Development of new codon optimisation tools and development of a synthetic gene expression system in the green alga Chlamydomonas reinhardtii

Francisco Navarro (Department of Plant Sciences, University of Cambridge), Marielle Vigouroux (John Innes Centre)

Most organisms share the same genetic code, based on three nucleotide codons that encode for one amino acid. However, synonymous codons (which specify a single amino acid) are not used at equal frequency by different species. Dr Francisco Navarro and Dr Marielle Vigouroux were interested in assessing the impact of codon usage in protein production in the green alga Chlamydomonas reinhardtii which has an unusually high GC content in its coding sequences. Chlamydomonas sequences have 68% GC content, while Arabidopsis is only 44% and human is 52%. The high GC content strongly biases codon selection towards GC-rich codons, making codon optimization a necessary step for the expression of genes from other species in the alga and hence an important consideration for any synthetic biology project.

The team performed sequence analysis of publicly available datasets to identify codon usage of genes transcribed under different conditions, at different expression levels, and along the length of coding sequences. The conclusion of this analysis was that codon selection in C. reinhardtii is very robust, characterized by a strong preference towards GC-rich codons, for most amino acids with only few exceptions. In addition, the team found that codon preference was not uniform along coding sequences. The results pointed out several considerations to take into account for the codon optimization of transgenes. In order to test gene variants with different levels of codon optimization, Francisco and Marielle developed an experimental platform for measuring the production of a reporter protein.

The platform was designed to test the expression of different gene variants of a fluorescent protein that differed in the choice of synonymous codons. Chlamydomonas transcription regulatory sequences (promoters, terminators and UTRs) were used to drive the expression of the reporter gene. The team were able to implement a versatile cloning strategy and tested the expression of several fluorescent reporters in different strain backgrounds. The DNA parts generated followed -specific MoClo DNA parts following the common syntax for gene assembly in plant synthetic biology co-authored by many members of OpenPlant. Some of these parts have been contributed to a Chlamydomonas-specific MoClo kit, created by a consortium of European labs, including from the Univeristy of Cambridge, which will be an open resource for the Chlamydomonas community.