Reprogramming the Cleavage Specificity of Botulinum Neurotoxin Serotype B1

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.