## 3 Discussion  
Astaxanthin is one of the strongest antioxidants and its natural format is being demanded by increasing market looking for natural products [1]. Thereby, the topic of improving astaxanthin production from natural sources is attractive for the industry. Lots of effort has been made by scientists trying to design an efficient protocol for astaxanthin production using natural source [7]. As a major natural source of astaxanthin, _Haematococcus phyvialis_ accumulated as high as 5% dry weight of astaxanthin [21] and its dairy supplements can be used human-friendly [22]. It was found that two specific enzymes, \(\beta\)-carotene hydroxylase (CrtR-b) and ketolase (BKT), are crucial for astaxanthin formation [23; 24; 25; 26] and many researches were focused on aiming to improve astaxanthin content [27; 28; 29]. However, as the important precursor to initial the carotenoids synthesis in _H. pluvialis_, limited research was conducted for the regulation of geranylgeranyl pyrophosphate (GGPP) on astaxanthin production. It was recorded that GGPP synthase (GGPPS) plays an important role on GGPP formation [6]. In _Arabidopsis_, a well-studied species, 12 _GGPPS_ genes were found and 10 of them have the ability to produce GGPP [30]. Research suggested that GGPP formation was regulated with different pattern in _Arabidopsis_[30]. Previously, there are three _GGPPS_ genes from _H. pluvialis_ in Genbank and one (Genbank accession no. KX236181) have the activity to catalyze GGPP formation [31]. Unfortunately, no further information was reported regarding the other two _GGPPS_ genes (Genbank accession nos. KX236182, KX236183). With the expectation of discovering more paralogs having enzymatic activity, this study screened an Iso-Seq data covering transcripts from the control and stressed _H. pluvialis_ cells. The stress condition was high light and salt which was normally used to induce the astaxanthin formation [20]. Eight candidates were found and their gene structures were illustrated in this study (Figure 1), using the available genome sequences (BIG Data Center GSA Database accession no. PRJCA000614) [20]. Generally, two conserved aspartate rich motifs, FARM and SARM, and flexible CxxxC motifs are presented in the domain of amino acids of _GGPPS_ genes [16; 31; 32]. Based on the domain analysis, FARM and SARM motifs were presented in all eight _HpGGPPS_ genes while CxxxC motifs was only found in _HpGGPPS1-1_, _HpGGPPS1-2_, KX236181, and XM_001703117 (Figure 2). It has been documented that FARM, SARM and CxxxC motifs are important for GGPP biosynthesis by affecting the IPP and DMAPP substrate binding process and by affecting the physical interaction between subunits of heteroimeric GPP, respectively [31; 32]. Proteins with features of conserved FARM, SARM, and CxxxC motifs displayed the function of catalyzing GGPP formation [33], indicating similar function might be employed by _HpGGPPS1-1_ and _HpGGPPS1-2_. Differently, the CxxxC motifs was not found in other six _HpGGPPS_ genes (Figure 2). Such proteins with conserved FARM and SARM motifs but without CxxxC motifs was shown to be involved in the production of medium (C25) to long (C45) chain isoprenoids [34]. It is noted that six _HpGGPPS_ genes isolated in this study (_HpGGPPS2-1_, _HpGGPPS2-2_, _HpGGPPS3-1_, _HpGGPPS3-2_, _HpGGPPS3-3_, and _HpGGPPS3-4_) and two previously reported _GGPPS_ genes (KX236182 and KX236183) have modified FARM motifs (DDxx-D instead of DDxxxxD). The effect of this type of modification on their bio-function has not been reported. The feature of modified FARM motifs, conserved SARM motifs, and lacking CxxxC motifs was also observed in _GGPPS_ genes from other species, such as _Linus usitatissimum_, _Ricinus communis_, _Physcomitrella patens_, _Zea mays_, and _Vitis vinifera_[16]. According to the nucleotide sequence similarity and phylogenetic tree analysis constructed from putative amino acids, eight candidates can be grouped into three classes with more than one member in each class (Figure 3).