## 8 Highly Branched Isoprenoids in _Haslea_
### Terpenoid and HBI Biosynthetic Pathways
FPP serves as a substrate to squalene synthase (SQS), the first enzyme of the sterol biosynthesis pathway, which condenses two molecules of FPP to form squalene [172]. [PERSON] and co-workers showed that the overall quantity of HBIs produced by _R. setigera_ decreases in a dose-dependent manner when using the FPP inhibitor risedronate, suggesting that C\({}_{25}\) and C\({}_{30}\) HBIs are the result of a _head-to-middle_ condensation of an FPP and a C\({}_{10}\)- or a C\({}_{20}\)-terpene, respectively [168]. Whether FPPS takes part in the biosynthesis of HBIs in _Haslea_ is still under investigation. At the genetic level, a high degree of homology is observed among PTSs found in all domains of life. Protein sequence alignments reveal many conserved domains, among them the First and Second Aspartic Rich Motifs (FARM and SARM). Both domains are responsible for the binding of magnesium ions and are essential for enzymatic activity. In addition to the FARM/SARM, conserved residues located upstream of the FARM have been shown to regulate PTS product chain length [161]. Recent whole transcriptome analysis of the _H. ostrearia_ NCC 153.8 strain unveiled the presence of five PTS homologues. Phylogenetic analysis, subcellular localization prediction and functional characterization in the heterologous host expression systems of these PTS homologues provided the means to propose an original model for the central steps of terpenoid biosynthesis in _Haslea_ sp. First, an FPPS sharing 57% similarity with the _R. setigera_ FPPS was identified and named HoPTS1. This enzyme is probably localized in the endoplasmic reticulum membrane and produces FPP as a precursor to sterols. In addition to PTS1, four polyprenyl diphosphate synthases (HoPTS2-5) with orthologues present in other pennate and centric diatom species were identified. Among them, two were GGPP synthases--one likely to be cytosolic (HoPTS3) and a second one chloroplastic--which are possibly involved in carotenoid biosynthesis (HoPTS5). In addition to PTSs, a phytoene synthase (HoPSY) sharing 72% similarity with _P. tricorntum_ PSY was identified and predicted to be located in the chloroplast. Last, an SQS fused to an isoprenyl diphosphate synthase (HoSQS-IDI) was identified and predicted to be located in the cytosol [75]. Homologues of SQS-IDI fusions have been reported in other diatom species, such as _P. tricorntum_ and _T. pseudonana_, but also in some haptophyte and heterokontophyte algae, and have been proposed to act as multifunctional enzymes [167; 173]. Aiming to assess the suitability of _Haslea_ strain HoB4--isolated from the same location as strain NCC 153.8--as a model for the study of HBI biosynthesis in _Haslea_ sp., we investigated the possible presence of HBIs in this strain. The _H. ostrearia_ strain HoB4 was grown under standard laboratory conditions in non-axenic batch cultures to a total of 6 L. Cells were harvested by centrifugation and submitted to saponification according to [PERSON] et al., 1996 [153]. Subsequently, the presence of HBIs in the non-saponifiable hexane extract was investigated by GC-MS analysis (Figure 10a, unpublished results). Under our culture conditions, the main HBI produced was the C\({}_{25}\) tetraene (_E_)-2,6,14-trimethyl-10-methylene-9-(3-methylpent-4-en-1-yl)pentadeca-2,6-diene. The structure of this HBI was verified after HPLC purification, \({}^{1}\)H NMR analysis (Figure 10b) and comparison of its spectroscopic data with those previously reported by [PERSON] et al. from an _H. ostrearia_ strain isolated near the coast of England [153]. Thus, strain HoB4 produces HBIs and can be considered suitable to study HBI biosynthesis in _Haslea_ sp. Further experiments addressing the possible effects of environmental growth conditions, such as temperature, salinity and nutrient availability, on the quantity and diversity of HBIs produced by NCC527 may provide insights into the genes and functional roles of HBI biosynthesis in _Haslea_ diatoms.