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Supplementary materials for
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Characterizing yeast promoters used in Kluyveromyces marxianus
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Chun Yang1, 2, ShengLin Hu1, 2, Songli Zhu1, 2, Dongmei Wang1, 2, Xiaolian Gao1, 2, 3, Jiong Hong1,
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1. School of Life Science, University of Science and Technology of China, Hefei, Anhui, 230027,
P. R. China
2. Hefei National Laboratory for Physical Science at the Microscale, University of Science and
Technology of China, Hefei, Anhui 230026, P. R. China
3. Department of Biology and Biochemistry, University of Houston, Houston, TX 77004-5001,
USA
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1. Plasmid construction
In this study, 45 plasmids were constructed and consecutively named from pSL003 to pSL048. First,
the promoters to be evaluated were amplified by PCR. The P KmADH1 (GenBank X60224) and PKmTDH3
(GenBank DQ681075) promoters of K. marxianus were amplified from the genomic DNA of K.
marxianus NBRC 1777 with the primer pairs Bsu15I-pADH-KM-F/MunI-pADH-KM-R and
Bsu15I-pGAP-KM-F/MunI-pGAP-KM-R, respectively, and the products were digested by Bsu15 I and
Mun I. The PKmPGK (GenBank AP014599) promoter of K. marxianus was amplified with the primer pair
BstBI-pPGK-KM-F/EcoRI-pPGK-KM-R and digested by BstB I and EcoR I. The three promoters were
inserted into the BstB I- and EcoR I-digested YEGAp plasmid. The resulting plasmids were named
pSL003, pSL004, and pSL005, respectively. The PScADH1 (GenBank AB012284) promoter of S.
cerevisiae was amplified from the genomic DNA of S. cerevisiae with the primer pair
BstBI-pADH-SC-F/EcoRI-pADH-SC-R, and was also digested by BstBI and EcoRI. The PScADH1
promoter was also inserted into the BstBI- and EcoRI-digested YEGAp plasmid, and the resulting
plasmid was named pSL006. The PScPGK (GenBank KC562906) promoter of S. cerevisiae was amplified
from the genomic DNA of S. cerevisiae with the primer pair EcoRI-pPGK-SC-F/SalI-pPGK-SC-R, was
digested by EcoR I and Sal I, and was then inserted into the EcoRI- and SalI-digested YEGAp plasmid.
The resulting plasmid was named pSL008. The PScTDH3 promoter in the YEGAp plasmid was used
directly.
Three genes, GUSA, KlLAC4, and SH BLE, were used to evaluate the promoter activity. GUSA
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was amplified from the pBAV1K plasmid with the primer pair SalI-GUSA-F/NotI-GUSA-R. KlLAC4
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was amplified from the genomic DNA of K. lactis using the primer pair SalI-LacZ-F/NotI-LacZ-R. The
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primer pair SalI-Zeocin-F/NotI-Zeocin-R was employed to amplify SH BLE from the pGAPZα-A
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plasmid (Invitrogen, Carlsbad, CA, USA).
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The DNA obtained from these three genes was digested by SalI and NotI, and was then inserted
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into SalI- and NotI-digested pSL003, pSL004, pSL005, pSL006, pSL008, and YEGAp plasmids,
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respectively. The resulting plasmids were named with consecutive numbers from pSL009 to pSL026,
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as shown in Table S2. All the fragments, including the promoter, gene, and terminator, were amplified
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with the primer pair GAP-stuI-F/GAP-stuI-R and inserted into the pKmLEU2 MscI site. The resulting
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plasmids were named consecutively from pSL027 to pSL044 and they were used as the template for the
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amplification of integrated expression cassette. A map of the constructed plasmids was shown in Fig.
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S1 and the selection marker, promoter and gene used for evaluation of the plasmids were shown in
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Table S2.
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An α-amylase gene from Aspergillus oryzae was expressed under promoters PScTDH3 and PKmPGK to
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confirm the obtained information. The ORF of AoAMY gene (GenBank Acesssion No.
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XM_003189571.1) was amplified from YEGAp-AoAMY (pSL045) (Wang et al. 2014) with primer
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pair AoAMY- EcoRI-F/ AoAMY-NotI-R (Table S1). After the DNA fragment was digested by EcoR I
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and Not I, it was inserted into EcoR I and Not I digested pSL005. The resulting plasmids were named as
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pSL046. The expression cassettes from pSL045 and pSL046 were amplified using primer pair
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GAP-StuI-F/GAP-StuI-R and inserted into the pKmLEU2 MscI site. The resulting plasmids were
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named pSL047 and pSL048, and were used as the templates for the amplification of integrated
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expression cassette.
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2. Construction of the strains integrated the expression cassettes at KmLEU2 locus of genome
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After the integrative plasmids listed in Table 2S were constructed and the expression cassettes
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including KmLEU2, promoters and genes were amplified by PCR using the primer pair
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KmLEU2-F/KmLEU2-R, the expression cassettes were transformed into K. marxianus YHJ 010 by the
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lithium acetate method (Abdel-Banat et al. 2010; Yanase et al. 2010). The transformants were selected
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on SD medium supplemented with uracil and tryptophan. The obtained transformants were inoculated
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into 5 mL YPD, and the genomic DNA was extracted as previously described (Zhang B et al. 2011).
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The strains in which expression cassettes were homologously integrated on the KmLEU2 locus were
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screened through PCR with genomic DNA of transformants as template (Fig. S2). For SH BLE, GUSA,
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KlLAC4
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Leu961F/Leu2881R and EcoRI-AoAMY-F/GAP -StuI-R were used, respectively. The primer pairs and
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the corresponding strains are listed in Table S1.
and
AoAMY,
the
primer
pairs
SalI-Zeocin-F/Leu260R,
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SalI-GUSA-F/Leu260R,
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Fig. S1 Plasmid map used to evaluate the promoter. The “Promoter” indicated in the map was P KmADH1,
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PKmTDH3, PKmPGK, PScADH1, PScTDH3, or PScPGK, and the “Gene” in the map was SH BLE, GUSA, KlLAC4 or
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AoAMY. The expression cassettes were inserted into the KmLEU2 (black arrow line) and divided the
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KmLEU2 into two parts. The fragments including KmLEU2, promoter, gene, and terminator T ScTDH3 can
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be amplified by the primers KmLEU2-F and KmLEU2-R (small arrow) for integration. The plasmids
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with various combinations of promoters and genes are listed in Table S2.
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Fig. S2 Confirmation of site-directed integration of expression at KmLEU2 locus. The arrow indicates
the results of positive clones. The integration at KmLEU2 locus of SH BLE (A), GUSA (B), KlLAC4 (C)
and AoAYMY (D) were confirmed by PCR and detected by the size change of amplified products. A 1.8
Kb PCR products confirmed that SH BLE expression cassette correctly integrated using primers
SalI-Zeocin-F/Leu260R; for GUSA, the size was 3.4 Kb products which confirmed the correctly
integrated using primers SalI-GUSA-F/Leu260R. For the longest KlLAC4 gene, the primers
Leu961F/Leu2881R were used, and the size of PCR product was 6.0 Kb due to the insertion of KlLAC4
expression cassette in KmLEU2 locus，otherwise both 2.8 Kb and 6Kb products would appeared. 1.7
Kb PCR products using primers EcoRI-AoAMY-F/GAP-StuI-R should be detected if the AoAMY
expression cassette was correctly integrated. Other products with various fragments were unspecific
PCR product. For each strain, at least three clones were obtained for analysis
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Fig. S3 Determination of the amylase activity through halo assay on soluble starch plate and
quantitative determination of the activity in culture supernatant. (A) Halo assay at 30°C; (B) Halo assay
at 42°C; (C) the amylase activity in culture supernatant.
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Table S1 Primers used in this study
Primers
Sequences (5’->3’)*
Application
Bsu15I-pADH1-KM-F
ATCGATCGATCCGAGTGGCATGATTCGCGCAG
K. marxianus ADH1 promoter amplification
MunI-pADH1-KM-R
ATCGCAATTGTGTGTGTATGATTTTGTTT
K. marxianus ADH1 promoter amplification
Bsu15I-pGAP-KM-F
ATCGATCGATGCATGCCCATTACCCGGAATAT
K. marxianus TDH3 promoter amplification
MunI-pGAP-KM-R
ATCGCAATTGTGTGATGTGTAAAAGTGTGTGT
K. marxianus TDH3 promoter amplification
BstBI-pPGK-KM-F
ATCGTTCGAAGGAGCACGATATCTTGGTCATTGC
K. marxianus PGK promoter amplification
EcoRI-pPGK-KM-R
ATCGGAATTCCATTTTTGTATCTTTATATAGGTAG
K. marxianus PGK promoter amplification
BstBI-pADH1-SC--F
GGGTTCGAATCTAGCTCCCTAACATGTAGGTGG
S. cerevisiae ADH1 promoter amplification
EcoRI-pADH1-SC-R:
GGGGAATTCTGTATATGAGATAGTTGATTGTATG
S. cerevisiae ADH1 promoter amplification
EcoRI-pPGK-SC-F:
GGGGAATTCCATGGCGGGCATGTATCATTGCCCT
S. cerevisiae PGK promoter amplification
SalI-pPGK-SC-R:
GGGGTCGACTGTTTTATATTTGTTGTAAAAAGTA
S. cerevisiae PGK promoter amplification
SalI-GUSA-F
GGGGTCGACGAAACGATGGGCAGCAGCCATCACCAT
GUSA amplification
CATC
NotI-GUSA-R
ATGCGCGGCCGCCTACCGGCCGCATAGGCCTTGTTTG
GUSA amplification
SalI-Lac-F:
GGGGTCGACGAAACGATGTCTTGCCTTATTCCTGAGA
KLLAC4 amplification
ATT
NotI-Lac-R:
ATGCGCGGCCGCTTATTCAAAAGCGAGATCAAACTCA
KLLAC4 amplification
SalI-Zeocin-F
GGGGTCGACGAAACGATGGcCAAGTTGACCAGTGCC
SH BLE amplification
GTTC
NotI-Zeocin-R
ATGCGCGGCCGCTCAGTCCTGCTCCTCGGCCACGAA
SH BLE amplification
G
AoAMY-EcoRI-F
ACGTGAATTCATGATGGTCGCGTGGTGGTCTC
AoAMY amplification
AoAMY-NotI-R
ATAAGAATGCGGCCGCTTACGAGCTACTACAGATCTT
AoAMY amplification
GCTACC
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GAP-stuI-F
GAGAAGGCCTACCAGTTCTCACACGGAACACCAC
Expression cassette amplification
GAP-stuI-R
GAGAAGGCCTTCAATCAATGAATCGAAAATGTC
Expression cassette amplification
KmLEU2-F
AGAAGAGTCATATAGCCAAAGAACC
Expression cassette amplification
KmLEU2-R
CATGGTTTTGCGAGTTAAGTC
Expression cassette amplification
RT-kmACT1-F
CCCAATGAACCCAAAGAATAACAG
For RT-PCR of ACT1
RT-kmACT1-R
GATAGCATGAGGCAAGGAGAAACC
For RT-PCR of ACT1
RT-Zeocin-R
GCGCTGATGAACAGGGTCACGTCG
For RT-PCR of SH BLE
RT-Zeocin-F
CCAAGTTGACCAGTGCCGTTCCGGT
For RT-PCR of SH BLE
RT-LAC4-F
CAGGAAGACTGGAAGTACGGTAAAC
For RT-PCR of KLLAC4
RT-LAC4-R
CCATTAACATAAAGCTCGTAACAATTGTC
For RT-PCR of KlLAC4
RT-GUSA-R
ACATCGGCTTCAAATGGCGTATAGC
For RT-PCR of GUSA
RT-GUSA-F
ATCAGTTCGCCGATGCAGATATTCG
For RT-PCR of GUSA
Leu260R
CATATGCGAGTCCATTATGTCCTTCT
For integration confirmation
Leu961F
GAATCACCAGAATGGCTGCATTTTTAGC
For integration confirmation
Leu2881R
GTTGGAAGAGGCTATCAGACCAGAC
For integration confirmation
*underlined sequences indicate the restriction enzyme site
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Table S2 Plasmids used in this study
Plasmids
Selection
marker
Promoter
Gene
Reference
YEGAp
pKmLEU2
pSL003
pSL004
pSL005
pSL006
pSL008
pSL009
pSL010
pSL011
pSL012
pSL013
pSL014
pSL015
pSL016
pSL017
pSL018
pSL019
pSL020
pSL021
pSL022
pSL023
pSL024
pSL025
pSL026
pSL027
pSL028
pSL029
pSL030
pSL031
pSL032
pSL033
pSL034
pSL035
pSL036
pSL037
pSL038
pSL039
pSL040
pSL041
pSL042
pSL043
pSL044
ScTRP1
KmLEU2
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
ScTRP1
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
KmLEU2
PScTDH3
PKmADH1
PKmTDH3
PKmPGK
PScADH1
PScPGK
PKmADH1
PKmADH1
PKmADH1
PKmTDH3
PKmTDH3
PKmTDH3
PKmPGK
PKmPGK
PKmPGK
PScADH1
PScADH1
PScADH1
PScTDH3
PScTDH3
PScTDH3
PScPGK
PScPGK
PScPGK
PKmADH1
PKmADH1
PKmADH1
PKmTDH3
PKmTDH3
PKmTDH3
PKmPGK
PKmPGK
PKmPGK
PScADH1
PScADH1
PScADH1
PScTDH3
PScTDH3
PScTDH3
PScPGK
PScPGK
PScPGK
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
GUSA
KLLAC4
SH BLE
(Hong et al. 2007)
(Hong et al. 2007)
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pSL045
pSL046
pSL047
pSL048
ScTRP1
ScTRP1
KmLEU2
KmLEU2
PScTDH3
PKmPGK
PScTDH3
PKmPGK
AoAMY
AoAMY
AoAMY
AoAMY
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(Wang et al. 2014)
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Table S3 Yeast strains used in this study
Strains
Relevant genotype
Reference
K. marxianus
NBRC 1777
YHJ 010
Wild type
NBRC1777;
(Hong et al.2007)
ΔKmUra3::Kanr,
ΔKmLeu2::HisG,
(Hong et al.2007)
ΔKmTrp1::HisG
YYC 027
YHJ 010; KmLEU2; PKmADH1-GUSA
This study
YYC 028
YHJ 010 ; KmLEU2; PKmADH1-KLLAC4
This study
YYC 029
YHJ 010; KmLEU2; PKmADH1-SH BLE
This study
YYC 030
YHJ 010; KmLEU2; PKmTDH3-GUSA
This study
YYC 031
YHJ 010; KmLEU2; PKmTDH3-KLLAC4
This study
YYC 032
YHJ 010; KmLEU2; PKmTDH3-SH BLE
This study
YYC 033
YHJ 010; KmLEU2; PKmPGK-GUSA
This study
YYC 034
YHJ 010; KmLEU2; PKmPGK-KLLAC4
This study
YYC 035
YHJ 010; KmLEU2; PKmPGK-SH BLE
This study
YYC 036
YHJ 010; KmLEU2; PScADH1-GUSA
This study
YYC 037
YHJ 010; KmLEU2; PScADH1-KLLAC4
This study
YYC 038
YHJ 010; KmLEU2; PScADH1-SH BLE
This study
YYC 039
YHJ 010; KmLEU2; PScTDH3-GUSA
This study
YYC 040
YHJ 010; KmLEU2; PScTDH3-KLLAC4
This study
YYC 041
YHJ 010; KmLEU2; PScTDH3-SH BLE
This study
YYC 042
YHJ 010; KmLEU2; PScPGK-GUSA
This study
YYC 043
YHJ 010; KmLEU2; PScPGK-KLLAC4
This study
YYC 044
YHJ 010; KmLEU2; PScPGK-SH BLE
This study
YYC 045
YHJ 010; KmLEU2
This study
YYC 046
YHJ 010; KmLEU2; PScTDH3-AoAMY
This study
YYC 047
YHJ 010; KmLEU2; PKmPGK-AoAMY
This study
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