*3.6. Functional Complementation Experiment in E. coli*

*E. coli* JM109 (Table 2) was used as a host for complementation experiments by cotransformation of the plasmid pUC-psy with pACCRT-E (Table 2). *E. coli* JM109 harboring only plasmid pACCRT-EB (Table 2) was cultured as a positive control, and only plasmid pUC-*psy* was cultured as a negative control for PSY functional analysis. The different strains were cultivated in 100 mL LB medium containing 100 μg·mLƺ1 ampicillin and 50 μg·mLƺ1 chloramphenicol at 28 °C and 180 rpm. IPTG (1 mM) was added when the optical density at 600 nm (OD600) reached 0.5, and the culture was kept at 28 °C for 2 days. The *E. coli* cells were collected by centrifugation at 12,000 rpm and used for 

high-performance liquid chromatography (HPLC) analysis. 


**Table 2.** Strains and plasmids used in this study*.*

Pigments in the bacteria were extracted according to procedures described by Breitenbach *et al.* [38]. *E. coli* JM109 cells harboring different plasmids were collected by centrifugation and freeze dried. Pigment extraction was carried out in acetone (80%, v/v) using ultrasonication, and the solvent was removed by blowing with nitrogen gas. The carotenoids were then resuspended in acetone for subsequent HPLC analysis. All operations were carried out on ice under dim light to prevent photodegradation, isomerizations, and structural changes of the carotenoids. The samples were prepared for HPLC by dissolving the dried residues in 1 mL of acetone and filtered through a polycarbonate 0.22 μm filter (Millipore, Carrigtwohill, Ireland). The extracted pigments were separated on a Kromasil KR100-5C18 analytical column (250 mm × 4.6 mm, 5 μm) using an UltiMate3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA). The procedures described by Huang [39] were used with the following modifications: the mobile phase consisted of solvent A (acetonitrile/methanol/0.1 M Tris-HCl (pH 8.0), 84:2:14, v/v/v) and solvent B (methanol/ethyl acetate, 68:32, v/v). Pigments were eluted at a flow rate of 1 mL·minƺ1 with a linear gradient from 100% solvent A to 100% solvent B over a 5 min period, followed by 25 min of solvent B. The column temperature was maintained at 30 °C and the sample volume was 20 μL. The pigments were monitored by diode array detector, and the targeted products were identified by their absorption spectra and typical retention times compared with the control. 
