Artificial Caprolactam-Specific Riboswitch as an Intracellular Metabolite Sensor

Abstract

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.

Recent trends in biocatalysis

“…During SELEX, a library of oligonucleotides specifically binding a target ligand or ligands are produced, selected, and enriched in vitro. 73,125,126 Furthermore, natural riboswitches can be engineered like TFs and enzymes. 127 Examples include the engineering of a set of riboswitch-based genetic devices to enable the control of gene expression according to changes in the environmental pH 128 and the switching of a thiamine pyrophosphate-sensing riboswitch from a device for the repression of downstream genes to an activator.…”

Configurable Compartmentation Enables In Vitro Reconstitution of Sustained Synthetic Biology Systems

“…5a). Lactam is an essential class of Nylon precursor and could be detected in the environment as an industrial chemical pollutant 46 . The concentration of lactam residual in the waste falls within 100 mM range 47 .…”

Developing a Riboswitch-Mediated Regulatory System for Metabolic Flux Control in Thermophilic Bacillus methanolicus

“…Popular engineering strategies involve the use of riboswitches to regulate the expression of the genes in the biosynthetic pathways competitive to the one of the chosen product [ 11 , 12 ], to deactivate riboswitch-controlled feedback inhibition in selected biosynthetic pathways [ 13 ] or to achieve dynamic control of the parts of biosynthetic pathways that can constitute metabolic burden for the cells, for example membrane proteins such as product transporters [ 14 ]. The desired riboswitches can either be detected through screening of sequences selected based on transcriptomic and functional studies [ 12 , 15 ], or designed using screening [ 2 , 11 , 16 , 17 ] or based on the pre-existing data [ 13 ].…”