## 2 Results
### IUP Enhanced the C5 Hemiterpene Diphosphate Production  
Of all the vector constructs, the transformants which contained the V4 construct, showed relatively higher IPP productivities, with the strains V4-49 and V4-68 being the best (Figure 3a). Strain V4-49, which co-expressed both enzymes individually and at the N-terminal end of the fusion (i.e., ScCK-GSGSGS-YFP and AtIPK-GSGS-CFP), gave the greatest increase in production, achieving 5.3 mg/L IPP, which was 8.6-fold higher than that produced by the parental UVM4 strain (Figure 3a). In addition, we also noticed that the fusion order had a significant impact on the folding and function of the fusion enzymes. Thus, strain V4-49 gave a 3-fold higher IPP yield than that produced by the strain V3-2 in which the individual enzymes were at the C-terminal end of the fusion proteins, i.e., YFP-ScCK and CFP-AtIPK (Figure 3a). These results indicate that the individual expression of each enzyme and an N-terminal orientation in the fusion with YFP/CFP gives a superior product generation. Figure 2: Overview of the expression and screening strategy for the C5 hemiterpene diphosphate production in _C. reinhardtii_. (**a**) Expression vectors for the fluorescent reporters YFP and CFP (V1 and V2, respectively); fusion of the codon-optimized choline kinase (CK) and isopentenyl phosphate kinase (IPK) to the C-terminus (V3) or N-terminus (V4) of mCerulean3 and the mVenus reporters in the pOpt_mCerulean3_Paro and pOpt_mVenus_Hyg expression vectors; fusion of CK and IPK with YFP or CFP in various arrangements (V5, 6, 7, and 8). All constructs in this work were transformed into _C. reinhardtii_ strains and recovered under selective antibiotic conditions, as depicted. (**b**) Microplate reader-screened strains expressing fluorescent fusion proteins. Three biological replicates were used to calculate the fluorescence intensity averages and the standard deviations (SD) in all cases. Statistical significance (one-way ANOVA) compared to UVM4 is represented by asterisks (** indicates a difference at the \(p\leq 0.01\) level). Among the V5, V6, V7 and V8 constructs, in which the ScCK and AtIPK are expressed together in a single fusion protein, the V7-3 and V7-28 strains gave a higher IPP yield. ScCK-GSGSGS-YFP-AtIPK (strain V7-3) exhibited the highest production, achieving 2.7 mg/L IPP, which was 4-fold higher than that of the parental UVM4 strain. Similar to the UVM4, the V8 (AtIPK-GSGS-CF-ScCK) strains had a low fluorescence intensity compared with other strains, suggesting that V8 strains may have lower expression levels of ScCK and AtIPK, resulting in lower IPP productivities. The results indicate that individual expression of the two enzyme activities, albeit as fusion proteins with fluorescent proteins, is better than the incorporation of both activities in the same protein. Some previous studies also showed that the direct fusion of proteins can result in an impaired biological activity, probably because the functional domains are brought too close together to properly interact with their corresponding binding proteins (i.e., receptors or ligands) [23] or because of other steric effects. To determine the toxicity of the reaction substrates in _C. reinhardtii_, UVM4 cells were cultured in a TAP medium supplemented with different concentrations of isoprenol or prepnol. _C. reinhardtii_ can survive, but not grow, in a TAP medium supplemented with a maximum of 30 mM isoprenol or 20 mM phenol, while a concentration of 20 mM isoprenol or 10 mM prepnol is suitable for the growth of _C. reinhardtii_ (Supplementary Figure S1). On the one hand, the concentration of isoprenol affects the growth of _C. reinhardtii_; on the other hand, it will affect IPP production. Therefore, it is necessary to explore the optimal substrate concentration. The transformant was cultured in a TAP medium supplemented with different concentrations of isoprenol until the mid-logarithmic phase or stationary phase was reached, and the IPP content was quantified.