Expanding the Genetic Code of a Photoautotrophic Organism

The photoautotrophic freshwater cyanobacterium Synechococcus elongatus is widely used as a chassis for biotechnological applications as well as a photosynthetic bacterial model. In this study, a method for expanding the genetic code of this cyanobacterium has been established, thereby allowing the incorporation of unnatural amino acids into proteins. This was achieved through UAG stop codon suppression, using an archaeal pyrrolysyl orthogonal translation system. We demonstrate incorporation of unnatural amino acids into green fluorescent protein with 20 ± 3.5% suppression efficiency. The introduced components were shown to be orthogonal to the host translational machinery. In addition, we observed that no significant growth impairment resulted from the integration of the system. To interpret the observations, we modeled and investigated the competition over the UAG codon between release factor 1 and pyl-tRNA_CUA. On the basis of the model results, and the fact that 39.6% of the stop codons in the S. elongatus genome are UAG stop codons, the suppression efficiency in S. elongatus is unexpectedly high. The reason for this unexpected suppression efficiency has yet to be determined.