# Chlamydomonas chloroplast genes tolerate compression of the genetic code to just 51 codons

## TL;DR

Scientists successfully compressed the genetic code in Chlamydomonas chloroplast genes to 51 codons, maintaining viability and photosynthesis.

## Contribution

A novel codon compression scheme was developed and tested on multiple essential genes in the Chlamydomonas chloroplast genome.

## Key findings

- Codon-compressed genes in Chlamydomonas chloroplasts remained functional and viable.
- Multiple essential genes, including rpoA and ycf1, were successfully recoded without affecting fitness.
- Over 70 functional sequences were recovered for the Rubisco subunit under the 51-codon system.

## Abstract

We demonstrate the use of the Chlamydomonas chloroplast genome as a synthetic biology platform to test radical compression of the universal genetic code. Genome engineering was used to generate strains in which 13 of the 64 possible triplet codes (codons) were eliminated from several essential and/or highly expressed genes, representing ~20% of the protein coding sequence in the genome. In all cases, viable and photosynthetically active strains were obtained, confirming the functional expression of the codon-compressed genes. These results pave the way for compression of the entire chloroplast genome to 51 codons, as well as the opportunity for more extreme modification or expansion of the genetic code.

Genome scale engineering has enabled codon compression of the universal genetic code to eliminate seven codons in Escherichia coli, 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 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 transfer RNAs (tRNAs), are absent, compressing the genetic code to 51 codons. 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 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 ycf1, encoding a subunit of the chloroplast translocon, psaA and psbA, 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 rbcL 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.