## 1 Introduction
1.1.1.34) is one of the key enzymes in the MVA pathway and catalyzes the reduction of HMG-CoA to mevalonate [27]. HMGR is known as main regulator and rate-limiting enzyme in early biosynthesis of sterol and non-sterol isoprenoids in MVA-harboring eukaryotic cells and it is highly regulated at the transcriptional, translational, and post-translational levels [28]. In yeasts and mammals, HMGR contains a sterol sensing domain (SSD) that is responsible for detecting sterol levels in the cell and maintaining sterol homeostasis [29]. The SSD is located in the N-terminal membrane binding domain of the HMGR enzyme [28]. Moreover, it has been reported that genetic manipulations on HMGR, including truncation of N-terminal domain, led to considerable accumulation of terpen in transgenic plants and yeast [30; 31; 32]. Although HMGR has been extensively characterized in model eukaryotic organisms, little is known about its features in diatoms. MVA products IPP and DMAPP are subsequently used for the synthesis of squalene, the first committed intermediate in the formation of sterols [33] (Figure 1). In plants, fungi, and animals, squalene is converted into 2,3 epoxysqualene. This reaction is conventionally catalyzed by the enzyme squalene epoxidase (SQE, E.C. 1.14.17) [33]. Several studies indicate that SQE is a control point in cholesterol synthesis modulated by sterol levels and post-translationally regulated by cholesterol-dependent proteasomal degradation [33; 34]. However, diatoms do not possess a conventional SQE, and instead this step is catalyzed by a recently characterized alternative squalene epoxidase (AltSQE) [16]. The diatom AltSQE belongs to the fatty acid hydroxylase superfamily and differs from the conventional flavoprotein SQE. Whether AltSQE has a similar role to SQE in sterol regulation is not known. While selective inhibitors for AltSQE are not known, the treatment of diatoms with statins, known to inhibit _HMGR_ enzymes, has resulted in perturbation of isoprenoids metabolism that included an overall decrease of total sterols content [24; 35], suggesting that a conventional _HMGR_ enzyme  
Figure 1: Upstream reactions and conserved core of sterol biosynthesis pathway in diatoms and genetic targets overexpressed in this study, highlighted in orange. Mevalonate pathway, MVA; 3-hydroxy-3-methylglutaryl-coenzyme A reductase, HMGR; truncated HMGR, tHMGR; alternative squalene epoxidase, AltSQE; squalene epoxidase from _N. oceanica_, NoSQE; oxidosqualene cyclase, OSC. might be involved in the pathway. In this work, we genetically targeted the HMG CoA reduction and squalene epoxidation steps in _P. tricornutum_ to provide further insights into the nature of the diatom sterol biosynthesis pathway and its regulatory constraints. Considering the demonstrated challenges in genetically down-regulating the essential genes involved in _P. tricornutum_ sterol metabolism [14; 16], we chose to investigate these pathway nodes by gene overexpression and subcellular localization. We generated independent diatom exconjugant lines constitutively expressing (i) either the full-length _HMGR_, (ii) an N-terminal truncated version of _HMGR_ (_HMGR_), or (iii) a heterologous SQE from _Nannochloropsis oceanica_ (_NoSQE_) over the background of the endogenous AltSQE. By phenotyping these transgenic diatom cell lines, we describe specific changes in several nodes of the sterol biosynthesis pathway and provide evidence for regulatory mechanisms unique to diatom sterol metabolism.