Development of Caprolactam-Specific RNA-Based Biosensor

Abstract

Caprolactam is a monomer that is used in synthesis of value-added chemical, Nylon-6. Production of caprolactam in recombinant microbial strain entails unwanted byproduct, valerolactam with five carbons. To engineer the strain as an efficient caprolactam producer, development of biosensor that reports the intracellular concentration of caprolactam is required. Here, we selected caprolactam-specific aptamer from random RNA library and engineered it into a riboswitch that regulates translation initiation. Iterative selections in controlled conditions in vitro and in vivo allowed enrichment of aptamers with target-specific binding affinity and riboswitches with target-dependent conformation-changing ability from randomized oligonucleotide pool. Screened riboswitch successfully discriminated caprolactam from valerolactam, which reported a proportional fluorescence signal only to intracellular caprolactam concentration and high fold-activation ratio. The modular design of riboswitch circuit could drive diverse cellular responses by swapping reporter genes, which broadens its scope of application. We expect that novel synthetic biology toolkit sparks the evolutionary engineering of value-added producer strains.