2.1. Transformation of the Chlamydomonas chloroplast with a codonoptimized CcCLS gene (CO-CcCLS) The CDS of the C. creticus CcCLS gene was codon-optimized for improved expression in the C. reinhardtii chloroplast (CO-CcCLS) (Supplementary Fig. 1). CO-CcCLS was cloned without the transit peptide as an N-terminal fusion with the FLAG–tag in the pCG2-FLAG chloroplast expression vector, under the control of the PSBD promoter/ 5′UTR and PSBA terminator/3′UTR (Fig. 2A). The obtained construct (pCG2-FLAG- CO-CcCLS) was introduced in the chloroplast of the cell wall-less cw15 strain using glass beads transformation (Demurtas et al., 2013). Chloroplast transformants were selected on TAP agar plates supplemented with 100 μg/ml spectinomycin and subcultured for 10 rounds in the same medium to eliminate all wild-type copies of the chloroplast genome, a condition known as homoplasmy. Correct integration in the chloroplast genome and homoplasmy were verified by PCR with appropriate primers (Fig. 2) (Demurtas et al., 2013). In total, 79 transformants were tested by PCR and 49 of them showed correct integration and homoplasmy. Fig. 2C shows the PCR results of seventeen representative transformants, where only twelve had correct integration and homoplasmy.