## 4 Discussion
Unannotated enzymes catalyze the conversion of zeinoxanthin to lutein and \(\alpha\)-carotene to \(\alpha\)-cryptoxanthin (Figure 4). CrtR-b catalyzes the conversion of \(\alpha\)-Carotene to zeinoxanthin and \(\alpha\)-Cryptoxanthin to lutein, respectively. Recently, this enzyme is found in the chloroplasts of tomato, tobacco and _Haematococcus pluvialis_[60]. It is an oxidoreductase, with reduced iron-sulfur protein as one donor, which can introduce hydroxyl groups to the donor molecule [60]. CrtZ catalyzes the conversion of \(\beta\)-carotene to zeaxanthin, \(\beta\)-carotene hydroxylase (CrtZ) acts as a hydroxylase. Meanwhile, \(\beta\)-carotene hydroxylase (CrtZ) is reported to be the rate-limiting enzyme in astaxanthin biosynthesis [61]. In eubacteria and cyanobacteria, multiple genes encoding \(\beta\)-carotene 3-hydroxylase, which are responsible for the conversion of \(\beta\)-carotene to zeaxanthin [61]. Furthermore, CrtZ adds a hydroxyl group to the 7,8-dihydro-\(\beta\) end group and the \(\beta\) end group in _Erwinia_[57]. There are also other types of tomato erythromycincisases have been reported to be CHXB and CHXE during the carotenoid biosynthesis in strawberry. The enzyme CHXB is responsible for the conversion of \(\alpha\)-carotene to zeinoxanthin, \(\beta\)-carotene to \(\beta\)-cryptoxanthin and \(\beta\)-cryptoxanthin to zeaxanthin. The enzyme CHXE is responsible for catalyzing the conversion of zeinoxanthin to lutein [62]. The homology and other characteristics of CrtZ, CHXB and CHXE need be further clarified. The carotenoid epsilon-hydroxylase (EC: 1.14.14.158) is found in the cytochrome enzyme family in _Arabidopsis thaliana_, and it is named the LUT1 responsible for catalyzing the conversion of zeinoxanthin to lutein. Although the hydroxylase LUT1 is not annotated in these two strains in this study, a new carotenoid epsilon-hydroxylase or gene may be discovered based on the prediction of metabolic process [63]. The MEP pathway is normally present in most bacteria, green algae and plant plastids. However, IPP and FPP molecules are also produced via the mevalonate (MVA) pathway in archaea, fungi, higher plant cytoplasm and other eukaryotes [64; 65]. In watercress and microalgae, the key steps are the conversion of three IPP molecules and DMPP by the enzyme (GGPPS/GGPS/FPPS) to generate GGPP by the condensation process [9; 11; 26]. In living organisms, the condensation of IPP with DMPP to form GPP is catalyzed by geranyl diphosphate synthase (GPS). Finally, GPP is condensed with one molecule of IPP to form FPP by farnesyl diphosphate synthase (FPS) [66]. In eukaryotes, especially higher plants, lutein and zeaxanthin are primarily synthesized from terpenoids. Two molecules of GGPP are used to synthesize phytoene. Phytoene is then converted to \(\zeta\)-carotene, after which \(\zeta\)-carotene is converted into lycopene. Lycopene is then converted to \(\alpha\)-carotene by lycopene cyclase (\(\varepsilon LCY/\beta\)LCY). Finally, the formation of lutein from \(\alpha\)-carotene is catalyzed by \(\alpha\)-carotene hydroxylase (\(\beta\)CHX/\(\varepsilon\)CHX) [67]. Recently, the biosynthetic pathway from GGPP to zeaxanthin is also found in various organisms, such as: _Echinicola marina_, _Acaryochloris_, _Prochlorococcus_ and _Synechococcus_ PCC7942 [12; 65; 68; 69]. _Splingomonas_ sp. SG73 is the same genus as _Splingomonas morindae_ sp. NBD5, the biosynthetic pathway from zeaxanthin to nostoxanthin is further discovered [70]. Based on Liquid Chromatography-UltraViolet-Mass Spectrometry (LC-UV-MS/MS) analysis, three biosynthetic pathways from GGPP to lutein, zeaxanthin, dielectospirilloxanthin are discovered in _Flavobacterium_[13]. Further, we optimized the strain NBD5 fermentation culture by temperature, carbon source and nitrogen source, and obtained lutein yield of 1.6 mg/g, 1.8 mg/g and 1.9 mg/g, respectively [15]. In this article, the biosynthetic pathways of lutein and zeaxanthin are obtained from the genome annotation data of _Splingomonas morindae_ sp. NBD5 and _Splingopyxis_ sp. USTB-05.