Cellodextrin Transporters Play Importantroles in Cellulase Induction in the Cellulolytic

Electronic Supplementary Material

Applied Microbiology and Biotechnology

Cellodextrin transporters play importantroles in cellulase induction in the cellulolytic fungus Penicillium oxalicum

Jie Li1

Email:

Guodong Liu1*

* Corresponding author

Email:; Fax: +86 531 88565610

Mei Chen1

Email:

Zhonghai Li1

Email:

Yuqi Qin1, 2

Email:

YinboQu 1,2*

* Corresponding author

Email: ; Fax: +86 531 88565234

1 State Key Laboratory of Microbial Technology, School of Life Science, Shandong University,27 Shanda South Road, Jinan, Shandong 250100, P. R. China

2 National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250100, P. R. China

Electronic Supplementary Material

Contents

1. Construction of cdtsingledeletants
2. Construction ofcdtdoubledeletants
3. Cellodextrin consumptionof yeasts
4. The effects of single cdt gene knockout
5. Oligonucleotide primers
6. Information of putative cellodextrin transporters
   

1. Construction of cdtsingle deletants

Electronic supplementary material Fig. S1 Targeted disruptions ofcdtC, cdtD and cdtGin P. oxalicum (a) Schematic illustration of the homologous integration of the Aspergillusoryzaeptra gene at the cdtC, cdtDor cdtG gene locus resulting in the deletion of the coding sequences. (b) Southern blot analysis of ΔcdtC. Genomic DNA was digested with XhoI, and a 4.5-kb XhoI fragment in the wild-type strain was replaced by a 6.5-kb fragment in ΔcdtCstrain when probed by a cdtC left arm sequence. (c)Southern blot analysis ofΔcdtD. Genomic DNA was digested withPstI,and a 3.1-kb PstI fragment in the wild-type strain was replaced by a 9.1-kb fragment in ΔcdtD strain when probed by a cdtD right arm sequence. (d) Southern blot analysis of ΔcdtG. Genomic DNA was digested withPstI,and a 3.2-kb PstI fragment in the wild-type strain was replaced by a 5.8-kb fragment in ΔcdtG strain when probed by a cdtG right arm sequence.

1. Construction ofcdtdoubledeletants

Electronic supplementary materialFig. S2 Construction ofcdtdouble knockout strains (a) Schematic illustrations of the homologous integrations of the E. coli gene hph expression cassettes at the cdtC,orcdtDgene locus in cdt single deletion mutants. (b-c) Southern blot analysis of ΔcdtDC and ΔcdtGC. Genomic DNA was digested with XhoI. A 4.5-kb XhoI fragment in the parental strain was replaced by a 6.4-kb fragment in ΔcdtDC and ΔcdtGC strains when probed by a cdtC left arm sequence. (d) Southern blot analysis ofΔcdtGD.Genomic DNA was digested withPstI. A 3.1-kb PstI fragment in the parental strain was replaced by a 2.6-kb fragment in ΔcdtGD strain when probed by a cdtDright arm sequence.

1. Cellodextrin consumption of yeasts

Electronic supplementary materialFig.S3Cellodextrin consumption by YCdtC (a), YCdtD (b) and YCdtG (c).G2-G6represents cellobiose,cellotriose, cellohexose, cellopentaose andcellohexose, respectively.The residual sugars at different time points were determined by HPLC analysis.

1. The effects of single cdt gene knockout

Electronic supplementary materialFig. S4The effects of cdt single gene knockout on cellobiose consumption and cellulase production(a) Cellobiose consumptions by P. oxalicum strains lacking cdtC, cdtDor cdtG, respectively. Strains were incubated with 1.5 mM of cellobiose. Error bars indicate standard deviations of three independent incubations. (b) Extracellular filter paper hydrolyase (FPase) activities inΔcdtC, ΔcdtDΔcdtGandwild strain 114-2.Data are means of results from three independent cultures. Error bars indicate standard deviations.

1. Oligonucleotide primers

Electronic supplementary materialTable S1Oligonucleotide primers used in this study

Primer name / Sequence (5′→3′) / Product
Construction of expression vectors
aF-SalI / GCGCGTCGACATGGCACCGTCGATGAATCCGA / cdtA ORF
aR-HindIII / GCGCAAGCTTCTAGGCCTTCTCCGTAGTGATC
bF-SmaI / GCGCCCCGGGATGCCTCTCGGAGTGGGAAGCA / cdtB ORF
bR-SmaI / GCGCCCCGGGTTAATACGGTCGGCGAAAGACC
cF-SalI / GCGCGTCGACATGTCGGAGAAATACATTACCC / cdtC ORF
cR-HindIII / GCGCAAGCTTAAGCGCCGTGGTCAATCTTC
dF-SalI/dF2 / GCGCGTCGACATGGGAAAGGATACTTTCACTC / cdtD ORF
dR-HindIII/dR2 / GCGCAAGCTTCACAGCTCAACCTGAGTAGTG
eF-SmaI / GCGCCCCGGGATGGGGTTGCGATTCAGGCCAC / cdtE ORF
eR-SmaI / GCGCCCCGGGTCATTTGACTTCGACCTCAACA
fF-SmaI / GCGCCCCGGGATGGTCTTGGTTAGCAAACACC / cdtF ORF
fR-SmaI / GCGCCCCGGGTTAAGCGACGTTCTTTGCATCT
gF-SalI / GCGCGTCGACATGTCAAAATACGAGGGTGCTG / cdtG ORF
gR-SmaI / GCGCCCCGGGCTACGCCTCCATGCCAACGACA
hF-SalI / GCGCGTCGACATGGCTGCAGACCATACAGGCC / cdtH ORF
hR-HindIII / GCGCAAGCTTCTAAACCCGCTCAACTTGCTCC
iF-SalI / GCGCGTCGACATGTCGCCTAAAGAACCGGTCA / cdtI ORF
iR-HindIII / GCGCAAGCTTCTATTCTTCGTACTTGATCACG
jF-HindIII / GCGCAAGCTTATGGCTCCTGCTGCAGCTCACG / cdtJ ORF
jR-SmaI / GCGCCCCGGGTCAAGCCAGCTTGTCTTCCACG
kF-HindIII / GCGCAAGCTTATGGCGAAGCAGGGCTCCCTGG / cdtK ORF
kR-SmaI / GCGCCCCGGGTCAACATGCCTCACTTCTTGTT
Single deletion of cdt genes in P. oxalicum
cupF / TCGACCTTTGACCGAAGGAG / 5’ flanking of cdtC
cupR-ptra / TGGGATCCCGTAATCAATTGCCCCCGATAGGACTCGGGG
cdF-prta / AGAGCGGCTCATCGTCACCCCATTTTGGGGGGGAATCTT / 3’ flanking of cdtC
cdR / AGCCGAGACTACCACCAGCC
ccF / ACGAGCCTCTAGCTCGATACCTATT / cdtC deletion cassette
ccR / TGGCAGGGCTAGTCGCCCTCAGTA
dupF/dF3 / AGTCACTCAGTAGCGAGTGCG / 5’ flanking of cdtD
dupR-ptra / ATGGGATCCCGTAATCAATTGCCCACCTCTCTTAGCGGCA
ddF-prta / AGAGCGGCTCATCGTCACCCCATTGCTCTACCAAGTCATT / 3’ flanking of cdtD
ddR/dR4 / TCTGAGCAGCGACGGCATTTC
dcF / AACCACGAAAAAAACTCAGGTCATA / cdtD deletion cassette
dcR / GGCGGTGTAATAAGGAGTCTGTGCG
gupF / GGATTGGACTGCTACCTATGT / 5’ flanking of cdtG
gupR-ptra / GATCCCGTAATCAATTGCCCTTCGAGTAGGAAGGTC
gdF-prta / AGCGGCTCATCGTCACCCCATTGGGGGAAGAAAGTT / 3’ flanking of cdtG
gdR/g-blotR / TCCGACACCCCTAACGTAAGT
gcF / ACTTGTCCTCAGCGAGCGATTGTGT / cdtG deletion cassette
gcR / ATCGTTTCCAAGGGTGGCTACAAGG
pR3 / CTCGTTAGCTCGTAATCCAC / Unique sequence inΔcdtD(with primer dF3)a
pF4 / CCAAACCAACCGACAATGTA / Unique sequence inΔcdtD(with primer dR3)a
pF1 / GGGCAATTGATTACGGGATC / ptracassette
pR1 / ATGGGGTGACGATGAGCCGC
Double deletion of cdt genes in P. oxalicum
cupR-hph / CTTCAATATCAGTTAACGTCGCCGATAGGACTCGGG / 5’ flanking ofcdtC(withcupF)
cdF-hph / ATTCCGTCACCAGCCCTGGGTTGTTTGGGGGGGAAT / 3’ flanking ofcdtC(with cdR)
dupR-hph / CTTCAATATCAGTTAACGTCGACCTCTCTTAGCGGCA / 5’ flanking ofcdtD(withdupF)
ddF-hph / ATTCCGTCACCAGCCCTGGGTTGTGCTCTACCAAGTC / 3’ flanking ofcdtD(withddR)
gupR-hph / CTTCAATATCAGTTAACGTCGTTCGAGTAGGAAGGTC / 5’ flanking ofcdtG(withgupF)
gdF-hph / TCCGTCACCAGCCCTGGGTTGTGGGGGAAGAAAGTT / 3’ flanking ofcdtG(withgdR)
c-blotF / GAGCAATACGCATCAACACG / probe of cdtCinSouthern blot
c-blotR / ACTCCAGCCATTTCACGACC
d-blotF / TGCTCTACCAAGTCATTCTC / probe of cdtD inSouthern blot
d-blotR / GTCAAGTCCCTGGAGCACATC
g-blotF / GCAGAAGAGCAAGTAACAATC / probe of cdtG inSouthern blot
hF1 / CGACCTTAACTGATATTGAAGGAGC
hR1/hR4 / GAAAATTCCGTCACCAGCCCTGGGTTG / hphcassette
hR3 / AGACTGAGGAATCCGCTCTTGG / Unique sequence inΔcdtGD(with primer dF3)b
Real-time quantitative PCR
cQF / CGCTCCCCATTGCTCAGACT / Partial cdtCsequence
cQR / GAGCCTCGGCAGGAACAACA
dQF / CTTGATTCTGAGTGCCGTGA / Partial cdtDsequence
dQR / CCACCTTCCTTTGCAATGAG
gQF / GACAACCCGTGGTGGGACTT / Partial cdtGsequence
gQR / GGACCATTGGCCGAACAGAG
cbh1QF / GTACTTGCGATCCTGATGGG / Partial cbh1 sequence
cbh1QR / CCACGGTGAAGGGAGACTTG
xyn1QF / GGTCTCCAGGCTCACTTCATC / Partial xyn1 sequence
xyn1QR / GTCGAGGGCAAGTTCATACG
act-F / CTCCATCCAGGCCGTTCTG / Partial actin sequence
act-R / CATGAGGTAGTCGGTCAAGTCAC
Over-expression of cdt genes in P. oxalicum
pcbhIF-EcoRI / GCGCGAATTCCTGAGGCGCGTCCGTT / promter of cbh1
pcbhIR-EcoRI / GCGCGAATTCTGTGATGGATTGGATCAAAGAT
OCF-SmaI / GCGCCCCGGGATGTCGGAGAAATACATTACC / cdtC gene and terminator
OCR-XbaI / GCGCTCTAGACTGTCTGTTGCTGTGGATTTA
ODF-XbaI / GCGCTCTAGAATGGGAAAGGATACTTTCACT / cdtD gene and terminator
ODR-XbaI / GCGCTCTAGACGGTAGTGAGACAAGCGAGC
OGF-SmaI / GCGCCCCGGGATGTCAAAATACGAGGGTGCT / cdtG gene and terminator
OGR-XbaI / CGCTCTAGACCAATAAACTTTCTTCCCCCA

aInformation of primer locations are indicated inElectronic supplementary materialFig. S1

bInformation of primer locations are indicated inElectronic supplementary materialFig. S2

1. Information of putative cellodextrin transporters

Electronic supplementary material Table S2Cellodextrin transporter candidates in P. oxalicum

Name / Protein ID / TCDB best hita / BLASTP
E-value / Gene transcription levelsb
Glucose / Cellulose
CdtA / PDE_04820 / CDT-2 (2.A.1.1.83) / 2.0E-87 / 0 / 0
CdtB / PDE_07257 / CDT-2 (2.A.1.1.83) / 0 / 9.82 / 1.11
CdtC / PDE_00607 / CDT-1 (2.A.1.1.82) / 0 / 3.05 / 98.23
CdtD / PDE_00753 / CDT-2 (2.A.1.1.83) / 2.0E-109 / 116.87 / 373.28
CdtE / PDE_08035 / CDT-2 (2.A.1.1.83) / 9.0E-105 / 12.20 / 30.99
CdtF / PDE_02109 / CDT-2 (2.A.1.1.83) / 1.0E-90 / 1.36 / 0.55
CdtG / PDE_09395 / Lac12(2.A.1.1.9) / 9.0E-102 / 19.65 / 5.26
CdtH / PDE_02900 / CDT-2 (2.A.1.1.83) / 4.0E-82 / 7.45 / 0.83
CdtI / PDE_09011 / CDT-2 (2.A.1.1.83) / 1.0E-80 / 2.37 / 0.55
CdtJ / PDE_09534 / CDT-2 (2.A.1.1.83) / 2.0E-97 / 0.68 / 0
CdtK / PDE_04857 / CDT-2 (2.A.1.1.83) / 7.0E-54 / 2.71 / 8.58

a TCDB family IDs are shown in parentheses.

b Reported in transcripts per million total sequencing tags. Data from the result of transcriptomic analysis of P. oxalicum 114-2 grown in different media (2% glucose for 41 h; 2% glcuose for 39 h and then 2% cellulose for 2 h) using RNA-Seq (NCBI GEO accession NO. GSE34288).

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