<i>Chlamydomonas</i> chloroplast genes tolerate compression of the genetic code to just 51 codons

Genome scale engineering has enabled codon compression of the universal genetic code to eliminate seven codons in <i><i>Escherichia coli</i></i>, but to allow more radical schemes for codon compression and reassignment to be tested at genome scale, while avoiding significant technical challenges, smaller, simpler genetic systems are needed. Here, we report a recoding scheme for the 205 kb <i>Chlamydomonas reinhardtii</i> 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 transfer RNAs (tRNAs), are absent, compressing the genetic code to 51 codons. Several recoding strategies were tested on the essential <i>rpoA</i> 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 sequence without affecting expression of a reporter protein or strain fitness under standard laboratory conditions. The same strategy was successfully used for codon compression of <i>ycf1</i>, encoding a subunit of the chloroplast translocon, <i>psaA</i> and <i>psbA</i>, intron-containing highly expressed genes encoding reaction center subunits of both photosystems, and an 8.5 kb operon encoding essential and nonessential 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 <i>rbcL</i> mutant strain. More than 70 functional sequences with diverse codons were recovered. For all recoded genes, viable homoplasmic lines were obtained, showing the efficacy of our codon compression scheme.