Abstract
Genome scale engineering has enabled codon compression of the universal genetic code of up to three codons in E. coli, providing the means for genetic code expansion. To go much beyond this number, smaller and simpler genetic systems are needed to avoid significant technical challenges. Chloroplast genomes offer multiple advantages for codon compression and reassignment. Here we report a recoding scheme for the Chlamydomonas reinhardtii chloroplast genome, in which two stop codons and one or more of the codons for arginine, glycine, isoleucine, leucine and serine, all of which have two cognate tRNAs, are absent, compressing the genetic code to 51 codons. Firstly, several recoding strategies were tested on the essential rpoA gene, encoding a subunit of the chloroplast RNA polymerase. A defined compression scheme, which relied on swapping the target codons with the permitted frequent codons, could replace the native protein coding sequence without affecting chloroplast protein expression levels or the strain fitness. The same strategy was successfully used for codon compression of ycf1, encoding a subunit of the chloroplast translocon, psaA and psbA, intron-containing highly-expressed genes encoding reaction centres subunits of both photosystems, and an 8.5 kb operon encoding essential and non-essential genes. Finally, we tested degeneracy of the 51-codon genetic code by exploring the combinatorial design for the large subunit of RuBisCO, relying on restoration of photosynthesis in an rbcL mutant strain. More than 70 functional sequences with diverse codon adaptation indices were recovered. In all codon-compressed genes there was no observable penalty on photosynthetic growth.
Competing Interest Statement
The authors have declared no competing interest.
