## Discussion
### Levels of End-Point Campesterol Increased after Heterologous Expression of NoSQE in P. tricornutum  
Diatoms possess a distinct AltSQE [16] catalyzing the conversion of squalene into 2,3 epoxysulene which is then transformed into cycloartenol, the first committed step towards the production of steroids (Figure 1). Whether the presence of an AltSQE confers diatoms and other microleukaryotes with specific biological advantages is not yet known. Similarly, AltSQE and SQE are mutually exclusive and, to date, no organisms have been found to naturally harbor both [16]. The genetic manipulation of SQE and squalene synthase, SQS, has been extensively used for enhanced production of squalene and triterpenoids [54; 55; 56]. Point mutations in the SQE gene (_ERG1_) in yeast resulted in accumulation of squalene [57]. Similarly, accumulation of squalene was observed in the green microalgae _Chlamydomonas reinhardtii_ after knocking down the SQE gene, while co-transformation lines with SQE-overexpression and SQE-knockdown yielded similar amounts of squalene [58]. This is the first study to investigate the response of diatoms to the expression of a conventional SQE. We did not observe any significant changes in growth and photosynthetic phenotypes of _T. pseudonana_ and _P. tricornutum_ expressing heterologous NoSQE (Figures S6-S11). This suggests that in diatoms there is no apparent toxicity or physiological reason for the mutual exclusivity between AltSQE and conventional SQE. Confocal microscopy images of lines expressing NoSQE-mVenus fusion proteins, revealed that heterologous NoSQE was proximal to the chloroplasts, indicating that diatoms could recognize the ER signal peptide on the heterologous NoSQE, localizing it in the ER membrane (Figure 3 and Figure S3), just as the endogenous AltSQE ([PERSON] et al., 2019) and native SQE enzymes are in other species [59; 60]. Significant accumulation of cycloartenol (1.8-fold) and obtusifoliol (1.8-fold) intermediates, but not of 2,3 epoxysulene, was obtained for _P. tricornutum_ lines expressing NoSQE-mVenus (Figure 4b). These intermediates occurred after the formation of 2,3 epoxysulene, which is the product of the reaction catalyzed by SQE (Figure 1). As expected, we did not observe increased accumulation of squalene, which is the substrate for SQE, contrary to accumulation obtained by expressing HMGR-mVenus which is upstream of squalene production (Figure 1). Nevertheless, heterologous expression of NoSQE-mVenus resulted in a 2-fold increase of campesterol, an end-point sterol. Accumulation of intermediates was higher in _P. tricornutum_ cell lines expressing HMGR compared to those expressing NoSQE-mVenus (Figure 4a). This indicates that intermediates accumulated by MVA pathway manipulation (i.e., HMGR) do not necessarily increase the flux to brassicared, suggesting that sterol regulation is occurring at the conserved core point and at other points further down the metabolic pathway. In particular, this might suggest that in _P. tricornutum_ the epoxidation of squalene might be involved in pathway flux modulation, as observed in mammals [33; 34], and that, to complete the scenario suggested by the results obtained by expressing NoSQE, it is plausible that an additional pathway checkpoint exists at the level of the C22-desaturation (E.C 1.14.19.41, Phatr3_J51757) [14]. When treated with funconazole and fenpropimorph, inhibitors targeting upstream of campesterol, the transcription of _Phatr3_J51757_ significantly increases [15]. This further supports the hypothesis that the last reaction in sterol synthesis could be a highly regulated point to maintain stable sterol levels in the cell. These results suggest that to observe changes in end-point sterol compounds, increasing the precursors pool is not enough; genetic manipulation should target other points further down in the metabolic pathway, such as committed steps in sterol synthesis. A future co-expression approach to increase end-point sterol compounds in _P.