Supplementary Material
Cellulase gene cloning
Copies of cellulase genes: egl1 (cel7b), cbh2 (cel6a) and bgl1 (cel3a) were generated
from cDNA prepared from T. reesei RNA using reverse primers listed in Table S1. The
accession numbers for egl1, cbh2 and bgl1 are M15665.1, M55080.1 and U09580.1
respectively. For RNA extraction T. reesei cultures were collected by filtration onto a
glass fiber pad (Whatman, Conrad, MA, USA). The resultant “cake” was immersed in
liquid nitrogen and ground using a mortar and pestle until a flour-like consistency was
obtained. RNA was extracted using the hot-phenol method as previously described (1).
For cDNA synthesis, the extracted RNA (10 μg) was first treated with DNaseI (Promega,
Madison, USA) as per manufacturer’s instructions and then reversed transcribed into
cDNA using the one-step High-capacity cDNA Reverse Transcription (RT) Kit from ABI
(Applied Biosystems, Foster City, CA, USA) as per the manufacturer’s instructions.
The cDNAs corresponding to each gene were amplified as overlapping DNA fragments.
For each gene, the native secretory signal sequence was included in the amplified cDNA.
A 6xHIS tag was incorporated into the C-terminal end of the egl1 and cbh2 coding
sequences (Table S1). PCR amplification was performed using the high fidelity DNA
Polymerase Verizyme™ (York-Bio, York, UK) as per the manufacturer’s instructions.
The PCR products were electrophoresed on 1% (w/v) agarose gels. DNA was extracted
from the gels at 55ºC using gel dissolving solution (5.5 M guanidine thiocyanate, 10 mM
Tris-HCl; pH 7.5). The agarose solution was transferred to a silica resin spin column
(Epoch LifeScience, Texas, USA) and DNA fragments isolated as per manufacturer’s
instructions.
Cloning cellulase insert into low and high copy number plasmids
The cellulase genes, with the exception of egl2, were initially cloned into the low copy
number plasmid pGREG586, which contains a GAL1 promoter, recombination sites Rec1
and Rec2, a CYC transcription terminator sequence (229 nts) immediately downstream of
the Rec2 site and a kanMX cassette (2). The primers corresponding to the 5’ and 3’ ends
of the cellulase genes (Fig. 2) contain sequences homologous to the Rec1 (34 nts) and
Rec2 (35 nts) sites of the pGREG vector (Table S1). The pGREG586 vector was
linearized with the restriction enzyme SalI. The amplified DNA fragments together with
the linearised plasmid were introduced into S. cerevisiae strain S150-2B using the lithium
acetate method (2). The plasmid and inserts were reconstituted by homologous
recombination in vivo. Transformants were selected on YPD agar containing G418.
The original endogenous GAL1 promoter (461nts) in pGREG586 was replaced with the
S. cerevisiae PGK1 promoter (719 nts) [accession number: AB304875.1]. Briefly,
pGREG586 was digested with SpeI and SacI restriction enzymes to excise the GAL1
promoter. The PGK1 promoter was amplified from S. cerevisiae genomic DNA using
primers pGREGPGK_F and pGREGPGK_R (Table S1), which contain sequences
homologous to the pGREG vector, and was inserted into the plasmid by homologous
recombination in vivo.
The genes bgl1, cbh2 and egl1 genes, under the control of the PGK promoter were
subsequently cloned into the high copy number plasmid by homologous recombination in
vivo. The high copy number plasmid, pRSH42 (3) is referred to herein as pRSH. Briefly,
the bgl1 gene cassette, spanning from the PGK1 promoter to the CYC terminator was
amplified from the plasmid pGREGbgl1using primers pRSPGK_F and pRSCYC_R
(Table S1), which each contain 35 nts homologous to the multicloning site (MCS) of the
pRSH plasmid (3). The amplified DNA fragment was mixed in a molar ratio of 10:1 with
pRSH, linearised with KpnI and SacI within the MCS, and introduced into strain S1502B as described above. The resultant plasmid was called PGKbgl1. The high copy
number egl1 and cbh2 plasmids were generated as follows; briefly, plasmid PGKbgl1
was digested with SalI to remove the bgl1 gene. The egl1and cbh2 gene cassettes were
amplified from the corresponding pGREG vectors with primers egl1_F1 and egl1_R2 and
cbh2_F1 and cbh2_R2 (Table S1) respectively and were inserted into linearised
PGKbgl1plasmid by homologous recombination in vivo.
Cloning of egl2
Genomic DNA was extracted from T. reesei as previously described (4). The egl2 gene
(cel5a, accession number DQ178347) was amplified from T. reesei (QM1923) genomic
DNA in two fragments, fragment one was amplified using egl2_F1 and egl2_R1 and
fragment two was amplified using egl2_F2 and egl2_R2 primers (Table S1). Both
fragments were then inserted into SalI digested PGKbgl1 via homologous recombination
in vivo in the yeast strain S. pastorianus CM-51, placing the gene downstream of the
PGK promoter and upstream of the CYC terminator as described above for the other
genes. For generation of the co-expressing endoglucanase clone, the egl2 cassette
(PGKegl2) was amplified in two fragments. Fragment one was amplified using
PsiPGK_F and egl2_R1 primers (Table S1). Primer PsiPGK_F contains 35bp with
homology to sequences upstream of the PsiI restriction enzyme site in the pRSH plasmid.
Fragment two was amplified using egl2_F1 and Psi1CYC_R primers (Table S1),
generating a fragment containing the remaining half of the egl2 gene, the CYC terminator
followed by a 35bp 3’end extension homologous to sequences downstream of the PsiI
restriction enzyme site in the pRSH plasmid. The PGKegl1 plasmid was linearized using
the restriction enzyme PsiI. The amplified gene fragments were inserted into the PsiI site
by homologous recombination in vivo using the yeast strain S. pastorianus CM-51,
placing the egl2 gene cassette 562bp upstream of the egl1 cassette.
Promoter swaps
The PGK promoter upstream of the bgl1 gene in the pRSH plasmid was replaced with the
high affinity glucose transporter promoter (HXT7, 973nt, accession number
BK006938.2) or the TEF-alpha 1 (TEF1, 402nt, accession number BK006949.2)
promoter. Likewise, the PGK promoter upstream of cbh2 in pRSH was replaced with the
TEF1 promoter. Original promoters were excised from plasmids by digestion with PsiI
and SpeI restriction enzymes. The digested plasmid was ethanol precipitated. DNA
sequences corresponding to the promoters were amplified from S. cerevisiae genomic
DNA (Novagen) using promoter specific primers (Table S1), which contained 35bp 5’
and 3’ extensions homologous to sequences upstream of the PsiI and downstream of the
SpeI restriction enzyme sites respectively. The HXT7 promoter was amplified using
PsiIHXT_F PsiIHXT_R, the TEF1 promoter was amplified using PsiITEF_F and
PsiITEF_R primers (Table S1). The amplified DNA fragments and the linearised plasmid
were reconstituted by homologous recombination in vivo following transformation into
the yeast strain S. pastorianus CM-51.
1.
2.
3.
4.
Campbell SG, Li Del Olmo M, Beglan P, Bond U. 2002. A sequence element
downstream of the yeast HTB1 gene contributes to mRNA 3' processing and cell
cycle regulation. Mol Cell Biol 22:8415-8425.
Jansen G, Wu C, Schade B, Thomas DY, Whiteway M. 2005. Drag&Drop
cloning in yeast. Gene 344:43-51.
Taxis C, Knop M. 2006. System of centromeric, episomal, and integrative
vectors based on drug resistance markers for Saccharomyces cerevisiae.
Biotechniques 40:73-78.
Kaiser C, Michaelis S, Mitchell A. 1994. Methods in yeast genetics: A Cold
Spring Harbor Laboratory course manual, p. vii+234p-vii+234p, Methods in yeast
genetics: A Cold Spring Harbor Laboratory course manual.
Table S1 : Oligonucleotide primers used for gene isolation and plasmid construction
Primer name
Oligonucleotide primer sequence (5’→3’)
bgl1_F1
GAATTCGATATCAAGCTTATCGATACCGTCGCAATGCGTTACGAACAGCAGCTGCGC
bgl1_R1
GCAGGCCCAGGTGGTATTGACC
bgl1_F2
TGCCATGGGTCAAACCATCAACGGC
bgl1_R2
GCCTTTGTCGTTGCACGAGGGCG
bgl1_F3
GCCAACATCCTGCCGCTCAAGAAGCC
bgl1_R3
GCGTGACATAACTAATTACATGACTCGAGGTCGAC CTACGCTACCGACAGAGTGCTCGTCAGCCTGATATCCCG
cbh2
cbh2_F1
GAATTCGATATCAAGCTTATCGATACCGTCGCAA ATGATTGTCGGCATTCTCACC
cbh2_F2
AACATCCCGGTCGAGCTCCGC
cbh2_R1
GGAATATTCCGATATCCGGACC
cbh2_R2
GCGTGACATAACTAATTACATGACTCGAGGTCGACCTAGTGATGGTGATGGTGATGCAGGAACGATGGGTTTGCG
egl1
egl1_F1
GAATTCGATATCAAGCTTATCGATACCGTCGCA ATGGCGCCCTCAGTTACAC
egl1_R1
TGGGCGCTGGGGATGTCGACGCC
egl1_F2
AACTCGAGGGCGAATGCCTTGACC
egl1_R2
GCGTGACATAACTAATTACATGACTCGAGGTCGACCTAGTGATGGTGATGGTGATGAAGGCATTGCGAGTAGTAGTCGT
egl2
egl2_F1
GAATTCGATATCAAGCTTATCGATACCGTCGACAATGAACAAGTCCGTGGCTCCATTGC
egl2_R1
GGATAAACCTTCGAGGTAACGCAAGTGCCATCTGTGGTACAGCCAAAGTCAAAAC
egl2_F2
GCGGGTTTTGACTTTGGCTGTACCACAGATGGCACTTGCGTTACCTCGAAGGTTTATCCT
egl2_R2
GCGTGACATAACTAATTACATGACTCGAGGTCGACCTACTTTCTTGCGAGACACGAGCTG
PGK1
pGREGPGK_F
AGGGAACAAAAGCTGGAGCTCGTTTAAACGGCGCGCCCCAAGAATTACTCGTGAGTAAGG
pGREGPGK_R
GTGATGCGATCCTCTCATACTAGTGCGGCCGCTCTAGACCGCGGTGTTTTATATTTGTTG
pRSPGK1_F
CGTAATACGACTCACTATAGGGCGAATTGGGTACCCCAAGAATTACTCGTGAGTAAGG
PsiIPGK_F
TTTAACCAATAGGCCGAAATCGGCAAAATCCCTTACCAAGAATTACTCGTGAGTAAGGAAAGA
TEF1
PsiITEF1_F
CCAATAGGCCGAAATCGGCAAAATCCCTTAAATGTTTCTACTCCTTTTTTACTCT
PsiITEF1_R
GGTGATGGTGATGCGATCCTCTCATACTAGTGCGGCCGCTCTAGATTGTAATTAAAACTTAGATTAGATTGC
HXT7
PsiIHXT_F
CCAATAGGCCGAAATCGGCAAAATCCCTTAGGGGTTGTTAAGCATGCCCTGCTAAAC
PsiIHXT_R
GGTGATGGTGATGCGATCCTCTCATACTAGTGCGGCCGCTCTAGATTTTGATTAAAATTAAAAAAACTTTTTG
CYC1
pRSCYC_R
TTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCGGCCGCAAATTAAAGCCTTCG
PsiICYC_R
CACTCAACCCTATCTCGGTCTATTCTTTTGATTTAGGCCGCAAATTAAAGCCTTCGAGCG
KAN
Kan_F
TCTTTCCAGACTTGTTCAAC
Kan_R
CCACTGCGATCCCCGGCAAA
HYGRO Hygro_F
TGTTTATCGGCACTTTGCA
Hygro_R
AGTGTATTGACCGATTCCTT
The sequences homologous to the plasmid integration sites are highlighted in bold
Underlined sequences are homologous to sequence of pRSH plasmid
Gene
bgl1