## 3 Discussion
This is approximately 17-fold higher than the yield from UVM4 and demonstrates that the IUP significantly enhances the C5 hemiterpene titer in _C. reinhardtii_. In this study, the IPP titer was highly improved by the IUP, but the limonee yield was still far lower than other species. Microalgae naturally generate a turnover of isoprenoid products, the FPP synthase (FPPs) catalyzes the sequential condensation of IPP and DMAPP to the FPP (C15), which is the precursor of sterols and ubiquitin (UQ) [9]. The FPPs will compete with GPP synthase (GPPs) to use IPP and DMAPP, which will decrease the IPP and DMAPP flux into GPP. Moreover, microalgae naturally generate a turnover of isoprenoidsinvolved in photosynthesis, e.g., carotenoids and the phytol chain of chlorophyll molecules, which are derived from geranylgeranyl pyrophosphate (GGPP, C20). The GGPP synthase (GGPPs) catalyze the GPP to form the GGPP. The GGPPs will compete with LS in order to use GPP, which will decrease the GPP flux into limonene. What's more, the low catalytic activity of terpene synthases is another important challenge in addition to the precursor supply [39]. The GPPs and LS should have enough accumulation to outcompete the native metabolism for the use of IPP, DMAPP, and GPP. Most studies that have attempted to improve the limonene production have focused on increasing the terpene precursor titer [15], for example, by introducing a complete MVA pathway into yeast peroxisomes [40]; overexpressing rate-limiting enzymes of the MEP pathway in cyanobacteria [41]; using aryl diphosphate (cis-isomer of GPP) as a substrate in _S. cerevisiae_ and _E. coli_[24]; overexpressing genes in the pentose phosphate (PP) pathway to enhance the C5 hemiterpene levels in the cyanobacterium Synechocystis sp. PCC 6803 [13]; engineering a PDH bypass to enhance the biosynthesis of cytosolic acetyl-CoA in _S. cerevisiae_[5]; or improving the GPP synthesis by a degradative regulation of ERG20 and mutated ERG20 WW in _S. cerevisiae_[42]. These results demonstrate that improving the availability of the substrate is an alternative strategy for boosting the limonene production. It is also clear that the strains expressing the IUP can serve as a reliable chassis for a high-value terpenoid production. Growing the above strains for 7 d in the opt2 medium supplemented with 10 mM isopropanol and a 15 mL dodecane overlay under a L:D regimen resulted in the highest limonene production levels (Figure 6). Under the optimized fermentation conditions, a maximum limonene content of 117 ug/L was achieved, which is the highest reported to date in _C. reinhardtii_. However, this yield was still far lower than that of [PERSON] and colleagues [28], which was obtained by engineering the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Compared with C. _reinhardtii_, the cyanobacteria have an ease of overexpression, have more reliable genetic tools available, and have a higher growth rate and biomass, which may be the reason for the limonene yield gap. Compared with other studies, by only improving the IPP production may not be enough to significantly increase the limonene production. To further improve the production of limonene, we should improve GPP and/or NPP synthesis which will produce more limonene precursor GPP or NPP for producing limonene. Microalgae naturally generate a significant turnover of isoprenoid products, such as sterols and UQ [9] and pigments associated with their color and photosynthetic machinery [4]. Sterols and UQ are derived from FPP (C15), and isoprenoids involved in photosynthesis, e.g., carotenoids and the phytol chain of chlorophyll molecules are derived from GGPP, (C20). Taking this into account, we should decrease the metabolic flux of the compete pathway of GPP flowed to FPP to produce sesquiterpenes or GGPP to produce the photosynthetically associated isoprenoid products that are more beneficial for the GPP supply and the limonene biosynthesis.