Supplementary Table 1 - Transformants of T. reesei RUT-C30 constructed in this
study.
Supplementary Table 2 – Sequences of oligos.
Phosphinothricin purification
Transformation of T. reesei
Immobilized metal-ion affinity chromatography
RNA preparation and quantitative real-time reverse transcription PCR
Plasmid construction
Supplementary Figure 1. Restriction enzyme map of pPK2, pPK1s and two based
vectors pSB902 and pSB903.
Supplementary Figure 2. The transformation efficiency and relative hpt mRNA
expression level using five short promoters.
Supplementary Figure 3. Alkaline endoglucanase activities in s-pSB902-V3
transformants and RUT-C30 with no additional plasmids.
Supplementary Figure 4. Copy number of the deletion cassette in two Δcbh1
transformants (CBH1-rgf.1 and CBH1-rgf.7).
Additional Results: DNA sequences of plasmids
Additional References
Supplementary Table 1. Transformants of T. reesei RUT-C30 constructed in this study.
Strain
Relevant features
promoter
reporter gene
Source
RUT-C30
parent strain
-
-
ATCC
pBar
RUT-C30 harboring vector pBar
As. nidulans trpC
bar
This study
p9B
RUT-C30 harboring vector p9B
truncated xyn2
bar
This study
C1-s
RUT-C30 harboring vector C1-s
As. nidulans gpd
hpt
This study
C1-pAs
RUT-C30 harboring vector C1-pAs
As. Nidulans amdS
hpt
This study
C1-pbNs
RUT-C30 harboring vector C1-pbNs
As. nidulans trpC
hpt
This study
C1-p7s
RUT-C30 harboring vector C1-p7s
xyn1
hpt
This study
C1-p9s
RUT-C30 harboring vector C1-p9s
truncated xyn2
hpt
This study
s-pSB902-V3
RUT-C30 harboring vector s-pSB902-V3
truncated xyn2
egv3
This study
bns-pSB101-rfp
RUT-C30 harboring vector bns-pSB101-rfp
As. nidulans trpC, cbh1 hpt, rfp
This study
CBH1-rgf.1, CBH1-rgf.7
pG01-rfp transformant
cbh1
This study
rfp
RUT-C30::pG01-rfp--gfp
RUT-C30::pG01-rfp harboring vector 9B-pSB401-gfp
cbh1, cbh2
rfp, gfp
This study
CBH1-V3.1
pG01-V3 transformant
cbh1
egv3
This study
CBH2-H2.1
pG02-H2 transformant
cbh2
H. cbh2
This study
CBH1-V3--CBH2-H2.1
pG01-V3 and pG02-H2 transformant
cbh1, cbh2
egv3, H. cbh2 This study
Supplementary Table -2. Sequences of oligos.
Name
oligos Sequences (5’ to 3’)
bar-forward
cgaaCACGTG(PmlI)ATGAGCCCAGAACGACGCC
bar-reverse
cgaaCGCGCGCG(MauBI)TCAGATCTCGGTGACGGGCA
PPT-forward
cgaaTTAATTAA(PacI)GTTAACTCTAGA(XbaI)gatatcGTCGGAGACAGAAGATGATATT
PPT-reverse
cgaaATTTAAAT(SwaI)GAGCTCACTAGT(SpeI)TTTTTTTTAATTTGTTGGAGATTTCA
VF
cgaa CACGTG(PmlI)GCCGACGGCCGCAGCAC
VR
cgaa CGCGCGCG(MauBI)TTACAGGCACTGGTGGTACCAG
C15F
cgaaTTAATTAA(PacI)GATATCAGTATGTAAGTCCAA
C15R
cgaaACTAGT(SpeI)TGCTACTAGACACTGCTATCGG
C13F
cgaaTCTAGA(XbaI)CGTGGCGAAAGCCTGACG
C13R
cgaaATTTAAAT(SwaI)ATCAATTGCTGCTTCATACTA
C25F
cgaaTTAATTAA(PacI)CTAGTCAATGGTAGCAGATCAGTC
C25R
cgaaACTAGT(SpeI)GAGATATAAGGCAGAATGGATACGA
C23F
cgaaTCTAGA(XbaI)TAGATTCCAATTACTCCACCTCTTG
C23R
cgaaATTTAAAT(SwaI)ACTACGCTAATCGGATAGATATTCG
qsar-f
TGGATCGTCAACTGGTTCTACGA
qsar-r
GCATGTGTAGCAACGTGGTCTTT
qhpt-f
AGCGAGAGCCTGACCTATTGC
qhpt-r
CCATGTAGTGTATTGACCGATTCCTT
Phosphinothricin purification
Phosphinothricin (PPT, also called glufosinate), the active ingredient in basta, is
currently expensive in its pure form. Basta (Bayer Cropscience, Australia), an
herbicide with PPT as an active ingredient (~18%), was used in the present study.
Because PPT is highly soluble in water, a simple extraction separates it from the
pigment and other non-specific inhibitory ingredients. In the current experiment,
Basta was extracted three times with an equal volume of 1-butanol, the solution was
lyophilized and the resulting gel was then dissolved in water (final concentration, 200
mg/mL).
Transformation of T. reesei
Transformation of the hpt marker vectors was performed based on protocols
described by Covert et al. (2001). The PPT marker vectors pBar, p9B,
9B-pSB401-H2 and 9B-pSB401-gfp were introduced into RUT-C30 by
Agrobacterium-mediated transformation (Moon et al. 2008) with the following
modifications. The transformant was selected by transferring each filter paper
onto plates containing M-100 medium amended with 10 g glucose l-1 and 400 μg
purified PPT ml-1. The medium also contained 100 μg of cefotaxime (Generay,
Shanghai, China) ml-1 to kill bacteria. After one day of incubation at 27°C, the
filter paper was overlaid with 10 ml freshly prepared M-100 agar containing 300
μg purified PPT ml-1. Plates were incubated for 4 to 7 days at 27°C until the
transformants had grown to the surface of the agar. Each primary transformant
was transferred to a single well of a 24-well microtiter plate containing M-100
medium (300 μg purified PPT ml-1).
Immobilized metal-ion affinity chromatography
Fermentation broth culture supernatant (9 ml; centrifuged at 7,000 g for 10 min
at 4°C) was collected for chromatography by adding 1 ml 10× NPI-10 (50 mM
NaH2PO4, 300 mM NaCl, 10 mM imidazole) and adjusting the pH to 8.0 using
NaOH. Mycelia were harvested by filtration and then washed twice with distilled
water. Excess moisture was removed by pressing the mycelia between sheets of
Whatman 3mm filter paper (Whatman), after which the mycelia were immediately
frozen in liquid nitrogen and ground into a fine powder. The powder was suspended in
an appropriate volume of NPI-10 and the pH was adjusted to 8.0 using NaOH.
Nitrilotriacetic acid (Ni-NTA) chromatography was carried out according to the
Ni-NTA Superflow Cartridge Handbook (www.qiagen.com). All chemicals were
purchased from Merck, except for Ni-NTA-Superflow (Qiagen).
RNA preparation and quantitative real-time reverse transcription PCR
About 100 mg of T. reesei mycelium was harvested. Total RNA was
extracted using the FastRNA Pro Red Kit (MPbio, U. S. A.), according to the
manufacturer’s instructions. Reverse transcription was performed with 1000 ng
of total RNA using TransScript All-in-One First-Strand cDNA Synthesis
SuperMix for qPCR (TransGen, China), according to the manufacturer’s
instructions. For qRT-PCR, the TransStart TipTop Green qPCR SuperMix
(TransGen, China) was used with 200 nM of forward and reverse primers (see
Supplemental Material Supplementary Table -2) and 1 μl of 10-fold diluted
cDNA in a final volume of 20 μl. The conditions of qPCR: 30s at 94oC, 45 cycles
of 5s at 94 oC and 30s at 60 oC，followed by dissociation stage. For hpt
transcription analysis, a SYBR green assay with reference to the small GTPase
gene (sar1) was performed (Steiger et al. 2010). Thermocycling was performed
in an ABI StepOne Plus thermocycler (Applied Biosystems, U. S. A.).
Plasmid construction
RUT-C30 genomic DNA was prepared according to the method described by
Penttilä et al. (1987). The expression vector pWEF31-V (Zhang et al. 2014) was used
as the source of the alkaline endoglucanase, egv. The template for the 0.64-kb
Aspergillus nidulans amdS promoter was the plasmid pMS-HALS (Steiger et al. 2011).
The template for the 0.56-kb bar, 1.0-kb PPT-resistant box and 0.36-kb As. nidulans
trpC promoter was the plasmid pTJK1 (Jones et al. 2007). The template for the
1.02-kb hpt was the plasmid pPK1s (Zhang et al. 2014). The vectors were built by
exploiting the compatibility of cohesive ends generated by XbaI and SpeI in
pPK2-derived BioBrick base vector, as previously described (Covert et al. 2001;
Wang et al. 2014). All primers used in this section are listed in Supplemental Material
Supplementary Table -2.
Expression vectors pSB902 and pSB903: The expression vectors pSB902 and
pSB903 (Fig. S1) (Supplemental Data File) were constructed using the truncated xyn2
promoter (0.24-kb), with or without native signal peptide, respectively, and the nos
terminator (GenBank: KF499077.1). pSBa01 and pSBbN (Supplemental Data File)
were constructed using the amdS or trpC promoter, respectively, and the nos
terminator. The gene expression device pSB701 (Supplemental Data File) was
constructed with the core promoter region of the gene xyn1 (Martinez et al. 2008) and
the nos terminator.
Hygromycin B resistance vectors pAs, pbNs, p7s and p9s: The 1.02-kb hpt was
subcloned into the PmlI/MauBI site of pSBa01, pSBbN, pSB701 and pSB903 to
generate the hygromycin B resistance vectors pAs, pbNs, p7s and p9s,
respectively (Fig. 1).
PPT resistance vectors pBar and p9B: The PPT-resistant box was amplified
with the PPT-forward/PPT-reverse primers. The 1.0-kb PPT-resistant box was
subcloned into the PacI/SpeI sites of pPK1s to produce the PPT resistance vector
pBar. The 0.56-kb bar gene was amplified with the bar-forward/bar-reverse
primers, then subcloned into the PmlI/MauBI sites of pSB903 to develop the PPT
resistance vector p9B (Fig. 1).
Alkaline endoglucanase EGV expression vector s-pSB902-V3: PCR
amplification of egv was carried out using the VF/VR primers. The 0.87-kb egv
gene was subcloned into the PmlI/MauBI sites of pSB902 to generate
pSB902-V3. The 2.7-kb hygromycin-resistant part (the smaller fragment from
pPK1s digested with PacI/SpeI) was subcloned into the PacI/XbaI site of the
precursor vector to generate s-pSB902-V3 (Fig. 1).
Alkaline endoglucanase EGV expression vector bns-pSB101-V3: The
1.7-kb hygromycin-resistant part (the smaller fragment from pbNs digested with
PacI/SpeI) was subcloned into the PacI/XbaI site of pSB101-V3 to generate
bns-pSB101-V3 (Fig. 1).
Alkaline
cellobiohydrolase
Humicola
CBH2
expression
vector
9B-pSB401-H2: The 1.1-kb PPT-resistant part (the smaller fragment from p9B
digested with PacI/SpeI) was subcloned into the PacI/XbaI site of pSB401-H2 to
generate 9B-pSB401-H2 (Fig. 1).
cbh1 knockout vectors C1-s, C1-pAs, C1-pbNs, C1-p7s and C1-p9s: In addition,
1.8 kb of the upstream non-coding region and 2.1 kb of the downstream non-coding
region from T. reesei cbh1 were amplified using the primer pairs C15F/R and C13F/R,
respectively. PCR fragments were digested with PacI/SpeI or XbaI/SwaI, respectively,
and ligated into the compatible sites (pPK1s, pAs, pbNs, p7s and p9s) to generate
C1-s, C1-pAs, C1-pbNs, C1-p7s and C1-p9s (Fig. 1).
Reporter gene expression vectors 9B-pSB401-gfp and bns-pSB101-rfp: The
green fluorescent protein expression vector pSB401-gfp (Wang et al. 2014) was
digested using XbaI/SwaI. The 1.9-kb DNA fragment was purified and subcloned into
the SpeI/SwaI sites of p9B to generate 9B-pSB401-gfp. The red fluorescent protein
expression vector pSB101-rfp (Wang et al. 2014) was digested with XbaI/SwaI, and
the 1.9-kb DNA fragment was purified and subcloned into the SpeI/SwaI sites in pbNs
to generate bns-pSB101-rfp.
Versatile vectors pG01-rfp and pG01-V3 for heterologous gene expression and
knockout of cbh1: In addition, 1.8 kb of the upstream non-coding region and 2.1 kb of
the downstream non-coding region from T. reesei cbh1 were ligated into
bns-pSB101-gfp and bns-pSB101-V to generate pG01-rfp and pG01-V3 (Fig. 1),
respectively.
A versatile vector pG02-H2 for heterologous gene expression and knockout of
cbh2: Finally, 1.8 kb of the upstream non-coding region and 2.1 kb of the downstream
non-coding region from T. reesei cbh2 were amplified using the primer pairs C25F/R
and C23F/R, respectively. PCR fragments were digested with PacI/SpeI or
XbaI/SwaI, respectively, and ligated into 9B-pSB401-H2 to generate pG02-H2
(Fig. 1).
Supplementary Figure 1. Restriction enzyme map of pPK2 (Covert et al. 2001), ppk1s (Zhang et al. 2014) and two based vectors pSB902
and pSB903.
Transformation efficiency
Ralative expression level of hpt
100
8
6
60
4
40
X fold expression
Transformation efficiency
80
2
20
0
0
C1-s
C1-pAs
C1-pbNs
C1-p7s
C1-p9s
strains
Supplementary Figure 2. The transformation efficiency and relative hpt mRNA expression level using five short promoters.
Alkaline endoglucanase activity (IU/mL)
3.0
s-pSB902-V3 transformant
RUT-C30
2.5
2.0
1.5
1.0
0.5
0.0
xylan
xylose
lactose
glucose
Carbon source
Supplementary Figure 3. Alkaline endoglucanase activities in s-pSB902-V3 transformants and RUT-C30 with no additional plasmids.
After cultivation with four different carbon sources: xylan, xylose, lactose and glucose, alkaline endoglucanase activity was determined in 50
mM sodium phosphate buffer, pH 9.0 at 50°C. Error bars represent the mean ± SEM (n= 3 samples) from the same experiment.
Supplementary Figure 4. Copy number of the deletion cassette in two Δcbh1 transformants (CBH1-rgf.1 and CBH1-rgf.7).
Additional Results
DNA sequences of plasmids
>pSB902
TTAATTAAGTTAACTCTAGAgatatcAACAACTACTAGACTGGGTAAATTGGTC
AATGGCCAGCCGCTCGGCCGTGCGGAGACGAGGCAAGCTTGATGAGGCCA
AATTATCCGTCAACTGTCTTATAAAGGAGCCCATGCCAAACCCCCCCTAAA
GACTCAAGAAGCCAAACCTGAACAACCCCAGCACCTGAACAGTCATACAA
CCCCTCCAAGCCCAAAAGACACAACAACTCCTACTAGCTGAAGCAAGAAG
ACATCAACATGGTCTCCTTCACCTCCCTCCTCGCCGGCGTCGCCGCCATCTC
GGGCGTCTTGGCCGCTCCCGCCGCCGAGGTCGAATCCGTGcacgtgAAAcgcgc
gcgCcaccaccaccaccaccactaatagGATCGTTCAAACATTTGGCAATAAAGTTTCTTA
AGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAA
TTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGAT
GGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAAC
AAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCCGTGTCATCTATGT
TACTAGATCACTAGTGAGCTCATTTAAATAAGCTT
>pSB903
TTAATTAAGTTAACTCTAGAgatatcACAACTACTAGACTGGGTAAATTGGTCA
ATGGCCAGCCGCTCGGCCGTGCGGAGACGAGGCAAGCTTGATGAGGCCAA
ATTATCCGTCAACTGTCTTATAAAGGAGCCCATGCCAAACCCCCCCTAAAG
ACTCAAGAAGCCAAACCTGAACAACCCCAGCACCTGAACAGTCATACAAC
CCCTCCAAGCCCAAAAGACACAACAACTCCTACTAGCTGAAGCAAGAAGA
CATCAACATGGTCcacgtgAAAcgcgcgcgCcaccaccaccaccaccactaatagGATCGTTCA
AACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGA
TGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGT
AATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTA
TACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAAT
TATCGCGCGCCGTGTCATCTATGTTACTAGATCACTAGTGAGCTCATTTAAAT
AAGCTT
>pSBa01
TTAATTAAGTTAACTCTAGAgatatcTCTACGCCAGGACCGAGCAAGCCCAGAT
GAGAACCGACGCAGATTTCCTTGGCACCTGTTGCTTCAGCTGAATCCTGGC
AATACGAGATACCTGCTTTGAATATTTTGAATAGCTCGCCCGCTGGAGAGCA
TCCTGAAtgcaagtaacaaccgtagaggctgacacggcaggtgttgctagggagcgtcgtgttctacaaggccaga
cgtcttcgcggttgatatatatgtatgtttgactgcaggctgctcagcgacgacagtcaagttcgccctcgctgcttgtgcaat
aatcgcagtggggaagccacaccgtgactcccatctttcagtaaagctctgttggtgtttatcagcaatacacgtaatttaaac
tcgttagcatggggctgatagcttaattaccgtttaccagtgccgcggttctgcagctttccttggcccgtaaaattcggcgaa
gccagccaatcaccagctaggcaccagctaaaccctataattagtctcttatcaacaccatccgctcccccgggatcaatga
ggagaatgagggggatgcggggctaaagaagcctacataaccctcatgccaactcccagtttacactcgtcgagccaaca
tcctgactataagctaacacagaatgGCCcacgtgAAAcgcgcgcgCcaccaccaccaccaccactaatagGAT
CGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTC
TTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTA
ACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCG
CAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGG
ATAAATTATCGCGCGCCGTGTCATCTATGTTACTAGATCACTAGTGAGCTCAT
TTAAATAAGCTT
>pSBbN
TTAATTAAGTTAACTCTAGAgatgtgTGATATTGAAGGAGCACTTTTTGGGCTT
GGCTGGAGCTAGTGGAGGTCAACAATGAATGCCTATTTTGGTTTAGTCGTC
CAGGCGGTGAGCACAAAATTTGTGTCGTTTGACAAGATGGTTCATTTAGGC
AACTGGTCAGATCAGCCCCACTTGTAGCAGTAGCGGCGGCGCTCGAAGTG
TGACTCTTATTAGCAGACAGGAACGAGGACATTATTATCATCTGCTGCTTGG
TGCACGATAACTTGGTGCGTTTGTCAAGCAAGGTAAGTGAACGACCCGGT
CATACCTTCTTAAGTTCGCCCTTCCTCCCTTTATTTCAGATTCAATCTGACTT
ACCTATTCTACCCAAGCCTCGATCATGGCAcacgtgAAAcgcgcgcgCcaccaccaccac
caccactaatagGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCC
TGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGC
ATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGA
TTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCG
CGCAAACTAGGATAAATTATCGCGCGCCGTGTCATCTATGTTACTAGATCAC
TAGTGAGCTCATTTAAATAAGCTT
>pSB701
TTAATTAAGTTAACTCTAGAgatatcAAGCAACTACGTAAAACTCCATGAGATT
GCAGATGCGGCCCACTGGAATACAACATCCTCCGCAAGTCCGACATGAAGC
CCCTTGACTTGATTGGCAGGCTAAATGCGACATCTTAGCCGGATGCACCCC
AGATCTGGGGAACGCGCCGCTTGAGGCCCGAAGCGCCGGGTTCGATGCAT
TACTGCCATATTTCAGCAGTTAACTAGGACCGGCTTGTGTCGATATTGCGGG
TGGCGTTCAATCTATTCCGGCACTCCTATGCCGTTTGATCCGATACCTGGAG
GGCGTGCTTTAGGCAAAATGCCAAGCTTCGAGGATACTGTACGAGCCGCTT
TCAACCTCACTTGATGATGTCTGAGTTTCATCAAGAGAATTGAAGTCAAAG
CTCAAATCATGATGTGAAGAGGTTTTGAATGTGGAAGAATTCTGCATATATA
AAGCCATGGAAGAAGACGTAAAACTGAGACAGCAAGCTCAACTGCATAGT
ATCGACTTCAAGGAAAACACGCACAAATAATCATCATGGCCcacgtgAAAcgcg
cgcgCcaccaccaccaccaccactaatagGATCGTTCAAACATTTGGCAATAAAGTTTCTT
AAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGA
ATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGA
TGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAA
CAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCCGTGTCATCTATG
TTACTAGATCACTAGTGAGCTCATTTAAATAAGCTT
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