## Results
### Expression and Subcellular Localization of Putative HMGR and tHMGR  
While the core reactions in sterol synthesis being conserved in _T. pseudonana_ and _P. tricornutum_[15], both diatoms produce a distinctive profile of sterol compounds which variates differently upon changing environmental conditions and chemical inhibitors treatment [15; 20]. Additionally, the sterol metabolism of the centric diatom _T. pseudonana_ has not been explored to the same depth as the model pennate _P. tricornutum_. To investigate the subcellular localization and evaluate the effect of overexpression of the rate-limiting enzyme HMGR on sterol accumulation, _T. pseudonana_ and _P. tricornutum_ were transformed with episomes containing their respective putative HMGR copy fused to mVenus, driven by a constitutive promoter (Figure 1, Figures S1 and S2). Episomes are maintained extra-chromosomally and therefore enable more consistent expression required for metabolic studies [37]. The trans-membrane domains of HMGR enzymes in mammals and yeast have been reported to contain a sterol sensing domain (SSD) that regulates expression and degradation of the enzyme [38]. Although our results suggest that diatom HMGRs lacks an SSD (Figure 2), we designed an N-terminal truncated version, tHMGR, to evaluate whether an unknown regulatory sequence is present in N-terminal region affecting activity of HMGR in diatoms. This tHMGR sequence encoded solely the C-terminal catalytically active region of the enzyme.  
The mVenus fluorescence was measured by flow cytometry and used as an indirect proxy of enzyme expression, since each expression system was C-terminal fused with mVenus protein. Three clones per expression system with the highest median mVenus signal were chosen for full scale experiments. In _P. tricornutum_, time course median mVenus fluorescence in mVenus control clones was 10-fold compared to WT, which confirms the effectiveness of the chosen promoter (Figure S4). Conversely, median mVenus fluorescence clones expressing HMGR-mVenus and tHMGR-mVenus was  
Figure 2: Maximum likelihood phylogenetic tree of diatom HMGR proteins and its domains. Numbers at the nodes represent bootstrap support (1000 replicates). HMGR from yeast, mammals and plants were used as outgroups. Arrows represent start of N-terminal truncated version for _P. tricornutum_ and _T. pseudonana_ used in this study.  
1.3-fold compared to WT, indicating an apparent regulation process occurring over the fused proteins (Figure S4). In _T. pseudonana_, median mVenus fluorescence in HMGR-mVenus, tHMGR-mVenus, and mVenus control clones appear similar to WT signal, suggesting that low expression was achieved using the _EF2_ promoter (Figure S5). However, confocal microscopy images confirmed expression of mVenus in both diatom species (Figure 3). Different cellular localizations were observed for each genetic construct. Images of control cell lines showed mVenus expression localized in the cytoplasm (Figure 3), while no mVenus fluorescence was detected in WT diatoms. mVenus fluorescence in exconjugants overexpressing HMGR-mVenus was detected around the chloroplast, suggesting that putative HMGR is localized in the endoplasmic reticulum, ER, which tightly surrounds the chloroplasts in diatoms [39; 40]. Conversely, tHMGR-mVenus localizes in the cytoplasm, consistent with truncation of the \(N\)-terminal membrane domain (Figure 3).