## 8 Highly Branched Isoprenoids in _Haslea_
### Terpenoid and HBI Biosynthetic Pathways  
Terpenoid biosynthesis can be divided in three distinct stages, i.e., the early, central and late enzymatic phases. Despite their structural diversity, all terpenoids are derived from the C\({}_{5}\) isopentenyl pyrophosphate (IPP) unit and its isomer dimethyl allyl pyrophosphate (DMAPP), which are synthetized in the early enzymatic steps [161]. IPP and DMAPP are produced by two distinct pathways: the cytosolic mevalonate pathway (MVA) and the plasidic methylerythriotl 4-phosphate pathway (MEP). The MVA pathway is present in archaebacteria and in the cytosol of higher plants, while the MEP pathway is present in  
Figure 9: Structure of different terpenoids produced by diatoms: (**a**) carotenoids; (**b**) sterols; and (**c**) highly branched isoprenoids. Figure adapted from [143]. eubacteria and in the chloroplast of higher plants [162]. The MVA pathway produces only IPP, while the MEP pathway produces both IPP and DMAPP with an IPP:DMAPP ratio of 85:15 [163]. The IPP:DMAPP ratio is regulated by the enzyme isopentyl diphosphate isomerase (IDI), which catalyzes the interconversion of IPP into DMAPP, marking the starting point of terpenoid biosynthesis in plants [164]. In the central steps, IPP and DMAPP are condensed by chain-elongating _trans_ and _cis_-premyltransferases (PTS) to give rise to prenyl chains of different lengths, i.e., C\({}_{10}\) mono-, C\({}_{15}\) sesqui-, C\({}_{20}\) di-, C\({}_{30}\) tri-, C\({}_{40}\) tetra- and C\({}_{n}\) poly-terpenes [141]. Subsequently, the C\({}_{10}\) monoterpene geranyl diphosphate (GPP), C\({}_{15}\) sesquiterpene farnesyl diphosphate (FPP), and C\({}_{20}\) diterpene geranylgeranyl1 diphosphate (GGPP) are further condensed to give rise to higher terpenoids (e.g., the C\({}_{30}\) squalene and the C\({}_{40}\) phytoene, which are formed by the condensation of two FPP and GPP molecules, respectively). In the final steps, linear terpenoids are subjected to modifications, including cyclization, reduction, isomerization, substitution, and conjugation [165]. Knowledge of the enzymes involved in HBIs biosynthesis in diatoms is still in its infancy. Recent advances in the elucidation of the early and central steps of terpenoid biosynthesis in diatoms have, however, shed some new light on this topic [166; 167; 168; 169]. Di-atoms seem to have retained both the MVA and MEP pathways, but there are differences across species in the allocation of the precursors towards the synthesis of different isogenoids. In _P. tricornutum_, _N. ovalis_ and _R. setigera_ the final products of the MVA pathway are directed toward the synthesis of sterols in the cytoplasm, while the final products of the MEP pathway are involved in the synthesis of plastic compounds, such as carotenoids and phytol. In contrast, _H. ostrearia_ and the centric diatom _T. pseudonana_ use MEP-derived precursors for the synthesis of their sterols [148]. This difference is likely to also apply to HBI biosynthesis. Using isotopic label experiments, [PERSON] and co-workers determined the involvement of the MVA pathway in the biosynthesis of C\({}_{25}\) and C\({}_{30}\) HBIs in _R. setigera_, while the MEP pathway appeared as the major route for the biosynthesis of isoprenoids in _H. ostrearia_. This result, however, could be explained by the inability of the diatom to undergo mixotrophic or heterotrophic growth or the existence of a compensation mechanism between the two pathways [170]. The identity of the specific enzymes involved in HBI biosynthesis downstream of the MVA and MEP pathways awaits characterization. The available information indicates that a farnesyl diphosphate synthase (FPPS) is likely to be involved in the synthesis of C\({}_{25}\) and C\({}_{30}\) HBIs in _R. setigera_[168]. FPPS is one of the most-studied _trans_-PTSs. It catalyzes the formation of FPP, first by a head-to-tail condensation of DMAPP and IPP, to which a second IPP is added, all in _trans_ geometry [171].