Artificial Caprolactam-Specific Riboswitch as an Intracellular Metabolite Sensor

Caprolactam is a monomer used for the synthesis of nylon-6, and a recombinant microbial strain for biobased production of nylon-6 was recently developed. An intracellular biosensor for caprolactam can facilitate high-throughput metabolic engineering of recombinant microbial strains. Because of the mixed production of caprolactam and valerolactam in the recombinant strain, a caprolactam biosensor should be highly specific for caprolactam. However, a highly specific caprolactam sensor has not been reported. Here, we developed an artificial riboswitch that specifically responds to caprolactam. This riboswitch was prepared using a coupled in vitro-in vivo selection strategy with a heterogeneous pool of RNA aptamers obtained from in vitro selection to construct a riboswitch library used in in vivo selection. The caprolactam riboswitch successfully discriminated caprolactam from valerolactam. Moreover, the riboswitch was activated by 3.36-fold in the presence of 50 mM caprolactam. This riboswitch enabled caprolactam-dependent control of cell growth, which will be useful for improving caprolactam production and is a valuable tool for metabolic engineering.