## Discussion
### HMGR Overexpression Lead to Accumulation of Sterol Pathway Intermediates in P. tricornutum  
In this study, we investigated the response of diatoms to genetic targeting of sterol biosynthesis, through manipulation of the MVA rate-limiting enzyme HMGR. The fluorescent localization of extra-chromosomally expressed HMGR-mVenus to the membrane surrounding the plastid is consistent with ER localization of proteins, including AltSQE, from previous studies in _P. tricornutum_[16; 39] and _T. pseudonana_[43]. Therefore, our study provides evidence that diatom HMGR is localized to the ER, just as in mammals, yeast, and higher plants [28; 44]. The truncation of the native HMGR sequence resulted in cytoplasmic localization, demonstrating that the signals for protein targeting are in the _N_-terminal portion of the protein sequence. In some cases, overexpression of HMGR has been showed to slightly increase sterol and intermediates production in other organisms. The overexpression of HMGR from the plant _Panax ginseng_ in _Arabidopsis thaliana_ resulted in a nearly two-fold increase of sitosterol, campesterol, and cycloartenol, while levels of squalene and stigmasterol did not significantly change [45]. In a more recent study, [PERSON] et al. [46] independently over-expressed all genes participating in the MVA pathway, obtaining a significant increase in total sterols when expressing HMGR (3.4-fold) and 3-hydroxy-3-methylglutaryl-co-enzyme-A synthase, HMGS (2-fold). The overexpression of native HMGR in _Arabidopsis_ (_HMG1_) led to high levels of HMGR mRNA, but only a slight increase in HMGR activity, and no changes in leaf sterol levels [47]. Heterologous expression of HMGR from _Heeva brasiliensis_ in tobacco, however, showed an increase in HMGR transcript and total sterol from leaves [48]. In this study, overexpression of endogenous putative HMGR-mVenus in _P. tricornutum_ resulted in an increased accumulation of the intermediates squalene, cycloartenol, and obtusifoliol and decrease in the end-point sterol campesterol. However, we did not detect 2,3 epoxysqalene, indicating possible differences on the catalytic rates of the enzymes squalene epoxidase and cycloartenol cyclase (Figure 1). While accumulation of squalene is detectable, 2,3 epoxysqalene seems to be rapidly converted into cycloartenol. Accumulation of 2,3 epoxysqalene has been reported before by chemically inhibiting cycloartenol cyclase with Ro 48-8071 [14; 15]. Accumulation of intermediates suggests that in _P. tricornutum_, overexpression of HMGR-mVenus boosted production of presumably MVA-derived intermediates, IPP, and DMAPP that are subsequently converted into squalene (Figure 1). Even though MVA products also serve as building blocks of other isoprenoids [49], perturbation of the rate-limiting step catalyzed by HMGR was sufficient to cause accumulation of downstream intermediates committed to sterol biosynthesis, such squalene, cycloartenol, and obtisofiloli. However, this metabolic bottleneck did not translate into overall increase of sterol compounds; on the contrary, levels of the end-point sterol campesterol were reduced and brassicasterol levels remained unchanged. Although fluorescence levels in _P. tricornutum_ exconjugants expressing the target enzymes were considerably lower compared to the expression of mVenus alone (Figure S5), phenotypic changes in terms of sterol profiles indicate that the level of expression was sufficient to cause perturbations in the metabolic pathway (Figure 4). Moreover, since all the enzymes expressed are membrane proteins, as confirmed by confocal microscopy (Figure 3 and Figure S3), fluorescent signal could have been hindered and no direct correlation with expression could be assumed. The absence of detectable sterol pathway intermediates in _T. pseudonana_ transformants (Figure S12) may be related with the promoter chosen for overexpression of the target enzymes, as higher expression may be necessary to trigger the accumulation of intermediates observed in _P. tricornutum_.