Applied Microbiology and Biotechnology
Supplementary materials
Fine-tuning of ecaA and pepc gene expression increases succinic acid
production in Escherichia coli
Names of authors:
Jing Wang1, Dandan Qin1, Baoyun Zhang2, Qiang Li3, Sha Li4, Xiaohua Zhou1, Lichun Dong1,
Dan Wang1,4*
Affiliations:
1
School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China
2
School of Bioengineering, Chongqing University, Chongqing 400044, China
3
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese
Academy of Sciences, Beijing 100190, China
4
State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of
Technology, Nanjing 210009, China
*Corresponding Author: Dan Wang
Tel: +86-23-65111179
Fax: +86-23-65111179
E-mail address: dwang@cqu.edu.cn (Dan Wang)
Mailing address: P. O. box 94#, Shazheng Street 174, Shapingba District, College of Chemistry and
Chemical Engineering, Chongqing University, Chongqing, 400044, China
Table S1
Primers, plasmids, plasmid libraries, strains and strain libraries used in this study.
Primers/Plasmids/
Plasmid
Description
Source/Restriction
libraries/
site
Strains/Strain
libraries
Primers
rbSSR-ecaA-up1
5’CGCGGATCCGCGCAGGAAAAAAAAAAAAAAA
This study/BamHI
AATGAGTAGTACTTTATATCGAAGGC3’
rbSSR -ecaA-up2
5’CGCGGATCCGCGCAGGACACACACACACACAC
This study/BamHI
ATGAGTAGTACTTTATATCGAAGGC3’
rbSSR -ecaA-up3
5’CGCGGATCCGCGCAGGATATATATATATATATATG
This study/BamHI
AGTAGTACTTTATATCGAAGGC3’
rbSSR -ecaA-up4
5’CGCGGATCCGCGCAGGCCCCCCCCCCCCCCCC
This study/BamHI
ATGAGTAGTACTTTATATCGAAGGC3’
rbSSR -pepc-up1
5’CGCGGATCCGCGCAGGAAAAAAAAAAAAAAA
This study/BamHI
AATGGTGGATTTGTTACGGCAGT3’
rbSSR -pepc-up2
5’CGCGGATCCGCGCAGGACACACACACACACAC
This study/BamHI
ATGGTGGATTTGTTACGGCAGT3’
rbSSR -pepc-up3
5’CGCGGATCCGCGCAGGATATATATATATATATATG
This study/BamHI
GTGGATTTGTTACGGCAGT3’
rbSSR -pepc-up4
5’CGCGGATCCGCGCAGGCCCCCCCCCCCCCCCC
This study/BamHI
ATGGTGGATTTGTTACGGCAGT3’
rbSSR -up
5’CTGTACGACGATGACGATAAGG3’
This study
rbSSR -ecaA-down
5’GGGAAGCTTCCCAGAAGTGTCTAGGGATTGAG
This study
3’
rbSSR -pepc-down
5’GGGAAGCTTCCCAACCTGTATTTCTCATCCCT3’
This study
CA-RT-up
5’AGTGTCTTGGGAACCTCAT3’
This study
CA-RT-down
5’CGTTTGTTTCTGCCTATT3’
This study
PC-RT-up
5’GCTTCTTGAGTAGCAATG3’
This study
PC-RT-down
5’GCCAAAATCGCCTCGTG3’
This study
AMP-RT-up
5’GCGACTTAGCCGTTATGGGT3’
This study
AMP-RT-down
5’TGTTGTGCTGGGAGAATTG3’
This study
pTrchisB
Expressing vectors with Trc promoter
Invitrogen
pTrc-ecaA1
ecaA gene cloned from Anabaena sp. PCC 7120 under
This study
Plasmids
the Trc promoter of pTrchisB with (A)16 as spacer motif
pTrc-ecaA2
ecaA gene cloned from Anabaena sp. PCC 7120 under
This study
the Trc promoter of pTrchisB with (Ac)8 as spacer motif
pTrc-ecaA3
ecaA gene cloned from Anabaena sp. PCC 7120 under
This study
the Trc promoter of pTrchisB with (AT)8 as spacer motif
pTrc-ecaA4
ecaA gene cloned from Anabaena sp. PCC 7120 under
This study
the Trc promoter of pTrchisB with (C)16 as spacer motif
pTrc-pepc1
pepc gene cloned from Anabaena sp. PCC 7120 under
This study
the Trc promoter of pTrchisB with (A)16 as spacer motif
pTrc-pepc2
pepc gene cloned from Anabaena sp. PCC 7120 under
This study
the Trc promoter of pTrchisB with (Ac)8 as spacer motif
pTrc-pepc3
pepc gene cloned from Anabaena sp. PCC 7120 under
This study
the Trc promoter of pTrchisB with (AT)8 as spacer motif
pTrc-pepc4
pepc gene cloned from Anabaena sp. PCC 7120 under
This study
the Trc promoter of pTrchisB with (C)16 as spacer motif
Plasmid libraries
pTrc-ecaA
ecaA gene cloned from Anabaena sp. PCC 7120 under
This study
the Trc promoter of pTrchisB with different base
sequences as spacer motif
pTrc-pepc
pepc gene cloned from Anabaena sp. PCC 7120 under
This study
the Trc promoter of pTrchisB with different base
sequences as spacer motif
Strains
DC1515
pflA::Cam ldhA::Tn 10 ptsG400::Kan in W1485
Clark, unpublished
Anabaena sp. PCC
Providing ecaA gene and pepc gene
Pasteur Institute in
7120
France
KCO111
DC1515 harboring plasmids pTrc-ecaA and pTrc-pepc
This study
which overexpressing ecaA gene 3.53-fold and pepc
gene 1.06 fold
Strain libraries
KCA1
DC1515 harboring pTrc-ecaA libraries, overexpressing
This study
ecaA gene
KPC1
DC1515 harboring pTrc-pepc libraries, overexpressing
This study
pepc gene
KCO1
DC1515 harboring pTrc-ecaA libraries and pTrc-pepc
libraries, overexpressing ecaA gene and pepc gene
This study