## 3 Discussion  
_C. reinhardtii_ is typical of Chlorophyta in solely relying on the MEP pathway in the chloroplast to supply C5 terpenoid precursors (IPP and DMAPP). Thus, enhancing C5 hemiterpene titers is crucial for producing high-value terpenoids. _C. reinhardtii_ chloroplasts can express foreign genes and accumulate heterologous products such as 13R(+) manoyl oxide [10]. However, the position effects and transgene silencing [34] can lead to large variations in the gene expression. As a result, it is usually necessary to screen many transformants to identify a strain with acceptable levels of the transgene expression [35]. Therefore, in this study, we expressed the foreign genes in the nuclear genome of _C. reinhardtii_ and targeted the protein products to the algal chloroplast to produce terpenoids. The V3 and V4 constructs generally showed a higher IPP yield than the V5, V6, V7 and V8 constructs, except that V3 was less productive than V7. Strain V4-49 produced IPP at 2-fold higher levels than strain V7-3 (Figure 3a). This indicates that, the fusion of ScCK and AtIPK in a single polypeptide is possible to express functional products, but the yield of the fusion enzyme is lower than that of the enzyme expressed individually. The expression of consecutive enzymes in a metabolic pathway as the multidomain fusion proteins can lead to a more effective substrate utilization and thereby increase production [9; 19; 20; 21; 22], but the nature of the linker peptides used may result in fusion proteins being misfolded, or in a low yield of the multidomain protein, or in a reduced bioactivity [23]. Although the fusion reduces the distance between the respective enzymes, the folding and natural conformation of the multidomain protein can be impaired compared to the free enzymes, and thus can hinder their catalytic ability [24]. The large size of such constructs can also cause false-positive transformants in _C. reinhardtii_[8], which would explain why the IPP yield was lower with the multidomain fusion enzymes. The limited supply of the precursors is one of the main challenges for the heterologous terpenoid synthesis in microbial hosts [36]. Enhancing the terpenoid titers has often relied on precursor pathway engineering, making both the MEP and MVA pathways critical engineering targets [10]. However, the MEP pathway is highly regulated at the genetic and metabolic levels [37], thus increasing the difficulty of engineering this precursor pathway. Previous studies have engineered the rate-limiting enzymes of the MEP pathway or have used the heterologous expression of the MVA pathway to increase the metabolic flux towards the intermediate metabolites IPP and DMAPP in algae, but with little improvement in the target production [10] or where improvements only emerged after multiple rounds of transformation and selection [38]. For _C. reinhardtii_, the relatively few selectable markers available limit the number of the engineering steps possible [8]; thus, it would be difficult to engineer the seven or six enzymatic steps of the MEP or MVA pathways, respectively, to enhance IPP and DMAPP production in this algae. Recently, an artificial IUP was developed to produce IPP or DMAPP via the sequential phosphorylation of the corresponding alcohol precursors [3; 16]. The IUP is much simpler than the natural MVA or MEP pathways, requiring only two reaction steps. It circumvents the intricate regulation required to achieve a high-level accumulation of C5 hemiterpenes via the natural routes and tremendously simplifies the biochemical engineering of the high-value terpenoid production [3; 15; 16]. However, the IUP needed additional isoprenol, which will increase cost. In this study, we heterologously expressed the key enzymes of the IUP in _C. reinhardtii_, achieving a maximum IPP production of approximately 10.5 mg/L in the V4-49 strain when grown in a TAP medium supplemented with 15 mM isoprenol (Figure 3b).