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.