Article Figures & Data
Figures
- FIG. 1.
(A) Growth of parental strain QM9414, the xyr1 retransformation strain (Rexyr1A), and the Δxyr1 strain on plates containing MA medium supplemented with 1% (wt/vol) l-arabinose (AN), l-arabitol (AL), xylitol (XL), and d-xylose (XO) and 2% (wt/vol) agar-agar at 30°C after 4 days. (B) Schematic presentation of the d-xylose catabolic pathway. In H. jecorina, d-xylose cannot be directly converted into d-xylulose because fungi do not have xylulose isomerase at their disposal (51).
- FIG. 2.
Enzyme activities of d-xylose reductase (A and C) and l-arabinose reductase (B and D) given in U/g, measured in cell extracts of QM9414 (QM) and the Δxyr1 strain. All strains were precultured in MA medium containing 1% (wt/vol) glycerol. For analysis of both reductase activities, mycelia were transferred to MA medium containing 1% (wt/vol) d-xylose (XO) (A and D) and l-arabinose (AN) (B and C) and grown for 5 and 10 h. One unit of activity is defined as the amount of enzyme responsible for the consumption of 1 micromole NADPH per minute due to its oxidation in the presence of d-xylose or l-arabinose under the defined assay conditions (5). Values are the means of results from three independent experiments. Error bars indicate standard deviations.
- FIG. 3.
Relative transcription levels of cbh1 (A), cbh2 (B), and egl1 (C). The parental strain QM9414 (QM) and the Δxyr1 strain were precultured with glycerol and thereafter transferred to MA medium containing 2 mM sophorose (SO) as inducer or 1% (wt/vol) cellulose (CL) as the sole carbon source and incubated 5 and 8 h or 8 and 24 h, respectively. After parallel extraction of RNA and DNA and reverse transcription of RNA, real-time PCR analysis of the respective genes was performed. Values are the means of results from three independent experiments. Error bars indicate standard deviations.
- FIG. 4.
Xylanase activities in culture supernatants of the parental strain QM9414 and the Δxyr1 strain. Both strains were precultured in MA medium containing 1% (wt/vol) glycerol. Detection of xylanase activity formation was accomplished after transfer of equal amounts of wet weight of mycelia to MA medium without a carbon source or to medium containing 2 mM sophorose or xylobiose as inducer and incubated for 5 and 8 h, or to medium containing 1% (wt/vol) d-xylose or glucose grown for 5 and 8 h, or to MA medium containing 1% (wt/vol) oat spelt xylan or carboxymethylcellulose and grown for 8 and 24 h. One unit of activity is defined as the amount of enzyme required to release 1 micromole of xylose reducing sugar equivalents per minute at 40°C. Data are the means of results from three independent experiments. Error bars indicate standard deviations.
- FIG. 5.
(A) Total (extracellular and mycelial-bound) β-glucosidase activity (+) and activity in the culture supernatant (−) of the parental and the Δxyr1 strains after replacement of glycerol pregrown mycelia into medium containing 2 mM sophorose (SO) and incubation for 5 and 8 h. (B) Activity of β-xylosidase in supernatants of the parental and the Δxyr1 strains after replacement of glycerol pregrown mycelia into medium containing 2 mM xylobiose (XB) and incubation for 5 h. All data are the means of results from three independent experiments. Error bars indicate standard deviations.
Tables
- TABLE 1.
Primers and probes used throughout the study
Primer name Sequence (5′-3′) Employed for Actf TGAGAGCGGTGGTATCCACG Actin real-time PCR Actr GGTACCACCAGACATGACAATGTTG Actin real-time PCR bgl1f CAGACAGTCACTCAACATCGGG bgl1 real-time PCR bgl1r ACCTTATCTTGGAGATTGAGCTTTGCC bgl1 real-time PCR bgl2f ACGGCTGCCTACCAGATCG bgl2 real-time PCR bgl2r AGCCGTCGGCGATCTTGC bgl2 real-time PCR bxl1f GCCAACTTCGCCACCAAGG bxl1 real-time PCR bxl1r CGGCAATCTGGTGGATCAATGTG bxl1 real-time PCR TaqMan cbh1 FAM CTGGACTCCACCTACCCGACAAACGAGACC cbh1 real-time PCR chb1f GATGATGACTACGCCAACATGCTG cbh1 real-time PCR cbh 1Df2 TGTTACAGTACTACGCCAACATGCTG cbh1 real-time PCR cbh 1r ACGGCACCGGGTGTGG cbh1 real-time PCR TaqMan cbh2 TR GACTTGCCGGATCGCGATTGCGCTGCCC cbh2 real-time PCR cbh2f CTATGCCGGACAGTTTGTGGTG cbh2 real-time PCR cbh2Dr GTCAGGCTCTGGAAGAAGG cbh2 real-time PCR cbh2r GTCAGGCTCAATAACCAGGAGG cbh2 real-time PCR TaqMan egl1 FAM CAACTCGAGGGCGAATGCCTTGACCCCTCACTC egl1 real-time PCR egl1f CTGCAACGAGATGGATATCCTGG egl1 real-time PCR egl1Dr GAGAGCGCCAGGAAAGGG egl1 real-time PCR egl1r GTAGTAGCTTTTGTAGCCGCTGC egl1 real-time PCR Xorf CTGTGACTATGGCAACGAAAAGGAG xyl1 real-time PCR Xorr CACAGCTTGGACACGATGAAGAG xyl1 real-time PCR TaqMan xyn1 FAM CGTCCAACCAACGCCCACAACAA xyn1 real-time PCR Taqxyn1f CAGCTATTCGCCTTCCAACAC xyn1 real-time PCR Taqxyn1Dr GAGGAGTCCTCCTACGCAGAA xyn1 real-time PCR Taqxyn1r CCAAAGTTGATGGGAGCAGAA xyn1 real-time PCR TaqMan xyn2 FAM CTGCCATCCCTTGCCGCC xyn2 real-time PCR Taqxyn2f GGTCCAACTCGGGCAACTTT xyn2 real-time PCR Taqxyn2Dr GGTAAGGGTAGGTAGTCTTACTTGTTC xyn2 real-time PCR Taqxyn2r CCGAGAAGTTGATGACCTTGTTC xyn2 real-time PCR TaqMan xyr1 FAM CGCGCTTGTGACCAGTGCAACCAGCTTCGTACC xyr1 real-time PCR xyr1f CCCATTCGGCGGAGGATCAG xyr1 real-time PCR xyr1Dr GATCAGTACATGTGCTTGAGCGC xyr1 real-time PCR xyr1r CGAATTCTATACAATGGGCACATGGG xyr1 real-time PCR Xyr1.1.f GGTACCAATTGTGAGCGCATCAC Construction of deletion cassette Xyr1.2.r CTGTCGACGATGGGAATTCGGGTCAAATGAC Construction of deletion cassette Xyr2.1.f CCGAATTCCCATCGTCGACAGGCACCTGG Construction of deletion cassette Xyr2.2.r GGTACCGAACATAGCCCAACG Construction of deletion cassette - TABLE 2.
Growth of the parental strain and strains Δxyr1 and Rexyr1 on plates containing MA medium supplemented with different carbon sourcesa
Avg mycelial growth zone diam (cm)b Carbon source After 3 days After 6 days QM9414 Δxyr1 Rexyr1A QM9414 Δxyr1 Rexyr1A Glucose 3.7 ± 0.29 3.9 ± 0.17 3.5 ± 0.19 8.0 ± 0.00 8.0 ± 0.00 8.1 ± 0.04 Glycerol 3.0 ± 0.15 3.4 ± 0.00 3.2 ± 0.12 7.2 ± 0.35 7.1 ± 0.10 7.2 ± 0.12 l-(+)-Arabinose 3.3 ± 0.10 3.5 ± 0.21 3.5 ± 0.17 7.4 ± 0.12 7.5 ± 0.40 7.3 ± 0.15 l-(−)-Arabitol 3.1 ± 0.17 3.7 ± 0.06 3.3 ± 0.08 7.7 ± 0.15 7.7 ± 0.12 7.6 ± 0.22 d-(+)-Xylose 4.1 ± 0.23 1.1 ± 0.10 4.2 ± 0.21 8.0 ± 0.00 2.4 ± 0.06 8.2 ± 0.16 Xylitol 3.3 ± 0.12 3.5 ± 0.06 3.5 ± 0.12 7.5 ± 0.06 7.5 ± 0.00 7.6 ± 0.17 Xylan 2.0 ± 0.17 2.5 ± 0.12 2.2 ± 0.19 5.5 ± 0.38 4.6 ± 0.12 5.6 ± 0.16 Cellulose 2.8 ± 0.17 3.3 ± 0.17 3.0 ± 0.14 5.7 ± 0.10 6.0 ± 0.00 5.9 ± 0.11 - TABLE 3.
Relative xyn1 transcription levels of the wild-type and the Δxyr1 strains
Carbon source (cultivation time, h) Amt of mRNA (cDNA) per gene dosea QM9414 Δxyr1 No carbon source (3) 1.0 ± 0.5 NDb No carbon source (5) 0.5 ± 0.1 ND No carbon source (8) 0.4 ± 0.2 ND Glucose (3) ND ND Glucose (5) ND ND Xylose (3) 1.9 ± 0.4 ND Xylose (5) 169.3 ± 16.5 ND Xylose (8) 0.0 ± 0.0 ND Xylan (8) 16.1 ± 2.6 ND Xylan (24) 9.8 ± 0.0 ND No carbon source (3) 1.0 ± 0.1 ND No carbon source (5) 3.8 ± 0.5 ND No carbon source (8) ND ND Sophorose (5) 97.9 ± 9.6 ND Sophorose (8) 6,701.7 ± 531.1 ND Xylobiose (3) 6,085.9 ± 637.1 ND Xylobiose (5) 265.6 ± 11.2 ND Xylobiose (8) 37.5 ± 3.2 ND Xylan (8) 4,305.9 ± 415.3 ND Xylan (24) 3,819.4 ± 277.6 ND - TABLE 4.
Xylanase and cellulase activities in supernatants from direct cultivations of the parental strain and the Δxyr1 strains with xylan or cellulose as respective carbon sources
Cultivation time (h) Xylanase activity (U/μg protein) in xylan supernatantsa,c Cellulase activity (U/μg protein) in cellulose supernatantsb,c QM9414 Δxyr1 QM9414 Δxyr1 24 NDd ND 0.003 ± 0.000 ND 48 0.206 ± 0.015 ND 0.655 ± 0.012 ND 72 0.142 ± 0.009 ND 0.885 ± 0.022 ND ↵ a One unit of activity is defined as the amount of enzyme required to release 1 micromole of xylose reducing sugar equivalents per min under the defined assay conditions.
↵ b One unit of activity is defined as the amount of enzyme required to release 1 micromole of glucose reducing sugar equivalents per min under the defined assay conditions.
↵ c Values are the means ± SD of results from three independent experiments.
↵ d ND, no detection.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Regulatory impact of Xyr1 on hydrolase expression in T. reesei.
Zipped TIF document, 206K.
- Supplemental file 1
-
Regulatory impact of Xyr1 on hydrolase expression in T. reesei.
