CSO – A sequence optimization software for engineering chloroplast expression in Chlamydomonas reinhardtii

Algal chloroplasts are promising hosts for expression of heterologous proteins. In contrast to the nuclear genome, these organelles lack transgene silencing mechanisms, making them more susceptible to genetic manipulation. While it is well established that codon selection greatly affects heterologous expression, a chloroplast-specific software for application of different sequence optimization algorithms is still missing. In this work we built CSO - a graphical user interface allowing users to engineer their genes for chloroplast expression in the model microalga Chlamydomonas reinhardtii. Within the software, users can choose between three different powerful codon selection algorithms which were built specifically for chloroplast expression: Optimal codons and folding, ChimeraMap, and Mimic original host. To provide users with general intuition regarding the performance of these algorithms, we designed different mCherry reporter genes, using each of the software algorithms. We transformed and expressed each of them in the chloroplast of C. reinhardtii, and quantified protein and mRNA abundance, alongside overall growth rates. For the reporter tested here, Mimic original host yielded the best results, though it had a lower Codon Adaptation Index score; while the algorithm supporting the highest expression could change depending on the target gene, our observations show that matching the chloroplast codon usage bias alone does not necessarily maximize expression, and that optimizing more complex sequence features could prove beneficial. CSO is freely available online; a link to its installation and download page is given in Section 3.1.