Submitted to Biotechnology and Bioengineering (Communication to the editor)
Direct biosynthesis of adipic acid from a synthetic pathway
in recombinant Escherichia coli
Supplementary Data
Jia-Le Yu,1 Xiao-Xia Xia,2 Jian-Jiang Zhong,1,2 Zhi-Gang Qian2
1
State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China
University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
2
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology,
Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai, 200240, China; telephone:
+86-21-34206968; fax: +86-21-34204831; e-mail: jjzhong@sjtu.edu.cn; zgqian@sjtu.edu.cn
Correspondence to: ZG. Qian and JJ. Zhong
Contract grant sponsor: National Basic Research Program of China (973)
Contract grant number: 2012CB721006
Contract grant sponsor: National High Technology R&D Program (863)
Contract grant number: 2014AA021201
Contract grant sponsor: Program for Professor of Special Appointment (Eastern Scholar) at
Shanghai Institutions of Higher Learning
Contract grant number: ZXDF080005
Running title: Adipic acid production from a synthetic pathway
1
Supplementary Method
Plasmid construction
Pfu DNA polymerase (TIANGEN, Beijing, China) was used for gene amplification. T4 DNA
ligase was purchased from TAKARA Biotechnology, Dalian, China. All of the restriction
enzymes were purchased from Fermentas (Thermo Fisher Scientific Inc., Waltham, MA).
Kits for plasmids extraction and DNA purification were purchased from TIANGEN. The
DNA sequences of the constructs that involved cloned fragments were confirmed by
sequencing. Plasmids used in this study are listed in Supplementary Table I.
The codon optimized ter gene of E. gracilis (without the transit peptide-coding region)
was assembled in plasmid pTRX-ter and purchased from 7Sea PharmTech Co., Ltd, Shanghai,
China). The ter sequence is:
5’-atggcgatgtttaccacgaccgcgaaagtgattcagccgaaaattcgcggctttatttgcaccacgacccatccgattggctgcga
gaaacgcgtgcaggaagagattgcgtatgcgcgcgcgcatccgccaaccagccctggcccgaaacgcgtgctggtgattggttgca
gcaccggctatggcctgagcacccgcattaccgcggcatttggctatcaggcggccaccctgggcgtgtttctggcgggcccaccga
ccaaaggccgcccggcggcagccggctggtataacaccgtggcgtttgagaaagcggcactggaagcgggcctgtatgcgcgca
gcctgaacggcgatgcgtttgatagcactaccaaagcgcgcaccgtggaagcgattaaacgcgatctgggcaccgtggatctggttg
tgtatagcattgcggcaccgaaacgcaccgatccggcgaccggcgtgctgcataaagcgtgcctgaaaccgattggcgcgacctata
ccaaccgcaccgtgaacaccgataaagcggaagtgaccgatgtgagcattgaaccggcgagcccggaagagattgcggataccgt
gaaagtgatgggtggcgaagattgggaactgtggattcaggcgctgagcgaagcgggcgtgctggcggaaggcgcgaaaaccgt
ggcgtatagctatattggcccggaaatgacctggccggtgtattggagcggcaccattggcgaagcgaagaaagatgtggagaaag
cagcgaaacgcattacccaacagtatggctgcccggcgtatccggtggttgcgaaagcgctggtgacccaggccagctcggcgatt
ccggtggttccgctgtatatttgcttactgtatcgcgtgatgaaagagaaaggcacccatgaaggctgcattgaacagatggtgcgctta
ctgaccacgaaactgtatccggaaaacggcgcgccgattgtggatgaagcgggccgcgtgcgcgtggatgactgggaaatggcgg
aagatgtgcaacaggcggtgaaagatctgtggagccaggtgagcaccgcgaacctgaaagatattagcgactttgcgggctatcaga
2
ccgaatttctgcgcctgtttggctttggcattgatggcgtggattatgatcagccggtggatgtggaagcggatctgccgagcgccgcg
cagcaataa-3’.
pMD19-T derivatives. The paaJ gene was amplified from the genomic DNA of E. coli
MG1655 with primers FpaaJ and RpaaJ (Supplementary Table II). The PCR products were
ligated into the T-cloning site of plasmid pMD19-T (Takara), generating pMD19T-paaJ.
Similarly, the hbd, crt, and ptb-buk1 genes of C. acetobutylicum ATCC 824 were cloned into
pMD19-T, leading to pMD19T-hbd, pMD19T-crt, and MD19T-ptb-buk1, respectively.
pET-28a(+)-based expression plasmids. The paaJ gene was amplified from pMD19T-paaJ
using primers FpaaJBam and RpaaJHind, digested BamHI and HindIII, and inserted into
pET-28a(+) at the same sites. The resulting plasmid, pET28a-paaJ allowed the expression of
PaaJ with an N-terminal hexahistidine tag. Similarly, the hbd, crt, ter, ptb, and buk1 were
amplified and cloned into BamHI-SalI site of pET-28a(+). Thus, pET28a-hbd, pET28a-crt,
pET28a-ter, pET28a-ptb, and pET28a-buk1 were constructed.
pTrc-ter-paaJ and pTrcbcd-etfBA-paaJ. The ter gene was amplified from pTRX-ter using
primers FterNco and RterKpn, and digested with NcoI and KpnI. The paaJ gene with its
native RBS sequence, was amplified from pMD19T-paaJ using primers FpaaJKpn and
RpaaJBam, and digested with KpnI and BamHI. The two restricted fragments were ligated
into plasmid pTrc99A at the corresponding sites, leading to plasmid pTrc-ter-paaJ, which
allowed the expression of ter and paaJ under the control of IPTG-inducible trc promoter.
Similarly, pTrcbcd-etfBA-paaJ was constructed except that the bcd-etfBA genes were
amplified from genomic DNA of C. acetobutylicum ATCC 824 with primers FbcdNco and
RetfBAKpn.
3
pZS*27ptb-buk1-hbd-crt. The ptb-buk1 genes flanked by synthetic RBS were amplified
from pMD19T-ptb-buk1 using primers FptbEco and Rbuk1Kpn, and digested with EcoRI and
KpnI. The hbd gene was PCR-amplified from pMD19T-hbd with primers FhbdKpn and
RhbdBam, and digested with KpnI and BamHI. The crt gene was PCR-amplified from
pMD19T-crt with primers FcrtBam-2 and RcrtMlu, and digested with BamHI and MluI. The
above three fragments were ligated into pZS*27mCherry at the appropriate sites to make
pZS*27ptb-buk1-hbd-crt, which allowed constitutive expression of the ptb, buk1, hbd, and
crt genes under the lacIQ promoter of moderate strength.
pZS*27ptb-buk1-paaH1-crt. The paaH1 gene was amplified from the genomic DNA of R.
eutropha H16 with primers FpaaH1Kpn and RpaaH1Bam. After restriction with KpnI and
BamHI,
the
paaH1
fragment
was
used
to
replace
the
hbd
fragment
in
pZS*27ptb-buk1-hbd-crt, resulting in plasmid pZS*27ptb-buk1-paaH1-crt.
pZS*27ptb-buk1-paaH1-ech. The h16_A3307 (ech) gene was PCR-amplified with primers
FechBam and RechMlu, using the genomic DNA of R. eutropha H16 as a template. After
digestion with BamHI and MluI, the ech fragment was used to replace the crt fragment in
pZS*27ptb-buk1-paaH1-crt, generating plasmid pZS*27ptb-buk1-paaH1-ech.
4
Table SI. Escherichia coli strains and plasmids used in this study.
Description
Source or
reference
DH5α
F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17
(rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1
Invitrogena
BL21(DE3)
F– ompT hsdSB (rB– mB– ) gal dcm (DE3)
Invitrogena
MG1655
Coli Genetic Stock Center strain (CGSC) No. 6300
CGSCb
QZ1111
MG1655 ΔptsG ΔpoxB Δpta ΔsdhA ΔiclR
Liu et al.
(2012)
pET-28a(+)
KmR, T7 promoter, pBR322 origin, 5.4-kb
Novagenc
pTrc99A
ApR, trc promoter, ColE1 origin, 4.2-kb
Pharmaciad
pMD19-T
ApR, vector with T-cloning site, pUC19 origin, 2.7-kb
Takarae
pZS*27mCherry
KmR, mCherry under the lacIQ promoter, pSC101* origin, 4.3-kb
Lab stock
pMD19T-paaJ
ApR, paaJ of E. coli MG1655 into pMD19-T, 3.9-kb
This study
pMD19T-hbd
ApR, hbd of C. acetobutylicum ATCC 824 into pMD19-T, 3.5-kb
This study
pMD19T-crt
ApR, crt of C. acetobutylicum ATCC 824 into pMD19-T, 3.5 -kb
This study
Strain/plasmid
Strains
Plasmids
pMD19T-ptb-buk1
ApR, ptb-buk1 of C. acetobutylicum ATCC 824 into pMD19-T,
4.7-kb
This study
pET28a-paaJ
KmR, paaJ into BamHI-HindIII site of pET-28a(+), 6.5-kb
This study
pET28a-hbd
KmR, hbd into BamHI-SalI site of pET-28a(+), 6.2-kb
This study
pET28a-crt
KmR, crt into BamHI-SalI site of pET-28a(+), 6.2-kb
This study
pET28a-ter
KmR, ter into BamHI-SalI site of pET-28a(+), 6.6-kb
This study
pET28a-ptb
KmR, ptb into BamHI-SalI site of pET-28a(+), 6.3-kb
This study
pET28a-buk1
KmR, buk1 into BamHI-SalI site of pET-28a(+), 6.4-kb
This study
pTrc-ter-paaJ
ApR, codon-optimized ter and paaJ under Ptrc, 6.6-kb
This study
pTrc-bcd-etfBA-paaJ
ApR, bcd-etfBA and paaJ under Ptrc, 8.4-kb
This study
pZS*27ptb-buk1-hbd-
KmR, ptb-buk1, hbd, and crt genes under the lacIQ promoter,
crt
7.2-kb
pZS*27ptb-buk1-paa
KmR, ptb-buk1, paaH1, and crt genes under the lacIQ promoter,
H1-crt
7.2-kb
pZS*27ptb-buk1-paa
KmR, ptb-buk1, paaH1, and ech gene under the lacIQ promoter,
H1-ech
7.2-kb
5
This study
This study
This study
Ap, ampicillin; Km, kanamycin; R, resistance.
a
Invitrogen, Corp., Carlsbad, CA.
bColi
c
Genetic Stock Center, New Haven, CT.
Pharmacia Biotech, Uppsala, Sweden.
d
Novagen, Inc., Madison, WI.
e
Takara Biotechnology, Dalian, China
6
Table SII. Primers used in this study
Name
Sequences (5’-3’)
Target DNA
FpaaJ
ATTACAGGAGAAGCCTGATG
RpaaJ
TCAAACACGCTCCAGAATCATGGC
Fhbd
Rhbd
ATGAAAAAGGTATGTGTTAT
TTATTTTGAATAATCGTAG
RBS-paaJ
hbd
Template
E. coli MG1655
genomic DNA
C. acetobutylicum
ATCC 824 genomic
DNA
C. acetobutylicum
Fcrt
AGGAGGATTAGTCATGGAAC
Rcrt
TTATCTATTTTTGAAGCCTTC
Fptb-buk1
ATGATTAAGAGTTTTAATGAAAT
Rptb-buk1
TTATTTGTATTCCTTAGCTTTTTC
FpaaJBam
TCCGGGATCCATGCGTGAAGCCTTTATTTG
RpaaJHind
CCGCAAGCTTTCAAACACGCTCCAGAATCA
FhbdBam
GCAGGGATCCATGAAAAAGGTATGTGTTAT
RhbdSal
FcrtBam
GCTGGTCGACTTATTTTGAATAATCGTAGA
GTAGGGATCCATGGAACTAAACAATGTCAT
RcrtSal
GCTCGTCGACCTATCTATTTTTGAAGCCTT
FterBam
GTAGGGATCCATGTTTACCACGACCGCGAA
RterSal
TAATGTCGACTTATTGCTGCGCGGCGCTCG
FptbBam
GCAGGGATCCATGATTAAGAGTTTTAATGAA
FptbSal
Fbuk1Bam
GCTCGTCGACTTATTTATTGCCTGCAACTA
GCAGGGATCCATGTATAGATTACTAATAAT
Rbuk1Sal
GTTCGTCGACTTATTTGTATTCCTTAGCTT
FterNco
RterKpn
AAGTCCATGGCGATGTTTACCACGAC
TACTGGTACCTTATTGCTGCGCGGCGCTCG
FpaaJKpn
RpaaJBam
CGTCGGTACCATTACAGGAGAAGCCTGATG
CTGCGGATCCTCAAACACGCTCCAGAATCAT
FbcdNco
AAGTCCATGGATTTTAATTTAACAAGAGAA
RetfBAKpn
TACTGGTACCTTAATTATTAGCAGCTTTAA
RBS-crt
ATCC 824 genomic
DNA
C. acetobutylicum
ptb-buk1
ATCC 824 genomic
DNA
paaJ
pMD19T-paaJ
hbd
pMD19T-hbd
crt
pMD19T-crt
ter
pTRX-ter
ptb
pMD19T-ptb-buk1
buk1
pMD19T-ptb-buk1
ter
pTRX-ter
RBS-paaJ
pMD19T-paaJ
bcd-etfBA
C. acetobutylicum
ATCC 824 genomic
DNA
FptbEco
GGCGGAATTCAAGAGGAGAAAAGATCTATGA
TTAAGAGTTTTAATGA
Rbuk1Kpn
GGTCGGTACCCCTCCTTTCCTTTATTTGTATTC
CTTAGCTT
FhbdKpn
RhbdBam
CGTCGGTACCATGAAAAAGGTATGTGTTAT
GAGCGGATCCTTATTTTGAATAATCGTAG
FcrtBam-2
GAGCGGATCCAGGAGGATTAGTCATGGAAC
RcrtMlu
FpaaH1Kpn
CGTCACGCGTTTATCTATTTTTGAAGCCTTC
TAGAGGTACCATGAGCATCAGGACAGTGGG
RpaaH1Bam
FechBam
CGGAGGATCCTTACTTGCTATAGACGTACA
TTCAGGATCCAGGAGGATTAGTCATGCCGTAC
RechMlu
GAAAACATCCT
GCTGACGCGTTTAGCGATGCTGGAAATTCG
7
RBS-ptb-bu
k1-RBS
pMD19T-ptb-buk1
hbd
pMD19T-hbd
RBS-crt
pMD19T-crt
paaH1
RBS-ech
R. eutropha H16
genomic DNA
R. eutropha H16
genomic DNA
Figure S1. MS spectrum of intermediates of the adipic acid biosynthetic pathway. 100 µM of
acetyl-CoA and 100 µM of succinyl-CoA were incubated with appropriate cofactors and the
purified pathway enzymes: (A) PaaJ, (B) PaaJ, Hbd, (C) PaaJ, Hbd, and Crt, (D) PaaJ, Hbd,
Crt, and Ter. The formation of the pathway intermediates was suggested by peaks showing
desired molecular weights (Down panels), while these peaks were absent if the enzymes were
pre-inactivated before the setup of enzymatic reactions (Up panels).
8
Figure S2. GC-MS spectrum of silylated derivatives of culture medium from recombinant E.
coli. Cell-free supernatant was acidified, extracted with ethyl acetate, and derivatized with a
silylating reagent before GC-MS analysis. Total ion chromatograph of the sample was
separated by GC (Up). Adipic acid, bis(trimethylsilyl) ester had a retention time of 16.72 min
with MS/MS spectrum shown below (Down).
9
Figure S3. Standard curve for adipic acid quantification. Authentic adipic acid was dissolved
in ethyl acetate and diluted to form a range of concentrations. The standards were derivatized
to form adipic acid, bis(trimethylsilyl) ester, and analyzed by GC-MS. Adipic acid,
bis(trimethylsilyl) ester had a characteristic precursor ion/daughter ion of 111→55 (m/z), thus
allowing quantification.
10