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.
Highlights
IntroductionBacterial cells have a limited number of resources and there is a trade-off between microbial growth and all other cellular functions, including production of useful proteins or metabolites
Growth control is a crucial technique for applications in microbial biotechnology [1].Bacterial cells have a limited number of resources and there is a trade-off between microbial growth and all other cellular functions, including production of useful proteins or metabolites
We developed a simple growth control device based on a unnatural amino acid (Uaa)-regulated growth controller, which is conceptually similar to the Uaa-regulated translational controller
Summary
Bacterial cells have a limited number of resources and there is a trade-off between microbial growth and all other cellular functions, including production of useful proteins or metabolites This suggests that precise growth control is necessary to achieve maximum efficiency of microbial production. Multi-cell systems have many advantages over traditional clonal population systems, but each cell population must be regulated to optimize its percentage in the total population [6,7] This challenge has prompted studies of more precise growth control for a specific population. Several methods for growth control have been described, including synthetic growth switches based on controlled expression of RNA polymerase or methionine synthetase [8,9] These methods are effective, and have some inherent drawbacks, such as non-titratable regulation and use of dropout media [9,10]. Engineered cell–cell signaling systems have been proposed to enable autonomous coordination of subpopulation densities, but these sophisticated systems require construction of complex artificial genetic circuits [10,11,12,13,14]
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