Mechanisms governing codon usage bias and the implications for protein expression in the chloroplast of Chlamydomonas reinhardtii

Chloroplasts possess a considerably reduced genome that is decoded via an almost minimal set of tRNAs. These features make an excellent platform for gaining insights into fundamental mechanisms that govern protein expression. Here, we present a comprehensive and revised perspective of the mechanisms that drive codon selection in the chloroplast of Chlamydomonas reinhardtii and the functional consequences for protein expression. In order to extract this information, we applied several codon usage descriptors to genes with different expression levels. We show that highly expressed genes strongly favor translationally optimal codons, while genes with lower functional importance are rather affected by directional mutational bias. We demonstrate that codon optimality can be deduced from codon–anticodon pairing affinity and, for a small number of amino acids (leucine, arginine, serine, and isoleucine), tRNA concentrations. Finally, we review, analyze, and expand on the impact of codon usage on protein yield, secondary structures of mRNA, translation initiation and termination, and amino acid composition of proteins, as well as cotranslational protein folding. The comprehensive analysis of codon choice provides crucial insights into heterologous gene expression in the chloroplast of C. reinhardtii, which may also be applicable to other chloroplast‐containing organisms and bacteria.