HPLC
For carotenoid extraction and HPLC analysis, transformed E .coli DH5α harbouring pYS71, pYS69, or pYS76 was cultured in 250 mL flasks containing 50 mL of LB broth with 25 μg mL–1 gentamycin at 37°C for 24 h. After centrifugation at 10,000 × g for 10 min, the cultured cells were repeatedly extracted with 3 mL of acetone for lycopene and β-carotene or ethanol for phytoene until colour was completely lost. The extracted solution was centrifuged and filtered through a GHP membrane (0.45 μm pore size). HPLC was performed using 20 μL of prepared sample, with solvent A (60% acetonitrile, 38% ethyl acetate, 2% acetic acid) and solvent B (100% methanol) as the mobile phase, on a C18Shim-pack GIS-DOS column (4.6 × 250 mm, 5 μm; Shimadzu) as a fixed phase at a flow rate of 1.5 mL min–1. β-Carotene was measured at 450 nm using a photodiode array detector. Lycopene was measured at 470 nm and phytoene at 280 nm. Phytoene, lycopene, and β-carotene standards were purchased from Sigma-Aldrich, Inc.
Generation of lacZY-integrations and non-polar deletion mutants
lacZY transcriptional integration mutagenesis (Campbell insertion) was performed as previously reported (Xu et al ., 2013). An internal DNA fragment of eanI was amplified with EanI-1E-1 and EanI-2K (Supplementary Table S3). The partial eanIfragment was purified, cloned into pGEM-T Easy (Promega), and confirmed by sequencing. For recombinational mutagenesis, the EcoRI/KpnI-digestedeanI fragment was cloned into the pVIK112 suicide vector (Kalogeraki and Winans, 1997), creating pCOK153. The parent strain PA13 was conjugated with pCOK153, and kanamycin-resistant colonies were selected. The mutants were confirmed through PCR using a primer that anneals upstream of the truncated fragment and the primer LacFuse followed by sequencing. We constructed rpoS and crtE null mutants using the same method as described previously.
Non-polar deletion mutagenesis was performed as previously reported (Xuet al ., 2013). We amplified upstream and downstream fragments (approximately 450 bp) of the targeted gene region by PCR using the corresponding primer pairs (Supplementary Table S3). After purification, the fragments were fused by overlap PCR. The final PCR products were cloned into pGEM-T Easy and confirmed by DNA sequencing. The fragments were excised using appropriate restriction enzymes and ligated into the suicide vector pNPTS138-R6KT (Lassak et al ., 2010). The resulting plasmids were introduced into PA13 by conjugative mating, and mating cells were spread on LB medium containing kanamycin and rifampicin. Single-crossover integrates were selected on LB plates containing kanamycin and rifampicin. Single colonies were grown overnight in LB with rifampicin (25 μg mL–1) and plated on LB containing 5% (w/v) sucrose to select for plasmid excision. We checked kanamycin-sensitive colonies for targeted deletion with colony PCR using primers bracketing the location of the deletion.