An Unnatural Amino Acid-Regulated Growth Controller Based on Informational Disturbance

Abstract

We designed a novel growth controller regulated by feeding of an unnatural amino acid, Nε-benzyloxycarbonyl-l-lysine (ZK), using a specific incorporation system at a sense codon. This system is constructed by a pair of modified pyrrolisyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl). Although ZK is non-toxic for normal organisms, the growth of Escherichia coli carrying the ZK incorporation system was inhibited in a ZK concentration-dependent manner without causing rapid bacterial death, presumably due to generation of non-functional or toxic proteins. The extent of growth inhibition strongly depended on the anticodon sequence of the tRNApyl gene. Taking advantage of the low selectivity of PylRS for tRNApyl anticodons, we experimentally determined the most effective anticodon sequence among all 64 nucleotide sequences in the anticodon region of tRNApyl gene. The results suggest that the ZK-regulated growth controller is a simple, target-specific, environmental noise-resistant and titratable system. This technique may be applicable to a wide variety of organisms because the growth inhibitory effects are caused by “informational disturbance”, in which the highly conserved system for transmission of information from DNA to proteins is perturbed.

Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon

“…The procedure for plasmid curing using pylS ZK -pylT is shown in Figure 1 a. A typical example of the results is provided by a target plasmid containing the pylS ZK -pylT GCC counter-selectable marker with tRNA pyl GCC , which has a strong growth inhibitory effect [ 29 ]. The sequence of target plasmid is shown in Figure S1 .…”

“…The target plasmid contains the counter-selection marker, pylS ZK -pylT GGU +pylT CGC , including two tRNA pyl genes, tRNA pyl GGU, and tRNA pyl CGC , both of which show a strong growth inhibitory effect [ 29 ]. We attempted to exchange the target plasmid directly with the parent plasmid, pACYC184 [ 38 ].…”

“…The procedure of plasmid exchange is illustrated in Figure 2a. We prepared an E. coli strain with the target plasmid containing pylS ZK and its two cognate tRNA genes: pylT GGU and pylT CGC , both of which exhibit strong growth inhibition in the presence of ZK [29]. This strain was further transfected with pACYC184, the parent plasmid of the target plasmid.…”

“…Recently, we have reported a precise and titratable microbial growth controller regulated by the unnatural amino acid, N ε -benzyloxycarbonyl- l -lysine (ZK) [ 29 ]. This growth controller consists of a pair of genes: ZK-specific modified pyrrolysyl-tRNA synthetase (ZKRS, encoded by pylS ZK ) and its cognate tRNA (tRNA pyl , encoded by pylT ) [ 30 ].…”

“…The anticodon sequence of tRNA pyl is CUA, but this can be modified to other sequences because the pyrrolysyl-tRNA synthetase (PylRS) does not use the anticodon sequence of tRNA pyl for substrate tRNA recognition [ 31 , 32 ]. Growth inhibition by ZK incorporation is highly dependent on selection of the target codon, partially due to the tendency for substitution with physicochemically more distant amino acids to be more toxic [ 29 ]. Thus, the freedom to select the target codon is advantageous for maximizing toxicity.…”