Figure 3.

CsALDH3I1 mediates crocetin biosynthesis in E. coli. A to C, Accurate mass chromatograms of zeaxanthin (A), crocetin dialdehyde (B), and crocetin (C) extracted from bacterial clones harboring the pTHIO empty vector or overexpressing CsCCD2 alone or in combination with CsALDH3I1 or CsALDH2C4. Polar and nonpolar fractions were extracted from bacterial cells and run on an HPLC-PDA-HRMS system alongside authentic standards. The metabolites have an accurate mass and a chromatographic mobility identical to those of the authentic standard. Two peaks were detected of crocetin (1, trans-isomer, and 2, cis-isomer; Supplemental Fig. S4). D to F, Relative quantities of zeaxanthin (D), crocetin dialdehyde (E), and crocetin (F) detected by HPLC-atmospheric pressure chemical ionization-HRMS (D and E) and HPLC-electrospray ionization (ESI)-HRMS (F) in recombinant clones. Data are means ± sd of three independent growth batches. Asterisks indicate statistically significant differences (Student’s t test, P < 0.01) compared with CsCCD2 values. Fold IS, Fold internal standard.