## 2 Results
### Heterologous Expression of LS in the IUP Strain Improves Limonene Production  
We further demonstrated the utility of the IUP by combining it with a downstream module for the limonene synthesis. Limonene, a cyclic monoterpene, possesses citrus-like olfactory properties and multiple physiological functions [24]. Limonene synthase (LS) was chosen from spearmint (_Mentha spicata_) because of its high fidelity, i.e., >90% of the LS product is limonene with minimal levels of isomeric by-products [25]. To allow catalytic activity in the cytosol, the predicted chloroplast transit peptide (CTP) of LS was replaced by methionine for the translation initiation, while retaining residues R58/R59 [5,26]. The precursor of limonene is geranyl diphosphate (GPP), which is condensed from one molecule of dimethylallyl pyrophosphate (DMAPP) and one molecule of isopentenyl pyrophosphate (IPP). Thus, the IPP/DMAPP ratio plays an essential role in the regulation of limonene production. To balance the ratio of IPP and DMAPP, isopentenyl diphosphate isomerase (IDI) was chosen [16]: IDI is a key rate-limiting enzyme in terpenoid biosynthesis that performs the regulatory isomerization of IPP into DMAPP and vice versa [27]. The V4-49 strain, a high-level C5 hemiterpene diphosphate-producing background strain, was chosen for the limonene synthesis. As mentioned above, the co-expression of the individual enzymes (ScCK-GSGSGS-YFP and AtIPK-GSGS-CFP) and the N-terminal orientation in the fusion with YFP/CFP was determined to most favor product generation. Thus, we chose a new vector, pOpt2_PsaD_aadA_Min, which confers spectinomycin resistance on _C. reinhardtii_, for the selection of transformants, and allowed the construction of N-terminal orientations of the enzyme IDI in fusion with RFP [11] and MsLS in fusion with aadA (Figure 4a). We randomly selected at least 300 transformantsants of each gene expression construct, using a microplate reader to detect the fluorescence signal of the transformants in the mid-logarithmic phase. The V12-118, V12-203 and V12-209 strains showed higher fluorescence intensities than other strains (Figure 4b), and the V12-203 strains showed the highest fluorescence intensity. The fluorescence signals of V12-203 were visualized by confocal microscopy. The fluorescence signals of all the fusion proteins were completely overlapped with those of the chlorophyll (Figure 4c), which indicated that the fusion proteins were localized to the chloroplast as intended by the CTP. Furthermore, the fusion proteins exhibited appropriate molecular masses in western blotting (Figure 4d). In order to promote terpene production and accelerate its detection, dodecane was overlayed to the strain culture because of its relatively low volatility, compatibility with algal growth and ability to solvate terpenoids [8,26]. The dodecane phase was harvested and subjected to GC-MS analysis (Figure 4e,f). The standard calibration curves in the range of 0.5-5 ppm limonene in dodecane were used to quantify the amount of limonene, and the R\({}^{2}\) coefficient for the calibration was greater than 0.99 (standard curve in Supplementary Figure S7). The quantification of limonene was performed using a standard curve of the commercial (R)-limonene (Merck KGaA, Darmstadt, Germany). The strains, and the transformants which contained the V12 construct, gave the highest limonene production, achieving approximately 23 \(\mathrm{\SIUnitSymbolMicro g}\)/L limonene from the strain V12 fermentations, which was approximately 23-fold that of V11 (which is cultured in the TAP medium without isoprenol added, MsLS expressed alone, as a control). The results show that the expression of the artificial IUP in _C. reinhardtii_ carrying the MsLS gene leads to an enhancement of the limonene production by more than 23-fold, likely due to the increased carbon flux towards IPP and DMAPP. The marked improvement in limonene yield suggests that the MsLS enzyme activity was limited by the substrate availability in the V11 strain, which lacks the IUP.