Fungal strain and culture condition
A. cellulolyticus Y-94 (FERM Number BP-5826) was cultured in 10 ml of medium in 100 ml flasks at 30°C with shaking at 200 rpm. The composition of the culture medium for A. cellulolyticus was described previously (Fang et al. 2009). Sampling was performed at 1, 3, and 7 days for analysis of gene expression using real-time PCR and/or enzyme activity. A. cellulolyticus strains were cultured in potato dextrose (PD) medium for cloning and transformation.
Measurement of the amount of ATP, β-xylosidase and β-mannosidase activity, and saccharification efficiency
The amount of ATP measured based on fluorescence using a Rucifel-250 kit (Kikkoman, Tokyo Japan) and Lumitester C-100 (Kikkoman) according to the manufacturers' instructions.
Activities for β-xylosidase and β-mannosidase were measured using 100 μl of 10 mM 4-nitrophenyl-β-D-xyloopyranoside (PNP-Xyl, Sigma, MO USA) or 4-nitrophenyl-β-D-mannopyranoside (PNP-Man, Sigma) as the substrates (final concentration is 1 mM), respectively. Fifty μl of enzymes were incubated with 1 mM PNP-Xyl or PNP-Man at 45°C for 10 minutes in 850 μl of 50 mM acetic acid buffer (pH.5). After 10 minutes, 500 μl of 1 M NaCO3 was added. Because 4-nitrophenol which will be generated from substrates by enzymatic hydrolysis is a chromogenic substance, enzyme activities were assayed by measuring absorbance at 420 nm by UV-2550 Spectrophotometer (Shimadzu, Kyoto, Japan). One unit of the enzyme activity is defined as the amount of enzyme that produces 1 μmol of p-nitrophenol per minute. For analysis of saccharification efficiency, culture medium was centrifuged at 9,000 g for 10 min to collect the supernatant containing the secreted enzyme. Enzyme solutions were incubated at 45°C in 50 mM acetic acid buffer with 4% xylooligosaccharides (Wako Pure Chemicals, Osaka JAPAN). The xylose concentration was measured using a high performance liquid chromatography system (JASCO, Tokyo, Japan), under the conditions described previously (Buaban et al. 2010).
Cloning β-xylosidase gene from A. cellulolyticus
A putative β-xylosidase gene, bxy3A, was identified in A. cellulolyticus genome sequence information using A. nidulans xlnD sequence as the query for a homology search. In silico molecular cloning (in silico biology, Yokohama Japan) which is a software for gene analysis was used for homology search. Augstus 2.2 http://augustus.gobics.de/ which is a program for eukaryotic genome sequence was used for the prediction of genes. The β-xylosidase coding region was amplified using A. cellulolyticus CF-2612 genomic DNA as the template, and the cellobiohydrolase Ι (cbh1) promoter was amplified from Y-94 genome. For the extraction of genomic DNA, cells cultured in PD medium were collected by centrifugation, and 3 volumes of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) with 2% sodium dodecyl sulfate (SDS) were added to the cell pellet. The cell suspension was incubated at 50°C for 1 hr. Potassium acetate (5 M) was added to the cell suspension at one-tenth of total volume, and this mixture was incubated on ice for 1 hr. The mixture was centrifuged at 13,000 g for 10 min, and the supernatant was subjected to two rounds of phenol-chloroform treatment, and ethanol precipitation was performed to obtain genomic DNA. The DNA was incubated with RNaseA (Nippon gene, Toyama, Japan) at 37°C for 1 hr to degrade contaminating RNA.
To amplify β-xylosidase open reading frame from A. cellulolyticus DNA, the forward primer with engineered Spe Ι site (5'-GCACTAGTATGGTCTACACCACG) and the reverse primer with engineered Kpn Ι site (5'-GCGGTACCTCAATTAGAATCAGGC) were designed based on sequence from the A. cellulolyticus genome sequence information (unpublished data) using Augustus 2.2 software The promoter from the cellobiohydrolase Ι (cbh1) gene (GenBank Accession number; E39854) was amplified with the forward primer with an Xho Ι site (5'-GCCTCGAG AAGCTTGGAAGCT) and the reverse primer with a Spe Ι site (5'-TACCATGGCTGCACTAGT GTGTCGATTGCTT). The amplified fragment of the cbh1 promoter was connected to β-xylosidase gene in frame, and incorporated into a shuttle vector pLD10 provided by Dr. H. Corby Kistler (University of Minnesota, USA), and the resulting plasmid, pLcbX-1, was obtained. E. coli DH5α cells (Takara Bio, Shiga, Japan) were used to maintain the plasmid.
Transformation of A. cellulolyticus
The parental strain, Y-94, was transformed using a slightly modified protoplast-PEG method (Fincham et al. 1989). An overnight culture of A. cellulolyticus was treated with in 10 mM KH2PO4, 0.8 M NaCl, and 0.2% Yatalase (Takara Bio) to prepare protoplasts. The protoplasts were washed with 0.8 M NaCl and suspended in Solution A (1.2 M Sorbitol, 10 mM Tris-HCl, 10 mM CaCl2). Plasmid (10 μg) was added to the protoplast suspension, then 50 μl of Solution B (40% PEG4000, 10 mM Tris-HCl, 10 mM CaCl2) was added to the protoplast suspension, and the suspension was incubated on ice for 30 min and RT for 15 min. Then, 8.5 ml of solution A was added to the cell suspension to dilute Solution B. The protoplasts suspension was spread on YPSA plate (1% Bacto yeast extract, 1% Bacto tryptone, 1 M Sucrose, and 2% Agar) and incubated overnight at 30°C. PD medium with 0.2% agar with 500 μg hygromycin was piled on to the YPSA plate. A single colony was isolated 3 days after the addition of PD medium. To confirm the presence of hygromycin phosphotransferase (hph) gene, transformant was checked by PCR using the forward (5'-ATGCCTGAACTCACCGCGAC-3') and the reverse (5'-CTATTCCTTTGCCCTCGGAC-3') primers.
Measurement of FPU, xylanase, and mannanase activities
The FPU activity assay described by Ghose (1987) was performed as a reference for cellulase activity. Whatman NO.1 filter paper (paper size; 1 cm × 6 cm, Whatman, Kent UK) was used as the substrate. Culture medium was centrifuged at 9,000 g for 10 min to collect the supernatant with the secreted enzyme. Enzyme solution in 1 ml of 50 mM citric acid buffer, pH4.8, was incubated with the substrate at 50°C for 60 min. DNS solution (3 ml; 1% 3, 5-dinitrosalycilic acid (Sigma), 1.2% NaOH, 0.05% sodium sulfate, and 20% potassium sodium tartrate tetrahydrorate) was added to the enzyme solution, and the mixture was boiled for 5 min, then the reaction mixture was put on ice. The amount of glucose was measured with the absorbance at 540 nm with UV-2550 Spectrophotometer as a reducing sugar.
For assay of xylanase and mannanase, 2% birch-wood xylan (Sigma) or 1% Konjac Glucomannan (Megazyme, Wicklow, Ireland) were used as substrates, respectively. The incubation time for enzymatic reaction was 30 min and activities were assayed according to the previously described method (Bailey et al. 1991).
Measurement of gene expression by real-time PCR
For analyzing expression of the β-xylosidase gene, we performed real-time PCR. RNA was extracted with Fast RNA Pro Red kit (MP biomedicals CA USA) at one day after starting the cultures. The extracted RNA was cleaned using the RNeasy mini kit (Qiagen, Hilden Germany). cDNA was prepared using the M-MLV reverse transcriptase (Takara Bio) and oligo (dT) 20 (Toyobo, Osaka Japan). Samples were labeled by iQ SYBR Green (Bio-Rad, CA USA). Primers used for real-time PCR were as follows: For β-xylosidase: 5'-TTCCCGGTTAGGGTTTGATG-3' (forward) and 5'-GGGACACCATTCACCGAGTT-3' (reverse), for cellobiohydrolase I: 5'-ACTGCCTCCTTCAGCAAACAC-3' (forward) and 5'-GGCGTAGTCGTCCCACAAA-3' (reverse), for β-actin (the internal control): 5'-CAACTGGGACGACATGGAGA-3' (forward) and 5'-GTTGGACTTGGGGTTGATGG-3' (reverse).
Nucleotide sequence accession number
The DDBJ accession number of bxy3A sequence is AB613265.