## 4 Discussion  
The MYB TFs are a large transcriptional family found in plants and play an important role in the regulation of plant secondary metabolites and other functions [42; 43]. In previous studies, some MYB TFs have been described, which play key roles in the regulation of terpenoid biosynthesis in plants [44; 45] and green microalga [29; 46]. In _Tripterygium wilderii_, _BpMYB21_ TFs are involved in the regulation of squalene through the upregulation of squalene epoxidase a and Cycloartenol synthesis [47]. _CiMYB42_ TFs from citrus regulate the triterpenoids and transcript level of _SQS_ involved in squalene synthesis [48]. By comparison, evidence on the transcriptional regulation of triterpenoids synthesis by MYB TFs in _C. reinhardtii_ is extremely limited. In the current study, we have investigated the role of heterologous overexpression of _MYB36_ in the regulation of triterpenoids in _C. reinhardtii_ and its effect on the upstream and downstream of the MEP pathway and its derivative biosynthetic pathway. We have obtained the _R2R3 MYB36_ (KF059390.1) TFs from the National Center for Biotechnology Information (NCBI) website. R2R3 MYB TFs belong to the medicinal plant _Salvia milliorrhiza_. _S. miliorrhiza_ contains various biologically active compounds, which have great potential to treat different diseases, including cerebrovascular diseases and others [49]. MYB36 showed an imperative role in the regulation of terpenoids, especially tanshinones and phenolic acid [49]. In order to improve transgene expression, _MYB36_ transcriptional factors were re-designed synthetically by codon optimization for heterologous expression in _C. reinhardtii_, as previously described [33]. After codon optimization, the MYB gene's GC contents increased from 48% to 65%. In order to analyze the foreign transcriptional factor, R2R3-MYB36 overexpressed in _C. reinhardtii_. We have found that the genes encoding for the  
Figure 5: Concentration of squalene in MYB36-overexpression lines. The peak with 15.61 retention time is the squalene. upstream MEP biosynthetic pathway, including _CrDXS_, _CrDXR_, _CrCMS_, _CrCMK_, _Cr. HDS_, and _Cr. HDR_ genes, were upregulated in the MYB36-overexpression lines. Previously similar studies showed that _SmMYB97_ TFs belong to the medicinal plant _S. milliorrhiza_ and affect the MEP biosynthetic encoding genes in its overexpression lines [27]. SmMYB98 TFs also upregulate upstream MEP biosynthetic encoding genes in transgenic lines [28]. In higher plants, _DXS_ and _DXR_ have a significant task in the regulation of flux in the MEP pathway [35]. Overexpression of _DXS_ accumulated the isoprenoid production in transgenic Arabidopsis, suggesting that catalyzing of DXS is one of the rate-limiting steps involved in the MEP biosynthetic pathway [36]. _DXR_ and _HDR_ are other enzymes that play a function as rate-limiting factors in the formation of IPP and DMAPP [37]. Recently, investigations on DXS have attained more attention in order to modify terpenoid metabolisms. Methyl jasmonate (MeJ)-treated _C. reinhardtii_ cells increase the expression of _DXS_ leading to increase the triterpenoids' squalene synthesis [38]. Heterologous overexpression of _DXS_ from _S. pomifera_ in _C. reinhardtii_ improved flux toward diterpenoid production via the MEP biosynthetic pathway [24]. MEP plays a crucial function in the control of the MEP biosynthetic pathway. MEP triggers the MDS gene expression, which is the initial obligated precursor of the pathway [39]. MYB36-overexpression lines may accumulate geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) as a result of an altered metabolism toward IPP. The expression levels of FPP and GGPP were significantly higher in the MYB36-overexpression _C. reinhardtii_ lines compared to parent lines. Our results were similar with a previous study, which indicated that accumulation of GGPP expression in MeJ-treated cells of _C. reinhardtii_ improved the terpenoid production [38]. The FPP (C15) is formed by the condensation of IPP and DMAPP.