Bioethanol production by heterologous expression of
Pdc and AdhII in Streptomyces lividans TK24
Journal name: Applied Microbiology and Biotechnology
Jae Sun Leea, Won-Jae Chib, Soon-Kwang Hongb, Ji-Won Yanga,c,and Yong Keun
Changa#
a
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of
Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon, 305-701, South Korea
b
Division of Bioscience and Bioinformatics, Myongji University, San 38-2 Namdong, Yongin,
Gyeonggido, 449-728, South Korea
c
Advanced Biomass R&D Center, 373-1, Guseong-dong, Yuseong-gu, Daejeon, 305-701,
South Korea
# Corresponding author:
Yong Keun Chang, changyk@kaist.ac.kr
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of
Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon, 305-701, South Korea
TEL +82-42-350-3927, FAX +82-42-350-3910
Table S1. Strains and plasmids used
Designation
Relevant characteristics
Source or Reference
Strains
S. lividans TK24
Str-6
John Innes Centre, UK
supE44ΔlacU169(Ø80lacZΔM15)
E. coli DH5α
Stratagene
hsdR17 recA1 endA1 gyr96 thi-1 relA1
E. coli ET12567
dam-13::Tn9 dcm-6 hsdS cat tet
MacNeil et al. 1992
pUC18
pMB1 ori ampr
Genescript USA Inc
T & A vector
lacZ, T-cloning flank, ampr
Real Biotech Corporation
Plasmids
High-copy, tsrr, ampr, E. coli-Streptomyces
pUWL201PW
Doumith et al. 2000
expression vector
High-copy, tsrr, ampr, E. coli-Streptomyces
pHSEV-1
This study
expression vector
2.9-kb fragment containing codon-
pUWL201PW-pet
optimized pdc and adhII inserted into the
This study
NdeI-BamH1 site of pUWL201PW
3.0-kb fragment containing codonoptimized pdc, tipA promoter, and codonpUWL201PW-Tpet
This study
optimized adhII inserted into the NdeIBamH1 site of pUWL201PW
2.9-kb fragment containing pdc and adhII
pUWL201PW-Zpet
from Zymomonas mobilis inserted into the
NdeI-BamH1 site of pUWL201PW
This study
Table S2. Primers used for the pet operon and Tpet module construction
Primer
Target gene
Sequence (5' → 3')
1
F-pdc
cat atg atg tcc tac acc gtc ggc acc tac
2
R-pdc
ctg cag tca cag gag ctt gtt gac
3
F-adh II
cat atg atg gcc tcc tcg acc ttc tac
4
R-adh II
gga tcc tca gaa ggc cga gag gaa cag ctc
5
F-RBS +adh II
ctg cag gag gag cat atg atg gcc tcc tcg acc
6
R-RBS +adh II
gga tcc tca gaa ggc cga gag gaa cag
7
F-Zpdc
cat atg atg agt tat act gtc ggt acc
8
R-Zpdc
ctg cag tta gag gag ctt gtt aac agg ctt acg
9
F-ZadhII
cat atg atg gct tct tca act ttt tat
10
R-ZadhII
gga tcc tta gaa agc gct cag gaa gag
11
F-RBS +ZadhII
ctg cag gag gag cat atg atg gct tct tca
12
R-RBS+ ZadhII
gga tcc tca gaa gga tcc tta gaa agc gct
13
F-tipA
ctg cag ggg ctg agg gag
14
R-tipA
ctg cag tcc gct ccc ttc
Figure S1.
Detection of Pdc and AdhII proteins expressed in S.lividans TK24/pet by SDS-PAGE. (M,
Molecular size marker; 1, S. lividans TK24/pUWL201PW; 2, S. lividans TK24/pet; 3, S.
lividans TK24/Zpet; 4, S. lividans TK24/Tpet)