# Marine Microalgae for Potential Lutein Production
## 3 Putative Biosynthetic Pathway of Lutein in Microalgae  
Lutein is found within the members of Chlorophyta, Chlorarachniophyta, Cryptophyta, Euglenophyta, and Rhodophyta algal species [17]. Knowledge of the biosynthetic pathways for lutein biosynthesis in microalgae is limited. The current understanding is based on identified chemical structures of carotenoids found in microalgae. It is now believed that all types of carotenoids, including lutein, are obtained from common five-carbon (C\({}_{5}\)) starting molecules isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These common metabolic precursors (IPP and DMAPP) might be derived from either of the two independent pathways: (i) the cytosolic mevalonate (MVA) pathway starting from Acetyl-CoA, or (ii) the plastid (chloroplast in microalgae) methylerythitol 4-phosphate (MEP) pathway starting from pyruvate [18]. However, there is evidence to suggest that the precursors for microalgal carotenoids including lutein biosynthesis proceed from the MEP pathway in _Dunaliella salina_, _Chlorella vulgaris_, _Scenedesmus_ sp. [18], and _Haematococcus pluvialis_[19] species. Lutein biosynthesis in microalgae begins in the MEP pathway (Figure 2) through the 5-carbon building block molecule isopentenyl pyrophosphate (IPP), which isomerises to dimethylallyl pyrophosphate (DMAPP) by the action of the enzyme IPP isomerase. Elongation of the carbon chain then takes place through continuous head-to-tail condensation of IPP to DMAPP followed by growing of the polyprenyl pyrophosphate chain by the action of the enzyme phenyltransferase [20; 21; 22]. As a result, geranylgeranyl PP (GGPP, C\({}_{20}\)), the first immediate precursor of lutein, is synthesised following a condensation reaction of GPP (C\({}_{10}\)) by the action of GGPP synthase. Next, the colourless C\({}_{40}\) carotenoid phytoene is formed through condensation of two GGPP (C\({}_{20}\)) molecules by the action of phytoene synthase (PSY). Next, lycopene (a coloured carotenoid with 13 double bonds and a chromophore of 11 conjugated double bonds) is formed by the conversion of phytoene (nine double bonds molecule) through stepwise desaturation reactions (or dehydrogenation reactions) catalysed by phytoene desaturase (PDS) and zeta-carotene desaturase (ZDS) enzymes. Lycopene is the precursor to the formation of both \(\alpha\)-carotene and \(\beta\)-carotene after two cyclisation reactions. The enzyme lycopene \(\beta\)-cyclase (_lcy-b_) catalyses the cyclisation of both ends of lycopene to make \(\beta\)-carotene with two \(\beta\)-rings. Meanwhile, the action of lycopene \(\beta\)-cyclase (_lcy-b_) and lycopene \(\varepsilon\)-cyclase (_lcy-e_) enzymes catalyse the cyclisation of both ends of lycopene to make \(\alpha\)-carotene with a \(\beta\)-ring and an \(\varepsilon\)-ring. This is an important branching point of the carotenoid biosynthesis pathway in microalgae, where one branch  
Figure 1: Schematic showing multiple potential health benefits of lutein. leads to the biosynthesis of \(\alpha\)-carotene and the other branch leads to the biosynthesis of \(\beta\)-carotene. The former is then converted to lutein in two hydroxylation steps, while the latter is converted to zeaxanthin and subsequently, to other carotenoids. The hydroxylation of \(\alpha\)-carotene at the C-3 and C-3' positions results in the formation of lutein and the enzymes involved in these processes are \(\beta\)-carotene hydroxylase and \(\varepsilon\)-carotene hydroxylase, respectively [23]. From the biotechnological perspective, a number of chemical inhibitors have been tested to regulate the carotenoid biosynthetic pathway in microalgae. For examples, [PERSON] et al. [24] studied the effect of the addition of 2-methylimidazole in _Dunaliella salina_ and found an increase of 1.7-fold lutein content and a related decline in \(\beta\)-carotene content.