## Results
### Influence of HMGR and tHMGR Expression on Sterol Levels in T. pseudonana and P. tricornutum  
Although expression of transgenes appeared to be low according to mVenus fluorescence levels, we proceeded to identify changes in sterol profiles in the transgenic lines. Sterols were extracted from exconjugants in the mid-exponential phase, which was the time period with the maximum observed mVenus fluorescence (Figures S4 and S5) with enough biomass to sample for sterol extraction (determined to be 48 h growth for _T. pseudonana_ and 72 h for _P. tricornutum_). After 75 h, cell density of _P. tricornutum_ HMGR-mVenus and tHMGR-mVenus was 1.4 times lower than WT, while no growth impairment was observed for mVenus exconjugants (Figure S6). No differences in chlorophyll levels and in effective quantum yield of PSII were observed in _P. tricornutum_ exconjugants (Figures S7 and S8). Similarly, no growth impairment, chlorophyll levels, or differences in effective quantum yield of PSII compared to WT were observed for _T. pseudonana_ exconjugants (Figures S9-S11). In _P. tricornutum_ overexpressing HMGR-mVenus, squalene levels were ten times higher in HMGR-mVenus exconjugants than in WT and mVenus controls. Moreover, a 3-fold increase in  
Figure 3: Confocal microscopy images showing subcellular localization of the mVenus fusion with target proteins in representative transgenic (**a**) _P. tricornutum_ cells, scale bars correspond to 5 μm, and (**b**) _T. pseudonana_ cells, scale bars correspond to 10 μm. Wild type (WT) as negative control and control cell lines that only expressed mVenus. 3-hydroxy-3-methylglutaryl-coenzyme A reductase, HMGR-mVenus; truncated HMGR, tHMGR-mVenus; squalene epoxidase from _N. oceanica_, NoSQE-mVenus. Scale bars correspond to 10 μm. cycloartenol and a 2.5-fold obtusifoliol accumulation was detected compared to WT (Figure 4). However, we did not detect the intermediate 2,3 epoxysqualene. Levels of end-point sterol campesterol decreased 2-fold, whereas no significant differences were observed in brassicastrol, the most abundant sterol in _P. tricornutum_. We detected traces levels of end point sterol 24- methylcholesta-5,24(24\({}^{\prime}\))-dien-3\(\beta\)-ol in the WT and mVenus controls, which is typically found in centric diatoms and has not been reported in _P. tricornutum_[18]. Levels of this end-point sterol were 17 times higher in two independent exconjugant lines expressing HMGR-mVenus. Total sterol levels were not affected despite significant changes in individual sterols (Figure 4). Since expression of mVenus, HMGR-mVenus, and tHMGR-mVenus in _T. pseudonana_ did not appear to be effective based on flow cytometry data (Figure S5), observed changes on sterol profiles may not be directly related to the overexpression of the targeted enzymes. _T. pseudonana_ cell lines transformed with _HMGR-mVenus construct exhibited a decrease in the minor sterols fucosterol and isofucosterol relative to WT control (Figure S12). However, isofucosterol reduction was also detected in the control expressing only mVenus (Figure S12). No intermediates were detected. Total sterol levels remained similar in the three independent cell lines studied; no significant differences were observed in comparison to the WT control (Figure S12). Expressing the catalytically active region of their putative HMGRs (tHMGR) was expected to reduce regulatory mechanisms that may affect HMGR activity in diatoms. In _P. tricornutum_, sterol changes in cell lines expressing tHMGR-mVenus were similar to those expressing HMGR-mVenus, with campesterol levels 2-fold less abundant than in HMGR-mVenus transformants (Figure 4). Figure 4: Sterol levels in _P. tricornutum_ transformants. (**a**) End-point sterol (**b**) intermediates accumulation. Identical letters denote no statistically significant differences among groups using the Pairwise Wilcoxon Rank Sum tests (\(p<0.05\), \(n=9\)). Traces of 24-methylcholesta-5,24(24\({}^{\prime}\))-dien-3\(\beta\)-ol were detected as in HMGR-mVenus expressing lines, 10 times higher compared to WT.