPCR activation and light in SPARK [11,12]. In the recently reported CHOMP [5] and SPOC [6] tools, TEV is activated by inputs such as rapamycin or abscisic acid.Despite the exquisite sequence-specificity of TEV, a major limitation is its slow catalysis. With a kcat of 0.18 s -1 (for its best high-affinity substrate sequence, ENLYFQS [13]), TEV is slower than many other proteases used for biotechnology, such as trypsin (kcat 75 s -1 [14]) and subtilisin (kcat 50 s -1 [15]). This slow turnover fundamentally limits the performance of technologies that rely on TEV, such as FLARE [8]. In vivo, FLARE requires calcium and light stimulation for at least 30 minutes to give TEV sufficient time to release detectable quantities of membrane-anchored transcription factor [8]. Yet for the neuronal activity integration applications for which FLARE is designed, a temporal reso...