Appendix
Supplementary Table 1 Bacterial strains and plasmids used in this work
Plasmids
Relevant propertiesa
Source
pMD T19 Simple Vector
Cloning vector, AmpR
Takara
pET-22b
AmpR, inducible T7 promoter
Invitrogen
pET-E
AmpR, carrying crtE gene of D. wulumuqiensis R12
This study
pET-I1
AmpR, carrying crtI1 gene
This study
pET-I2
AmpR, carrying crtI2 gene
This study
pET-EB
AmpR, carrying crtE and crtB genes of D.
wulumuqiensis R12
This study
pET-EB1
AmpR, carrying crtE gene of D. wulumuqiensis R12
and crtB1 gene
This study
pET-EB2
AmpR, carrying crtE gene of D. wulumuqiensis R12
and crtB2 gene
This study
pET-EBI
AmpR, carrying crtE, crtB and crtI genes of D.
wulumuqiensis R12
This study
pET-EB1I1
AmpR, carrying genes crtE gene of D. wulumuqiensis
R12, crtB1, and crtI1 genes
This study
pET-EB1I2
AmpR, carrying genes crtE gene of D. wulumuqiensis
R12, crtB1, and crtI2 genes
This study
pET-EB2I1
AmpR, carrying genes crtE gene of D. wulumuqiensis
R12, crtB2, and crtI1 genes
This study
pET-EB2I2
AmpR, carrying genes crtE gene of D. wulumuqiensis
R12, crtB2, and crtI2 genes
This study
Aerobic, gram-stain-positive, non-spore-forming, non-
Wang et al.,
2010
Strainsb
Deinococcus
wulumuqiensis R12
motile, tetrad-forming cocci, reddish-orange, circular,
opaque colonies (approx. 1.8–3.8 mm in diameter) are
formed after incubation on TGY medium for 14 days at
37°C
Escherichia coli DH5α
AmpR, deoR endA1 gyrA96 hsdR17 (rK- -mK+) recA1
relA1 supE44 thi-1 Δ(lacZYA-argF)U169 Φ80lacZ
Vazyme
1
ΔM15 F -λEscherichia coli
BL21(DE3)
AmpR, F - ompT hsdS (rB- mB-) gal dcm (DE3)
Vazyme
EDWe
AmpR, containing plasmid pET-22b
This study
EDW
AmpR, containing plasmid pET-EBI
This study
EDW-I1
AmpR, containing plasmid pET-I1
This study
EDW-I2
AmpR, containing plasmid pET-I2
This study
EDW11
AmpR, containing plasmid pET-EB1I1
This study
EDW12
AmpR, containing plasmid pET-EB1I2
This study
EDW21
AmpR, containing plasmid pET-EB2I1
This study
EDW22
AmpR, containing plasmid pET-EB2I2
This study
a
The crtB1, crtB2, crtI1, and crtI2 genes are the same gene as crtB and crtI respectively but
SD and AS regions were added and varied with different primers.
b
D. wulumuqiensis R12 was grown in TGY medium (10 g tryptone l-1, 1 g glucose l-1, and 5 g
yeast extract l-1) at 30 °C. Recombinant E. coli cells were grown aerobically at 30 or 37 °C in
Luria Bertani (LB) medium, 2×YT+G medium (16 g tryptone l-1, 10 g yeast extract l-1, 5 g
NaCl l-1, and 10 g glycerol l-1), and synthetic medium (SM) [10 g glycerol l-1, 10 g glucose l-1,
7.5 g L-arabinose l-1; 11.2 g KH2PO4 l-1 , 3 g (NH4)2HPO4 l-1, 0.3 g NaCl l-1, 1 g MgSO4·7H2O
l-1, 1.1 g leucine l-1, 0.7 g isoleucine l-1, 0.4 g valine l-1, 1.5 g threonine l-1, 2 g lysine l-1, 3.3 g
phenylalanine l-1, 2.2 g glutamine l-1, and 3.3 g methionine l-1].
Supplementary Table 2 Primers used in this work
Gene
Sequencea
Enzyme site
Shine-Dalgarno+
Aligned spacing
crtE
F:5’ GATCCATATGCGTCCCGAACTG
NdeI
N
R:3’ CTTGAATTCTCACTTCTCCCGCGT
EcoRI
N
F: 5’ CCGGAATTCGTGACGGAATTTTCGCC
EcoRI
N
R:3’CCCAAGCTTTCAGCCGTGGGC
HindIII
N
crtB
2
crtI
F:5’
CCCAAGCTTATGACATCCCCTCTTCCCTG
HindIII
N
XhoI
N
F:5’CCGGAATTCTAAGGAGGATATACATGTG
ACGGAATTTTCGCC
EcoRI
SD+AS I
R:3’ CCCAAGCTTTCAGCCGTGGGC
HindIII
N
F:5’CCGGAATTCAGGAGGATTACAAAGTGAC
GGAATTTTCGCC
EcoRI
SD+AS II
R:3’ CCCAAGCTTTCAGCCGTGGGC
HindIII
N
F:5’CCCAAGCTTTAAGGAGGATATACATATG
ACATCCCCTCTTCCCTGG
HindIII
SD+AS I
R:3’ CCGCTCGAGTCAGCGCCGGATGT
XhoI
N
F:5’CCCAAGCTTAGGAGGATTACAAAATGAC
ATCCCCTCTTCC
HindIII
SD+AS II
R:3’ GACCTCGAGTCAGCGCCGGAT
XhoI
N
R:3’ CCGCTCGAGTCAGCGCCGGATG
crtB1
crtB2
crtI1
crtI2
a
Restriction sites are underlined and SD+AS sequences are double underlined.
3
Supplementary Fig.1HPLC profiles of the isolated carotenoids from the strain EDWe, EDW,
EDW11, EDW12, EDW21, EDW22, and the commercial lycopene
A, commercial lycopene dissolved in acetone; B, acetone supernatant isolated from the strain
EDWe; C, isolated lycopene from the strain EDW; D, isolated lycopene from the strain
EDW11; E, isolated lycopene from the strain EDW12; F, isolated lycopene from the strain
EDW21; G, isolated lycopene from the strain EDW22.
4
Supplementary Fig.2 The colour of the cell pellets of the strains EDW11, EDW12, EDW21,
and EDW22
5
Supplementary Fig.3 Comparison of the phytoene dehydrogenase expression and the growth
of the recombinant strain E. coli EDW-I1 and EDW-I2
a, M: Protein marker; Lane 1: EDW-I2; Lane 2: EDW-I1; b, The data are means of three
independent experiments. Error bars represent standard deviations.
6
Supplementary Fig.4 Comparison of the phytoene synthase expression and the growth of the
recombinant strain E. coli EDW-B1 and EDW-B2
a, M: Protein marker; Lane 1: EDW-B2; Lane 2: EDW-B1; b, The data are means of three
independent experiments. Error bars represent standard deviations.
7