Construction of plasmids expressing carotenoid biosynthesis and cbp genes

The PAC-ZEAX plasmid (a.pngt from Prof F. Cunningham)⁵⁰ containing the P15A origin of replication and crtE, crtY, crtI, crtB, and crtZ genes under the control of the crtE promoter from Erwinia herbicola Eho10 was used for Zea production in E. coli⁵¹. Genes including EZR1976 (crtR) from Euhalothece, tpr6673 (crtW) from Tolypothrix sp. PCC7910, and slr0088 (crtO) from Synechocystis were cloned into the PAC-ZEAX plasmid using the XhoI and KpnI restriction sites (ezr1976 and tpr6673) or the NdeI and XhoI restriction sites (slr0088). Two genes, EZR1179 and EZL1782 (cruA), from Euhalothece were cloned into pBAD Myc/HisC using XhoI and HindIII sites, whereas genes encoding EuOCP (Ezr1969), EuHCP (Ezr2074), EuCTDH (Ezr2075), and SyOCP (Slr1963) were cloned into pBAD Myc/HisC using PstI and EcoRI sites. Primers used for cloning and plasmid construction are listed in Supplementary Table ^S3.