Antonie van Leeuwenhoek
Supplementary Material
Heterologous expression of galbonolide biosynthetic genes in Streptomyces coelicolor
Chao Liu • Juanli Zhang • Chunhua Lu • Yuemao Shen
Chao Liu • Yuemao Shen (
)
State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan,
Shandong 250100, P. R. China
Juanli Zhang • Chunhua Lu • Yuemao Shen (
)
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences,
Shandong University, Jinan, Shandong 250012, P. R. China
e-mail: yshen@sdu.edu.cn; Tel.: +86-531-8838-2108
Received: 9 December 2014 / Accepted: 23 February 2015
© Springer International Publishing Switzerland 2015
1
Construction of Heterologous Expression Constructs
The heterologous expression vectorswere constructedidenticalto previously reported (Liu et al. 2015).
Briefly, the gbnA-E fragment was first excised from fosmid 1D39 by digestion with MfeI and AfeI. The
~11.8 kb target DNA fragment wascloned into the EcoRI/EcoRV-digested pSET152 vector to generate
pSET152-apkcr. Next, the cat-ermER* cassette was amplified from plasmid pUC19-Cm-PermER* (Li et
al. 2014) use for promoter replacement. Then, the native promoter of gbnA in pSET152-apkcr was
replaced with a modified ermE* promoter (ermER*) by PCR-targeting (Gust et al. 2003) to produce the
integrativeplasmid pSET152-ermER*-apkcr.
Furthermore, to construct thegbnD-E deletion plasmid, the gbnC fragment was amplified from fosmid
1D39and digested by KpnI, followed by ligated into KpnI-digested pSET152-ermER*-apkcr to generate
pSET152-ermER*-apk.
2
Heterologous expression of gbnA-C in Streptomyces coelicolor ZM12
a
b
NL: 2.71E7
ZM12 WT
NL: 1.93E7
ZM12 WT
NL: 2.24E7
ZM12::ermER*apk
0
4
8
12
16
20
24
28
32
0
4
8
12
16
20
Time (min)
Time (min)
m/z = 150.00-500.00
m/z = 150.00-200.00
24
28
d
c
0
NL: 1.63E7
ZM12::ermER*apk
4
8
12
16
20
Time (min)
24
28
NL: 1.51E7
ZM12 WT
NL: 1.40E7
ZM12 WT
NL: 1.53E7
ZM12::ermER*apk
NL: 1.22E7
ZM12::ermER*apk
0
32
4
8
12
16
20
24
28
m/z = 300.00-350.00
e
NL: 7.43E6
ZM12 WT
f
NL: 3.29E6
ZM12 WT
NL: 5.35E6
ZM12::ermER*apk
4
8
12
16
20
32
Time (min)
m/z = 200.00-300.00
0
32
24
28
32
NL: 3.65E6
ZM12::ermER*apk
0
4
8
12
16
20
Time (min)
Time (min)
m/z = 350.00-400.00
m/z = 400.00-500.00
24
28
32
Fig.S1HPLC-MS analysis of the metabolic profiles of Streptomycescoelicolor ZM12 (host strain, WT)
and ZM12::ermER*apk (mutant strain containing gbnA-C).a-f The extracted ion chromatogram (EIC) of
m/z150-500, 150-200, 200-300, 300-350, 350-400, and 400-500, respectively.
3
References
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HSAF. Angew Chem Int Ed 53:7524–7530
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