Chlamydomonas

Papaefthimiou, Dimitra, Diretto, Gianfranco, Demurtas, Olivia Costantina, Mini, Paola, Ferrante, Paola, Giuliano, Giovanni & Kanellis, Angelos K., 2019, Heterologous production of labdane-type diterpenes in the green alga Chlamydomonas reinhardtii, Phytochemistry 167, pp. 1-8 : 2-4

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.

Figures

Fig. 1. Proposed pathway of labdane-type diterpenes predominant in Cistus creticus L. resin. A protonation-initiated cyclization, catalyzed by CcCLS, converts GGDP to the stable bicyclic intermediate copal-8-ol diphosphate. The formation of labda-13-ene-8α,15-diol, ent-manoyl oxide, ent-13-epi manoyl oxide and sclareol are probably due to the activity of non-specific terpenoid phosphatases. Abbreviations: CcCLS, Cistus creticus copal-8-ol diphosphate synthase.

Fig. 2. Chloroplast transformation of C. reinhardtii with recombinant CO-CcCLS gene, and verification of transgene integration and homoplasmy. A. Schematic map of pCG2-FLAG-CO-CcCLS vector used for chloroplast transformation. The length of each genetic element is indicated in each box. atpA prom.: Promoter of the α- subunit of the chloroplast ATP synthase; aadA: CDS of spectinomycin resistance gene; rbcL Ter.: ribulose bisphosphate carboxylase large chain Terminator; psbA Ter.: Photosystem II protein D1 Terminator; CO-CcCLS: Codon adapted CLS gene; FLAG: Flag Tag; psbD prom.: Photosystem II protein D2 promoter. B. Integration scheme of pCG2-FLAG-CO-CcCLS plasmid in the chloroplast genome. Primers Chla1-for/Chla1-rev (amplicon 1: length 1358 bp) and Chla2-for/Chla2-rev (amplicon2: length 2519 bp) were used to test correct integration in the chloroplast genome while primers Chla3-for/Chla3-rev were used to check homoplasmy (amplicon 3: length 954 bp). The absence of amplicon 3 indicates that the transformants are homoplasmic for the presence of transgene. C. PCR results of seventeen representative transformants. Transformants indicated in red have correct plasmid integration and are homoplasmic. M: 1 kb DNA ladder (NEB, catalog number N3232S).