## 4 Materials and Methods
### Construction of Plasmid pCDFDuet-AtaPT-CstDXS  
pCDFDuet-1 coexpression vector (Novagen, Darmstadt, Germany) was chosen to construct the recombinant plasmid containing the two genes encoding AtaPT (GenBank accession no. KP893683) and CstDXS (GenBank accession no. AB354578.1) based on the procedure reported by [PERSON] and [PERSON] [65]. The initial insertion was performed by incorporating AtaPT in the MCS-1 (_BamHI_ and _NotI_) followed by insertion of CstDXS into the MCS-2 (_KpnI_ and _XhoI_) of the pCDFDuet-1 vector to yield pCDFDuet-AtaPT-CstDXS. The details were as follows: The plasmid pUC57-AtaPT obtained from the gene synthesis technology (Invitrogen, Waltham, MA, USA) was used as the DNA template to provide AtaPT. The PCR reaction consisted of Phusion High-Fidelity DNA polymerase (NEB, Ipswich, MA, USA); the forward primer was AtaPT-F: 5\({}^{\prime}\)-GGTGATCCGATGCTCCCCCCCCATCAGACA-3\({}^{\prime}\), and the reverse primer was AtaPT-R 5\({}^{\prime}\)-AAAGCCGGCGCTCACACACGTGCG-3\({}^{\prime}\) (underlines are the recognition sites for _BamHI_ and _NotI_, respectively). The PCR cycle included pre-denaturation at 98 \({}^{\circ}\)C for 1 min, followed by 30 cycles of 98 \({}^{\circ}\)C for 30 s, 60 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 30 s, and a final extension at 72 \({}^{\circ}\)C for 5 min. The obtained PCR product (~1275 bp) was purified by using a Gel Band Purification Kit (GE Healthcare, Chicago, IL, USA), digested with _BamHI_ and _NotI_, and ligated into a pCDFDuet-1 vector which had been treated with the same restriction enzymes. The ligation mixture was transformed into _E. coli_ DH5\(\alpha\) and the positive clones were selected by spreading on the LB-agar-contained streptomycin (50 \(\upmu\)g/mL). The resulting plasmids were extracted using PureYieldTM Plasmid Mini-prep System (Promega, Madison, WI, USA) and the gene insertion was confirmed by double digestion with _BamHI_ and _NotI_. The resulting pCDFDuet-AtaPT was used as the DNA backbone in the next step.  
Based on the previous finding, the gene-encoded 1-deoxy-D-xylose 5-phosphate synthase of _C. stellatopilosus_ (CsDXS: 2163 bp) was predicted to contain the putative chloroplast transit peptide (cTP) that should be removed before the gene expression in _E. coli_ systems [40]. Therefore, the truncated CsDXS (CstDXS) which was absent of the cTP coding sequence (171 bp) was cloned from the young leaves of _C. stellatopilosus_. The PCR reaction consisted of Pfu DNA Polymerase (ThermoScientific, Waltham, MA, USA), CstDXS-F: 5\({}^{\prime}\)-TGCGCGTACCATGGCATACACTTTCAGAAA-3\({}^{\prime}\), and CstDXS -R: 5\({}^{\prime}\)-AGCCCTCGAGTGCTGACATAATTGCAGA -3\({}^{\prime}\) (underlines are the restriction sites for _KpnI_ and _XhoI_, respectively). The PCR condition was as follows: Pre-denaturation at 95 \({}^{\circ}\)C for 1 min, followed by 30 cycles of 95 \({}^{\circ}\)C for 30 s, 60 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 2 min, and a final extension at 72 \({}^{\circ}\)C for 5 min. The PCR product (1992 bp) was purified from the agarose gel by using the gel purification kit, double digested with _KpnI_ and _XhoI_, and was ligated to the pCDFDuet-AtaPT which was digested with the same restriction enzymes. The double digestion by _KpnI_ and _XhoI_ was performed to confirm the insertion of CstDXS in pCDFDuet-AtaPT (Figure S3). The nucleotide sequencing (IDT, Penang, Malaysia) was performed to verify the correct bases and the in-frame arrangement of both AtaPT and CstDXS in the pCDFDuet-AtaPT-CstDXS by using two pairs of primers: ACYCDuetUP1 primer 5\({}^{\prime}\)-GGATCTCGCGCCTCTCTCT-3\({}^{\prime}\), and DuctDOWN1 primer 5\({}^{\prime}\)-GATTAGCCGGTACAA-3\({}^{\prime}\) for AtaPT in the MCS-1, and DuctUP2 primer 5\({}^{\prime}\)-TTGTACACGGCCATAATC-3\({}^{\prime}\) and T7 term primer 5\({}^{\prime}\)-GCTAGTTATTGCCTCAGCG-3\({}^{\prime}\) for CstDXS in the MCS-2.