Supplementary materials
Transformation procedures for Synechocystis PCC 6803
The expression plasmids were transformed to Synechocystis according to the method published before
(Zang et al. 2007; Williams 1988) with minor modifications to achieve the highest transformation
efficiency. About 109 cells/ml Synechocystis cells (exponential phase) were harvested and washed twice
by fresh BG-11 medium. A total of 100 µl of the cell suspension was mixed with plasmid DNA to a final
concentration of 20 mg DNA/ml. Next, the mixture of cells and DNA was incubated for 5 h at 30 °C
under illumination of 30-50 µmol/m2 s, and then spread onto cellulose nitrate membranes filters resting
on BG11 agar plates (without antibiotic) for 18 hours. Finally, the membrane filters were moved onto
fresh BG11 agar plates containing 10 mg spectinomycin/ml, 10 mg kanamycin/ml or 10 mg
erythromycin/ml. Strains with double or triple antibiotic resistances were segregated on plates with the
corresponding antibiotics (Table S2). After about 1-2 weeks of incubation, single colonies were streaked
on plates and grown in the liquid BG11 medium supplemented with the corresponding antibiotics (20 mg
spectinomycin/ml, 20 mg kanamycin/ml, or 20 mg erythromycin/ml).
The genetic stability of foreign genes in Synechocystis mutant strains was checked by growing a
culture of the strain with periodic dilution and sub-culturing for about 5 generations (about 2 months).
Later, the cells from the culture were tested and confirmed for the presence of the foreign gene by PCR as
described above (Fig. S2). The mutant strains showed stable phenotypes under growing with the
antibiotic stress. For example, the galactosidase activity of the strains was stable after 5 months'
cultivation. Besides, the fatty alcohol strains showed stable fatty alcohol production after 3 month's
cultivation.
1
Supplementary Table 1 Primers used in this study
Primer
Sequence 5’-3’a
atpB-1
AGGTACCCTAATAAGTGCTCACCGCTCC
Purpose of primerb
Forward primer for PatpB-12,
PatpB-13, PatpB-14
GAGATCTGCTGATTGTCTAGGAATTTGTTTATAGT
atpB-2
Reverse primer for PatpB-12
TAG
atpB-3
GCATATGATTGTCTAGGAATTTGTTTATAGTTAG
Reverse primer for PatpB-13
CCATATGCTGATTGTCTAGGAATTTGTTTATAGTT
atpB-4
Reverse primer for PatpB-14
AG
Forward primer for PpsaD-12,
psaD-1
AGGTACCCTCCGCCATCTCGTTGAAG
PpsaD-13, PpsaD-14
CAGATCTAGGGATGAAAATGGAATTTCATAAGGT
psaD-2
Reverse primer for PpsaD-12
A
psaD-3
ACATATGGGATGAAAATGGAATTTCATAAGGTA
Reverse primer for PpsaD-13
psaD-4
ACATATGGATGAAAATGGAATTTCATAAGGTA
Reverse primer for PpsaD-14
psbA1-1
AGGTACCGGTCGGTTCCGTCAATGGC
Forward primer for PpsbA112, PpsbA1-13, PpsbA1-14,
GAGATCTAGCGAATAATTACGAAGTAAGATTTTG
psbA1-2
Reverse primer for PpsbA1-12
GG
psbA1-3
CCATATGCGAATAATTACGAAGTAAGATTTTGGG
psbB-1
AGGTACCCATCGGAAAGGCGTGTCATC
Reverse primer for PpsbA1-13
Forward primer for PpsbB-12,
PpsbB-13, PpsbB-14
psbB-2
GAGATCTTGACGCTCCTTCTAGTAACG
Reverse primer for PpsbB-12
psbB-3
GCATATGACGCTCCTTCTAGTAACG
Reverse primer for PpsbB-13
psbB-4
GCATATGCGCTCCTTCTAGTAACG
Reverse primer for PpsbB-14
psbD-1
CGGTACCCACCTTCAACAGTCTCCACG
Forward primer for PpsbD-12,
PpsbD13, PpsbD14
psbD-2
CAGATCTAAATGCAAATCCTCTTGCGTAGCTAGC
Reverse primer for PpsbD-12
psbD-3
CCATATGCAAATCCTCTTGCGTAGCTAGC
Reverse primer for PpsbD-13
psbD-4
CCATATGTGCAAATCCTCTTGCGTAGCTAGC
Reverse primer for PpsbD-14
2
Forward primer for PrbcL-12,
rbcL-1
GGGTACCGGGTTACATTCGCCTCAGTC
PrbcL-13, PrbcL-14, PrbcL-15
rbcL-2
GAGATCTCTAGGTCAGTCCTCCATAAACATTG
Reverse primer for PrbcL-12
rbcL-3
GCATATGGGTCAGTCCTCCATAAACATTG
Reverse primer for PrbcL-13
rbcL-4
CCATATGCGTCAGTCCTCCATAAACATTG
Reverse primer for PrbcL-14
rbcL-5
GCATATGGTCAGTCCTCCATAAACATTG
Reverse primer for PrbcL-15
PlacS1
GGGGTACCGCCTTTTTACGGTTCCTGGC
Forward primer for Plac
PlacA1
GGTAATCCATATGTGTTTCCTGTGTGAAATTGTT
Reverse primer for Plac
TTACTTCTGACACCAAACCAACTGGTAATGGTAG
Anti-sense primer located in
CGACC
the lacZ gene
lacZ-4c
Sense primer located 260 bp at
Ω-260c
GCTCACAGCCAAACTATCAGGTCAAG
the end of Ω fragment
Forward fusion PCR primer
XP-1
AGTGGTTCGCATCCTCGG
for XbaI mutation
ATGAATCCTTAATCGGTACCAAATAAAAAAGGGG
Reverse fusion PCR primer for
ACCTCTAGG
XbaI mutation
CCCTTTTTTATTTGGTACCGATTAAGGATTCATAG
Forward fusion PCR primer
CGGTTGCC
for XbaI mutation
XP-2
XP-3
Reverse fusion PCR primer for
XP-4
CCAGTGAATCCGTAATCATGGT
XbaI mutation
Forward primer for fusion
lacZ-m1
CCAGTGAATCCGTAATCATGGT
PCR cloning of lacZm
Reverse primer for fusion PCR
lacZ-m2
CCAGTGAATCCGTAATCATGGT
cloning of lacZm
Forward primer for fusion
lacZ-m3
CCAGTGAATCCGTAATCATGGT
PCR cloning of lacZm
Reverse fusion PCR primer for
M13-reverse
CCAGTGAATCCGTAATCATGGT
cloning of lacZm
Agp-1
GCACTGCCATAAAGTCAGAATAGGTT
Forward primer for agp-up
Agp-2
TGGATTCGGAACAGATTAGGTTC
Reverse primer for agp-down
at3g11980-
CATATGGTAGGTATGAAAGAAGGTCTGGG
Forward
primer
for
3
trunc-1
at3g11980-trunc
at3g11980-
Reverse primer for at3g11980GAATTCTTAGGCCCTTCCTTTTAAGACGTGC
trunc-2
trunc
agp-Fc
ACCAATGCCGACATAACCCT
Forward primer for agp-up
agp-Rc
AATGCGACTGCGAATGCCTA
Reverse primer for agp-down
TGGAGCCAGCATGGTAGGT
Forward primer for at3g11980
ATCAGCTTGGAGCCCGATA
Reverse primer for at3g11980
phaB-Fc
CGAAGGCATGTATGAACGGAAAG
Forward primer for phaAB-up
phaAB-4c
TGTTGATGGTGGGTATCGTGGTG
at3g11980_R
T_Fc
at3g11980_R
T_Rc
Reverse primer for phaABdown
a
The enzyme digestion site is underlined. The Shine-Dalgarno site of each promoter is framed. The
spacer sequences between the Shine-Dalgarno sequence and ATG start codon were dotted. The
complementary sequence of the start codon was bolded.
b
All native promoters were cloned with the digestion site of KpnI and BglII by using the forward primer
and the reverse primer named as “X-2” (X represents the gene name, eg: rbcL-2); the modified promoters
were respectively cloned with KpnI/NdeI enzyme digestion sites by using the reverse primer and forward
primer numbered as “X-3”, “X-4” and “X-5” (eg: atpB-3 for modified atpB promoter PatpB13); the Plac
(lactose promoter) was cloned with NdeI and KpnI for insertion to the same site of the expression vector
pXT37a.
c
Detect primers for genomic PCR in Synechocystis mutants.
4
Supplementary Table 2 Cyanobacteria strains and plasmids constructed and used in this study
Plasmids used for
Antibiotic
Genotypea
Strains
transformation
Source
resistance
Syn-FQ30a
pFQ30a
slr0168::Ω-PpsbD13-lacZ, -5 bp
Spr
This study
Syn-FQ30b
pFQ30b
slr0168::Ω-PpsbD12-lacZ, WD
Spr
This study
Syn-FQ30c
pFQ30c
slr0168::Ω-PpsbD14-lacZ, -6 bp
Spr
This study
Syn-FQ31a
pFQ31a
slr0168::Ω-PrbcL12-lacZ, WD
Spr
This study
Syn-FQ31b
pFQ31b
slr0168::Ω-PrbcL13-lacZ, -3 bp
Spr
This study
Syn-FQ31c
pFQ31c
Spr
This study
slr0168::Ω-PrbcL14-lacZ, -3 bp, 1 bp
mutation
Syn-FQ31d
pFQ31d
slr0168::Ω-PrbcL15-lacZ, -4 bp
Spr
This study
Syn-FQ32a
pFQ32a
slr0168::Ω-PatpB12-lacZ, WD
Spr
This study
Syn-FQ32b
pFQ32b
slr0168::Ω-PatpB14-lacZ, -1 bp
Spr
This study
Syn-FQ33a
pFQ33a
slr0168::Ω-PpsbA1-12-lacZ, WD
Spr
This study
Syn-FQ33b
pFQ33b
slr0168::Ω-PpsbA1-13-lacZ, -2 bp
Spr
This study
Syn-FQ34a
pFQ34a
slr0168::Ω-PpsaD-14-lacZ, -3 bp
Spr
This study
Syn-FQ34b
pFQ34b
slr0168::Ω-PpsaD13-lacZ, -2 bp
Spr
This study
Syn-FQ36
pFQ36
slr0168::Ω-PpsbB12-lacZ, WD
Spr
This study
Syn-FQ49
pFQ49
slr0168::Ω-Plac-lacZ
Spr
This study
pHB1567
slr0168::Ω-PpetE-lacZ
Spr
Syn-
(Gao et al.
HB1567b
2007)
(Tan et al.
Syn-LY2b
pLY2
slr0168::Ω
Spr
2011)
Syn-XT37a
pXT37a
slr0168::Ω-PpetE-lacZ
Spr
This study
Syn-XT37bb
pXT37b
slr0168::Ω-PpetE-lacZ
Spr
This study
5
Syn-LY43
pLY43
slr0168::Omega PpsbD13 at3g11980-trunc
Spr
This study
Syn-LY25
pLY25
ΔphaAB:: C.CE2 PrbcL12 far_jojoba
Emr
This study
Syn-LY21
pLY21
Δagp::C.K2 PpsbD13 at3g11980
Kmr
This study
Syn-LY10
pLY10
Spr
This study
Spr, Emr
This study
slr0168::Omega PpetE at3g11980 PpetE
far_jojoba
pXT14 (Tan et al.
slr0168::Omega Prbc far_jojoba Trbc
Syn-LY66
2011), pLY25
ΔphaAB:: C.CE2 PrbcL12 far_jojoba
pXT14 (Tan et al.
slr0168::Omega Prbc far_jojoba Trbc
Spr, Kmr,
Syn-XT14C
2011), pLY21,
Δagp::C.K2 PpsbD13 at3g11980 ΔphaAB::
This study
Emr
pLY25,
C.CE2 PrbcL12 far_jojoba
Abbreviations: Ω: the spectinomicin-resistance gene; WD: wild type; far_jojoba: the far gene from
jojoba; at3g11980-trunc: far gene from A. thaliana that truncated 114 aa from the N terminal; C.CE2:
antibiotic resistance gene for erythromycin and chloramphenicol; CK2: antibiotic resistance gene for
kanamycin. Emr: erythromycin-resistance; Kmr: kanamycin-resistance.
a
The modified promoters were mutated at the spacer region between SD site and the start codon. The -5
bp means deletion of 5 base pairs before the ATG start codon, other versa.
b
Strains Syn-HB1567 and Syn-XT37b were used as positive controls for Miller test; Syn-LY2 was used
as a negative control. The orientation of the expression cassette in pXT37b is reversed to that of pXT37a .
6
XbaI
SphI
PpetE
Ω
PpetE
Ω
XbaI
XhoI
slr0168
C- terminal
NdeI
EcoRV
Sal I
Pst I
Sph I
NdeI
EcoRI
XbaI
EcoRI
pHB1567
BglII
XbaI
EcoRV
NdeI
EcoRI
Xba I
pHB1536
Hind III
NdeI
slr0168
N- terminal
Fusion
PCR
NdeI
partial XbaI digestion
KpnI SphI
BglII
XbaI
SalI/HindIII,
blunt
XbaI
EcoRI
pMD18-T
slr0168
C- terminal
pMD18-T
BglII/SphI
slr0168
N- terminal
Nd e I
BglII
Kpn I
P petE
SphI
ori
lacZm
EcoRV
NdeI/T4 blunt
EcoRI/T4 blunt
ligation
EcoRI
XbaI
slr0168
C- terminal
slr0168
N- terminal
pQL17
Kpn I
Ppe tE
Ω
BglII
NdeI
XbaI
Xho I
EcoRV
Apr
pXT30
Nde I
EcoRI
XbaI
pQL18
pXT24a
ligation
Apr
ori
Ω
PpetE Bgl II
Nde I
EcoRV
XbaI
lacZ
ligation
slr0168
N- terminal
Ω
Ppe tE
NdeI
EcoRI
XbaI
XhoI
pXT36a
KpnI
BglII
Nde I
slr0168
C- terminal
XbaI
Apr
ori
Kpn I
Ω
Ppe tE
BglII
NdeI
Xba I
XhoI
lacZ
slr0168
N- terminal
pXT37b
EcoRI
XbaI
XhoI
lacZ
XbaI/
Self-ligation
slr016 8
C-terminal
slr0168
C- terminal
ori
EcoRI
XbaI
pXT37a
slr016 8
N- terminal
r
Ap
Apr
ori
Supplementary Fig. 1 The detailed procedures for construction of pXT37a. The platform pXT37a was
developed upon the synthetic parts from pHB1567 and pHB1536 and was designed with single enzyme
restriction sites flanking these biological parts through the following genetic modifications: (1) The
restriction site of XbaI between the promoter and the Ω fragment was mutated into KpnI by fusion PCR
using the pHB1536 as the template and using XP-1/XP-2 and XP-3/XP-4 as the primers (The SphI
between Ω and the backbone was removed). The cloned fragment was ligated into the BglII/SphI sites of
pHB1567 to obtain the plasmid pQL18. (2) The 5.4 kb fragment of slr0168N-Apr-ori-slr0168C was
recovered by digesting the plasmid pHB1567 with XbaI, and then the NdeI and EcoRI sites were blunted
to obtain the plasmid pXT24a. (3) The XbaI digested fragment Ω-PpetE-lacZ (from pQL18) and the XbaI
7
digested slr0168N-Apr-ori-slr0168C fragment (from pXT24a) were assembled to obtain the plasmid
pXT36a. (4) An NdeI site-mutated lacZ fragment, lacZm, was cloned by fusion PCR using pHB1567 as
the template and using lacZ-m1/ lacZ-m2 as well as lacZ-m3/M13-reverse as the primers pairs. Then
lacZm was ligated to the EcoRI/EcoRV site of pXT36a to obtain the plasmid pXT37b. (5) The orientation
of the Ω-PpetE-lacZ fragment to the slr0168N-Apr-ori-slr0168C fragment was reversed by digestion of
pXT37b with XbaI and ligating the resulting products. The obtained pXT37a was confirmed by PCR.
Abbrieviations: ori - the origin of replication; Amp - ampicillin-resistance gene sequence; laczm- mutated
lacZ gene fragment for removal of NdeI; PpetE – the copper inducible promoter for petE gene.
8
Supplementary Fig. 2 Genomic PCR results for the Synechocystis mutants.
A: Genomic PCR results for promoter parts in Synechocystis mutants. The genomic DNA from
Synechocystis PCC 6803 mutants was used as the templates. Primers: sense primer Ω-260 and antisense
primer of the promoter were used in (b) and (c) to check whether the DNA constructs were introduced or
not; Sense primer Ω-260 and antisense primer lacZ -4 located in the lacZ gene were used in (a), (d), (e),
and (f) to check whether the genomic integration of the expression cassette. M: DNA marker, 1 kb DNA
ladder was used in (b), while 200 bp ladder was used in (a), (c), (d), (e), and (f). (a) Lane 1-3: Syn-FQ49
that harbors Plac; (b) Lane 1-3: Syn-FQ30a that harbors PpsbD13; Lane 4: Syn-FQ30b that harbors
PpsbD12; lane 5: Syn-FQ30c that harbors PpsbD14; lane 6: Syn-FQ32a that harbors PatpB12; lane 7-9:
Syn-FQ31a that harbors PrbcL12; lane 10: Syn-FQ34b that harbors PpsaD13; (c) Lane 1-4: Syn-FQ33b
that harbors PpsbA1-13; (d) Lane 1: Syn-FQ36 that harbors PpsbA2-12; (e) Lane 1: Syn-FQ33a that
harbors PpsbA1-12; (f) Lane 1-3: Syn-FQ36 that harbors PpsbB-12; Lane 4: Syn-FQ31c that harbors
PrbcL14; Lane 5: Syn-FQ31c that harbors PrbcL14; Ω-260: sense primer located 260 bp upstream the
promoter.
B: Genomic PCR results for agp disruption and far expression in Synechocystis mutants. Templates:
Lane 0, negative control, PCR using ddH2O as a template; Lane 1, negative control, PCR using genomic
DNA from Synechocystis wild type as a template; Lane 2-3, PCR using genomic DNA from Syn-LY21
and Syn-XT14C respectively. Primer pairs: (a) agp-F and agp-R were used as primer pair to test the
partial segregation of the mutant strain; (b) psbD-1 and at3g11980_RT_R were used as primer pair to test
whether the DNA constructs were introduced or not; (c) at3g11980_RT_F and agp-1 were used as primer
pair to test whether the DNA constructs were introduced into agp locus.
C: Genomic PCR results for phaAB disruption and far expression in Synechocystis mutants.
Templates: Lane 0, negative control, PCR using ddH2O as a template; Lane 1, negative control, PCR
using genomic DNA from Synechocystis wild type as a template; Lane 2-4, PCR using genomic DNA
from Syn-LY25, Syn-LY66 and Syn-XT14C respectively. Primer pairs: (a) phaB-F and phaAB-4 were
9
used as primer pair to test the complete segregation of the mutant strain; (b) rbcL-1 and far_jojoba_RT_R
were used as primer pair to test whether the DNA constructs were introduced or not; (c) far_jojoba_RT_F
and phaAB-4 were used as primer pair to test whether the DNA constructs were introduced into phaAB
locus.
Supplementary Fig. 3 The comparison of the SD-ATG sequences in the modified promoters. The
symbol * represents one-base deletion; + represents one-base insertion; R represents one-base
replacement; all the mutated bases in the promoters were highlighted.
10
Supplementary Fig. 4 Maps of agp targeting plasmid pKC100 and the phaAB plasmid pKC104 (Tan et
al. 2013). pKC100 and pKC104 were used for deleting the agp gene and the phaAB gene by integrating
the expression cassette into the agp and phaAB locus of the Synechocystis genome, respectively. pKC104:
phaAB targeting vector that contain 1 kb fragment of the upstream phaA gene and 1 kb fragment
downstream phaB gene; pKC100: agp targeting vector that contain a 3.2-kb fragment including 934 bp
upstream agp, the agp gene itself, and 913 bp downstream agp; Apr: Ampicillin-resistance gene; ori:
origin of replicon.
10
5
gamolenic acid
hexadecanol
15
pentadecanol (IS)
20
icosane
heptadecane
Abandance
25
heptadecene
30
palmitic acid
35
cis-9-Hexadecenoic acid
heptadecanoic acid
stearic acid
oleic acid
9,12-octadecadienoic acid

octadecanol

0
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
Retention time (min)
11
36
Supplementary Fig. 5 GC–MS analysis of fatty alcohols in Synechocystis mutant strain Syn-XT14C.
The pentadecanol was used as the internal standard (IS) for quantification of the fatty alcohol yields in the
mutant strains. The peaks for fatty acids and long-chain alka(e)nes in the mutant strain were also shown
in the figure.
References
Gao H, Tang Q, Xu X (2007) Construction of copper-induced gene expression platform in Synechocysits
sp. PCC 6803. Acta Hydrobiologica Sinica 31 (2):240-244
Tan X, Yao L, Gao Q, Wang W, Qi F, Lu X (2011) Photosynthesis driven conversion of carbon dioxide
to fatty alcohols and hydrocarbons in cyanobacteria. Metabolic Engineering 13 (2):169-176.
doi:10.1016/j.ymben.2011.01.001
Williams JGK (1988) Construction of Specific Mutations in Photosystem-Ii Photosynthetic Reaction
Center by Genetic-Engineering Methods in Synechocystis-6803. Methods in Enzymology 167:766-778
Zang X, Liu B, Liu S, Arunakumara KK, Zhang X (2007) Optimum conditions for transformation of
Synechocystis sp. PCC 6803. Journal of microbiology 45 (3):241-245
12