## 4 Materials and Methods
### Plasmid Construction, Transformation, and Screening of Mutants  
All of the cloning in this work was performed with Thermo Fisher Scientific FastDigest restriction enzymes (Thermo Fisher Scientific, Shanghai, China) and the ClonExpress(r) II One Step Cloning Kit (Vazyme, Nanjing, China), according to the manufacturers' protocols. All PCRs were performed using the KOD OneTM PCR Master Mix -Blue- (TOYOBO, Shanghai, China), following the manufacturer's protocols, and the primers are listed in Supplementary Table S1. Following each cloning step, the vector sequences were confirmed by sequencing (Sangon Biotech, Shanghai, China). The plasmids were transformed into _Escherichia coli_ Top10, and the transformantsants were cultivated in Luria broth (LB) agar plates or in liquid medium with 100 mg L\({}^{-1}\) ampicillin. The amino acid sequences for the choline kinase from _Saccharomyces cerevisiae_ (ScCK, AAA34499.1), isopentenyl phosphate kinase from _Arabidopsis thaliana_ (AtIPK, AAN12957.1), isopentenyl-pyrophosphate delta isomerase from _E. coli_ (IDI, AAD26812.1) and 4S-limonene synthase from _Mentha spicata_ (MsLS, AAC37366.1) were codon optimized and synthesized de novo taking account of the nuclear codon bias of _C. reinhardtii_ (General Biosystem Company, Anhui, China). The MsLS was optimized for codon usage in _C. reinhardtii_ without the predicted chloroplast transit peptide (cTP) and is referred to in this work as MsLS. To minimize the exon lengths and improve the overall expression, the first  
Figure 6: Growth and limonene yield in 300 mL batch cultures of the limonene expression strains under optimized conditions. Strains were cultivated in 500 mL shake flasks containing a 300 mL opt2 medium supplemented with 10 mM isoprenol at 25 \({}^{\circ}\)C and 120 rpm under 16:8 h light: dark conditions, and using 15 mL dodecane as the overlay for 7 d. Three biological replicates were used to calculate the limonene yield averages and SDs. One-way ANOVA was used for the statistical analyses. Different letters (a–d) indicate significant differences (\(p\leq 0.05\)). intron of ribulose bisphosphate carboxylase small subunit 2 (rbcS2i1) was added to the synthetic transgene sequence, and the second intron (rbcS2i2) was incorporated into the last position between two guanine nucleotides (G/intron/G) [45]. All codon-optimized, intron-containing nucleotide sequences are listed in Supplementary Table S2 for _ScCK_ (GenBank accession no. OP046413), _AtIPK_ (GenBank accession no. OP046414), _IDI_ (GenBank accession no. OP046415), and _MsLS_ (GenBank accession no. OP046416). The sequences mentioned above were designed with the compatible restriction endonuclease sites and cloned into pOptimized (pOpt) Chlamydomonas expression vectors [46] in the fusion with either mVenus (yellow), mCerulean3 (cyan) or mRuby2 (red) fluorescent protein (Y/C/RFP) reporters and the selection markers as indicated. The pOpt vectors were modified to contain a 36-amino-acid photosystem I reaction center subunit II (PsaD) target peptide and thus can directly target the expressed proteins to the algal chloroplast. All constructs created are listed in Table 1. The key construct maps were shown in Supplementary Figure S3. The plasmids were introduced into _C. reinhardtii_ by glass bead agitation [47]. The positive transformants were selected on TAP agar plates supplemented with suitable antibiotics at 8 mg L\({}^{-1}\) (paroomycin), 9 mg L\({}^{-1}\) (hygromycin B), 10 mg L\({}^{-1}\) (zeocin) or 200 mg L\({}^{-1}\) (spectinomycin) at a light intensity of 150 umol photons m\({}^{-2}\) s\({}^{-1}\). The colonies were maintained on TAP agar plates with the respective antibiotic combinations. The colonies exhibiting fluorescence were picked for the TAP medium and cultivated with 150 umol photons m\({}^{-2}\) s\({}^{-1}\) light intensity at 120 rpm until sufficiently dense (mid-logarithmic phase or stationary phase). As [PERSON] et al.