Abstract
Proteases with reprogrammed specificity for nonnative substrates are highly desired in synthetic biology and biomedicine. However, generating reprogrammed proteases that are orthogonal and highly specific for a new target has been a major challenge. In this work, we sought to expand the versatility of protease systems by engineering an orthogonal botulinum neurotoxin serotype B (BoNT/B) protease that recognizes an orthogonal substrate. We designed and validated an orthogonal BoNT/B protease system in mammalian cells, combining mutations in the protease with compensatory mutations in the protease substrate and incorporating a truncated target sequence and then demonstrated use of this orthogonal BoNT/B protease-substrate combination to regulate complex transcriptional circuitry in mammalian cells. Transposing this platform into yeast, we demonstrated utility of this approach for in vivo protease evolution. We tested this platform with the newly designed orthogonal protease and then used it in a high-throughput screen to identify novel orthogonal protease/protease substrate combinations. While carrying out this work, we also generated new cleavage reporters that could be used to report botulinum toxin protease activity in mammalian cells using simple fluorescent readouts. We envision that these approaches will expand the applications of botulinum protease in new directions and aid in the development of new reprogrammed proteases.