## 4 Materials and Methods
### Plasmids Construction and Heterologous Expression  
The plasmid used for heterologous expression targeting gene functional analysis was adopted from pFZ153, a plasmid has astaxanthin synthetic gene cluster (\(Cr\)_E_, \(Cr\)_I_, \(Cr\)_B_, _idi_, \(Cr\)_Y_, \(Cr\)_Z_, and \(Cr\)_W_) and produces astaxanthin in _E. coli_[42]. To investigate the function of _HpGGPPS_ genes on astaxanthin synthesis, the pFZ153\(\Delta\)E plasmid was constructed (Figure 6b). Firstly, \(Cr\)_E_, \(Cr\)_I_, and \(Cr\)_B_ genes were removed from pFZ153 (Figure 6a) by _XbJ/EcoRI_ digestion. Secondly, the coding sequence  
\begin{table}
\begin{tabular}{c c c} \hline
**Primer ID** & **Sequences (\(\lx@sectionsign\)-3)** & **Used for** \\ \hline GGPPIF1 & CACTGCCTATCCCCCTTCCATC & Cloning of HpGGPPS1-1 and \\ GGPPIR1 & GCACCTCTGTGACCGCCTCTG & HPGGPPS1-2 \\ \hline GGPPI2F1 & GGCGACGGCGCAAATCAGT & Cloning of HpGGPPS2-1 and \\ GGPPI2R1 & AAGGCGCACGGAATACAAACATA & HpGGPPS2-2 \\ \hline GGPPI3F1 & GCTCTCCTCCTCCTCCTCGC & Cloning of HpGGPPS3-1, HpGGPPS3-2, \\ GGPPI3R1 & TGATCCAGAGCTCACT & HpGGPPS3-3 and HpGGPPS3-4 \\ \hline GGPPIPI2 & GTATCTGAAAAAAATGCGCAGGATGCACA \({}^{\dagger}\) & Subcloning of HpGGPPS1-1 \\ GGPPIR2 & CATAGAAGCTTTTGCGCGCGCATTCCTA & Subcloning of HpGGPPS1-2 \\ \hline GGPPI1F3 & GTATCTGAAATTCAAAAATGATGATGCCGGCGCAA & Subcloning of HpGGPPS2-1 and \\ GGPPIR3 & CATAGAAAGCCCCGCTATTCTTCTCCTCAGG & HpGGPPS2-2 \\ \hline GGPPI3F2 & GTATCTGAAAAAAAAATGATGATGATGATGATGAGAG & Subcloning of HpGGPPS3-1 \\ GGPPI3R2 & CATAGAAAAACTTTT  
of Crtl/CrtB gene was amplified from pFZ153 plasmid using primers CrtlB-F2/CrtlB-R2 containing _XbaI_ and _EcoRI_ restriction sites, respectively. Finally, the gel-purified fragment of Crtl/CrtB gene was digested with _XbaI_/_EcoRI_ restriction enzyme and then ligated with the pFZ153 fragment without _CrtE_, _Crtl_, and _CrtB_ genes to form the new plasmid pFZ153\(\Delta\)E.  
To complete the astaxanthin synthetic gene cluster, _HpGGPPS_ genes were inserted into pFZ153\(\Delta\)E to form plasmids pFZ153\(\Delta\)E/GGPPS. In detail, the coding sequences of _HpGGPPS_ genes were amplified from the sequenced plasmids containing _HpGGPPS_ genes, using primers listed in Table 1 containing either _EcoRI_/_Hind_III or _EcoRI_/_Not_I restriction sites. Similar as described above, the target PCR fragments were gel-purified and ligated into pFZ153\(\Delta\)E plasmid in either _EcoRI_/_Hind_III or _EcoRI_/_Not_I sites. Additionally, the coding sequence of _CrtE_ gene amplified from pFZ153 with primers CrtE-F1/CrtE-R1 harboring _EcoRI_/_Hind_III sites, was also ligated into pFZ153\(\Delta\)E plasmid to construct the positive control (pFZ153\(\Delta\)E/CrtE).  
The procedures of PCR amplification using Platinum SuperFi DNA polymerase and gel-purification using E.Z.N.A. Gel Extraction Kit are the same as described above. The ligation process was performed by T4 ligase (Promega, Madison, WI, USA), as described by the manual. _E. coli_ Top10 was used as a host during plasmids construction, as described above. Restriction enzymes used in this study were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).  
For the functional analysis of _HpGGPPS_ genes, plasmids of pFZ153\(\Delta\)E/GGPPS and pFZ153\(\Delta\)E/CrtE were transferred into an expression host, _E. coli_ strain BL21 (DE3). The transformed _E. coli_ colonies were selected on the LB agar plate with ampicillin (100 \(\mu\)g/mL) and then cultured in 15 mL culture tubes containing 3 mL LB liquid media supplemented with 100 \(\mu\)g/mL ampicillin (LB+Amp100 medium) overnight. The aliquot of 0.5 mL was added into a 250 mL flask containing 100 mL LB+Amp100 medium. The flasks were incubated at 30 \({}^{\circ}\)C under dark with shaking (200 rpm) until OD\({}_{600}\) reached 0.7-0.9, followed by adding IPTG to a final concentration of 0.1 mM. Three hours later, 50 mL of each culture was harvested and pigments were extracted as described below. For each plasmid, at least three independent _E. coli_ colonies were selected for pigment extraction as replicates.