Supporting Information
Enhanced production of coenzyme Q10 by self-regulating the engineered MEP
pathway in Rhodobacter sphaeroides
Wenqiang Lu, Lidan Ye, Haoming Xu, Wenping Xie, Jiali Gu, Hongwei Yu*
Institute of Bioengineering, Department of Chemical and Biological Engineering,
Zhejiang University, Hangzhou 310027, PR China
* Corresponding author:
Prof. Hongwei Yu
Tel: 86-571-8795-1873;
E-mail: yuhongwei@zju.edu.cn
Fax: 86-571-8795-1873;
Short running title: Self-regulation of metabolic pathway
SI-1
Table S1. Plasmids and bacterial strains used in this study
Plasmids and strains
References
Description
or source
Plasmids
pTrc99a
Expression vector; ampicillin resistant; trc Pharmacia
promoter;
pBBR1MCS2
Cloning vector; kanamycin resistant; (Kovach et
Broad-Host-Range Plasmid
pIND4tacG
al. 1995)
tac promoter, containing UbiG from R. (Lu et al.
sphaeroides 2.4.1, kanamycin resistance
pTsrid
2013)
dxs, dxr, idi and ispD genes cloned into This study
pTrc99a via biobrick standard
pBsrid
Fragment containing dxs, dxr, idi and This study
ispD genes with trc promoter and
terminator
of
pTrc99a
cloned
into
pBBR1MCS2
pBsrid-lacIq
lacIq with RBSa cloned into pBsrid
pBsrid-BlacIq
RBS of lacIq in pBsrid-lacIq replaced by This study
This study
RBSb
pBsrid-ClacIq
RBS of lacIq in pBsrid-lacIq replaced by This study
RBSc
pBsrid-DlacIq
RBS of lacIq in pBsrid-lacIq replaced by This study
RBSd
pBsrid-ElacIq
RBS of lacIq in pBsrid-lacIq replaced by This study
RBSe
pBsrid-pUbiG
UbiG operon cloned into pBsrid
This study
pBsrid-lacIq-pUbiG
UbiG operon cloned into pBsrid-lacIq
This study
pBsrid-BlacIq-pUbiG
UbiG operon cloned into pBsrid-BlacIq
This study
pBsrid-ClacIq-pUbiG
UbiG operon cloned into pBsrid-ClacIq
This study
pBsrid-DlacIq-pUbiG
UbiG operon cloned into pBsrid-DlacIq
This study
pBsrid-ElacIq-pUbiG
UbiG operon cloned into pBsrid-ElacIq
This study
strains
E.coli JM109
endA1, gyrA96, hsdR17(rk-mk+), recA1,
relA1, supE44, thi-1, del(lac-proAB),
e14−(McrA−), F’[traD36, proAB+, lacIq,
lacZdelM15]
E.coli S-17
recA, harbors the tra genes of plasmid (Simon et
RP4 in the chromosome; proA, thi-1
Rhodobacter
al. 1983)
sphaeroides Wild type Rhodobacter sphaeroides 2.4.1; ATCC
2.4.1
nalidixic acid resistance
BAA-808
RspMCS
R. sphaeroides 2.4.1 harboring plasmid This study
pBBR1MCS2
RspSRID
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid
RspSRIDL
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-lacIq
RspSRIDbL
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-BlacIq
RspSRIDcL
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-ClacIq
RspSRIDdL
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-DlacIq
RspSRIDeL
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-ElacIq
RspSRIDpG
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-pUbiG
RspSRIDaLpG
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-lacIq-pUbiG
RspSRIDbLpG
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-BlacIq-pUbiG
RspSRIDcLpG
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-ClacIq-pUbiG
RspSRIDdLpG
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-DlacIq-pUbiG
RspSRIDeLpG
R. sphaeroides 2.4.1 harboring plasmid This study
pBsrid-ElacIq-pUbiG
SI-2
Table S2 Primers and RBS sequences
Name
Nucleotide sequence
Description
Primers
dxsF
CGGAATTCATGACCAATCCCACCC
C
dxsacR
dxacF
GACGAGGCCGAACTCATCCACATG
ATCGCC
Site mutation and dxs cloning;
CATGTGGATGAGTTCGGCCTCGTC
EcoRI, HindIII, SpeI
GGCCGC
dxsR
AAGCTTCGGCCGCTACTAGTCGCC
GCAGGATCAGACCG
dxrF
dxrR
CCTCTAGACCTGGGGGAGGGTCAG
GATGCGCAGCCTGTCGATC
Cloning of dxr; XbaI, HindIII,
AAGCTTCGGCCGCTACTAGTCCTT
SpeI
TTCTGCTGGCGGAG
idiF
idiR
CCTCTAGACCTGGGGGAGGGTCAG
GATGACGGAAATGGTTCCCG
Cloning of idi; XbaI, HindIII,
AAGCTTCGGCCGCTACTAGTCGGC
SpeI
GCTTACTGCACGAC
ispDF
CCTCTAGACCTGGGGGAGGGTCAG Cloning
GATGACGACAGCCGCAATC
of
HindIII, SpeI
ispD;
XbaI,
ispDR
AAGCTTCGGCCGCTACTAGTCCGG
CCGCAAGCGCGAGCAG
trcsridF
CGAGCTCTCAGGCAGCCATCGGAA
Cloning of operon containing
GC
trc promoter and dxs, dxr, idi,
trcsridR
GGGGTACCGGCGACACGGAAATG
and ispD gene; SacI, KpnI
TTGAATAC
ispFF
CCTCTAGACCTGGGGGAGGGTCAG
GATGACGGAAATGGACATCCGCAC
CGGCAAC
ispFR
Cloning of ispF; XbaI, KpnI
GGGGTACCGGTGAGCCTCATGCCT
GCAGC
ispGF
CCTCTAGACCTGGGGGAGGGTCAG
GATGACGGAAATGTCGCTCAATCA
TGTCCG
ispGR
Cloning of ispG; XbaI, KpnI
GGGGTACCGCGACGGTGCGTTACT
CGGCCGC
lacaF
lacbF
laccF
GGACTAGTCCTGGGGGAGGGTCA
Forward primer for cloning
GGGTGGTGAATGTGAAACCAGTA
lacIq with RBSa; SpeI
GGACTAGTTCAGTGGAGGGATCA
Forward primer for cloning
GGGTGGTGAATGTGAAACCAGTA
lacIq with RBSb; SpeI
GGACTAGTGGAGGGGAGGCATCA
Forward primer for cloning
GGGTGGTGAATGTGAAACCAGTA
lacIq with RBSc; SpeI
lacdF
laceF
lacR
OubiGF
GGACTAGTGGAGGAGGGGGCTCA
Forward primer for cloning
GGGTGGTGAATGTGAAACCAGTA
lacIq with RBSd; SpeI
GGACTAGTCATCAACGGAGGTTCA
Forward primer for cloning
GGGTGGTGAATGTGAAACCAGTA
lacIq with RBSe; SpeI
GGGGTACCCTCACTGCCCGCTTTC
Reverse primer for cloning
C
lacIq; KpnI
CGCATGCATTTGGAATTACGTTATC
GA
OubiGR CGGGGTACCTTCTGAGGTCATTAC
Cloning UbiG operon from
pIND4tacG; NsiI, KpnI
TGG
RBS
RBSa
CCTGGGGGAGGG
(Huo 2011)
RBSb
TCAGTGGAGGGA
(Huo 2011)
RBSc
GGAGGGGAGGCA
(Huo 2011)
RBSd
GGAGGAGGGGGC
(Huo 2011)
RBSe
CATCAACGGAGGT
(Huo 2011)
SI-3
Cloning and expression of ADP-ribose pyrophosphatase
For
cloning
the
ADP-ribose
pyrophosphatase
gene,
primes
BsNudF-F
(CTAGCCATGGGCATGAAATCATTAGAAGAAAAAAC) with NcoI site and
BsNudF-R (CAGGGATCCTCATTTTTGTGCTTGGAGCG) with BamHI site were
used for PCR (Zheng et al. 2013). The PCR fragments amplified from Bacillus
subtilis 168 were digested by NcoI and BamHI and inserted into the corresponding
sites in the pET30a. The recombinant plasmids were transferred into E. coli BL21
(DE3) and screened on a LB agar plate containing 50 mg/L kanamycin. The positive
colony was selected and named as E. coli ARP. Then this strain was used to express
the ADP-ribose pyrophosphatase under induction of 0.1mM IPTG.
Figure S1 SDS-PAGE of the proteins from E. coli ARP. Lane 1: protein marker; lane
2: proteins from E. coli ARP
SI-4
Figure S2. The GC analysis results confirmed the production of prenol and isoprenol
Authentic prenol and isoprenol are purchased from Sigma-Aldrich (USA).
SI-5
Figure S3 The curves of cell growth, glucose consumption and CoQ10 accumulation of
RspMCS, RspSRID, RspSRIDL, RspSRIDbL, RspSRIDcL, RspSRIDdL and
RspSRIDeL. Data are shown as means±SD from three independent experiments.
SI-6
Figure S4 The curves of cell growth, glucose consumption and CoQ10 accumulation of
RspMCS,
RspSRIDpG,
RspSRIDaLpG,
RspSRIDbLpG,
RspSRIDcLpG,
RspSRIDdLpG and RspSRIDeLpG. Data are shown as means±SD from three
independent experiments.
References
Huo J. 2011. Design of a BioBrick Compatible Gene Expression System for Rhodobacter sphaeroides
USA: Utah State University.
Kovach ME, Elzer PH, Steven Hill D, Robertson GT, Farris MA, Roop II RM, Peterson KM. 1995. Four new
derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different
antibiotic-resistance cassettes. Gene 166(1):175-176.
Lu W, Shi Y, He S, Fei Y, Yu K, Yu H. 2013. Enhanced production of CoQ10 by constitutive overexpression
of 3-demethyl ubiquinone-9 3-methyltransferase under tac promoter in Rhodobacter
sphaeroides Biochemical Engineering Journal 72:42-47.
Simon R, Priefer U, Puhler A. 1983. A Broad Host Range Mobilization System for Invivo
Genetic-Engineering - Transposon Mutagenesis in Gram-Negative Bacteria. Bio-Technology
1(9):784-791.
Zheng Y, Liu Q, Li L, Qin W, Yang J, Zhang H, Jiang X, Cheng T, Liu W, Xu X. 2013. Metabolic engineering
of Escherichia coli for high-specificity production of isoprenol and prenol as next generation
of biofuels. Biotechnology for Biofuels 6(1):1-13.