## 2 Results
### Functional Identification of HpGGPPS Genes in Escherichia coli  
The function of isolated _HpGGPPS_ genes was analyzed by hetero-expression studies in _E. coli_ targeting the astaxanthin formation. The transformed _E. coli_ cells with pFZ153\(\Delta\)E/CrtE, a plasmid containing astaxanthin biosynthetic gene cluster originally from Bacteria as the positive control, displayed orange-red color in the cell pellet as a result of pigmentation of astaxanthin (Figure 4). Served as the negative control, _E. coli_ cells harboring pFZ153\(\Delta\)E (NCK), a plasmid without _GGPPS_ gene but with all rest genes (Crtl, CrtB, IPI, CrtY, CrtZ, CrtW) for astaxanthin production, displayed slight yellow color in the cell pellet. Similar as the positive control, the _E. coli_ transformants harboring pFZ153\(\Delta\)E/GGPPS1-1 (1-1) and pFZ153\(\Delta\)E/GGPPS1-2 (1-2) also displayed orange-red color, but relatively darker, which might be explained by more pigmentation. By contrast, the pigmentation was not observed in _E. coli_ pellets harboring pFZ153\(\Delta\)E/GGPPS2-1 (2-1), pFZ153\(\Delta\)E/GGPPS2-2 (2-2), pFZ153\(\Delta\)E/GGPPS3-1 (3-1), pFZ153\(\Delta\)E/GGPPS3-2 (3-2), pFZ153\(\Delta\)E/GGPPS3-3 (3-3), or pFZ153\(\Delta\)E/GGPPS3-4 (3-4) (Figure 4), indicating that they might not encode proteins associated with pigment production. Therefore, only proteins encoded by _HpGGPPS1-1_ and _HpGGPPS1-2_ have GGPP synthase activity to participate pigment formation. Figure 3: Phylogenetic relationships of the deduced amino acid sequences of _GGPPS_ genes obtained in this study and retried from Genbank. Numbers in parentheses are accession numbers of _GGPPS_ genes. The phylogenetic tree was constructed by MEGA version 7.0 using neighbor-joining method. Bootstrap values from the percentages of 1000 replications are indicated beside each node. To further confirm and quantify the astaxanthin production in _E. coli_ cells, a HPLC analysis was successfully conducted. The chromatographic profile of astaxanthin was generated using commercial standards (Supplemental Figure S2a). As expected, an absorption peak of astaxanthin was detected in the positive control and but not detected in the negative control. Similarly, the pigment in _E. coli_ cells harboring pFZ153 AE/GGPPS1-1 and pFZ153 AE/GGPPS1-2 have a peak at the same retention time as astaxanthin standard, indicating the existence of astaxanthin (Supplemental Figure S2b). Consisting with the color evaluation in cell pellets, astaxanthin was not detected in _E. coli_ cells harboring pFZ153 AE/GGPPS2-1, pFZ153 AE/GGPPS2-2, pFZ153 AE/GGPPS3-1, pFZ153 AE/GGPPS3-2, pFZ153 AE/GGPPS3-3, and pFZ153 AE/GGPPS3-4. Besides, no other absorption peaks were observed in their chromatographic profile. Meanwhile, the quantity of astaxanthin was measured using a standard curve constructed from a set of astaxanthin standards. Results showed that _E. coli_ cells harboring pFZ153 AE/GGPPS1-1 produced as high as 7.1 mg astaxanthin per gram of dried cells in average from three independent pigment extraction, which is slight more (but not significant at the level of 0.05) than _E. coli_ cells harboring pFZ153 AE/GGPPS1-2 (6.5 mg/g DW in average). Notably, the positive control has significant less yield of astaxanthin (2.4 mg/g DW in average), compared with _E. coli_ cells with _HpGGPPS_ genes (Figure 5). Figure 4: Hetero-expression experiments of pFZ153 AE/GGPPS in _E. coli_ targeting astaxanthin synthesis. _E. coli_ strain BL21 (DE3) was used as the host. _CrIE_ refers to the _GGPPS_ gene from _Pantoea ananatis_, served as the positive control producing astaxanthin; 1-1, 1-2, 2-1, 2-2, 3-1, 3-2, 3-3, and 3-4 refers to corresponding _HpGGPPS_ genes isolated in this study; NCK refers to the negative control, which is the BL21 (DE3) containing pFZ153 AE without _GGPPS_ gene for production of no astaxanthin. Colors of _E. coli_ pellet are representative of at least five transformantsants with similar results. Figure 5: Astaxanthin content in _E.