Kanna et al. AMB Express 2011, 1:15 http://www.amb-express.com/content/1/1/15
To amplify b-xylosidase open reading frame from A. cellulolyticus DNA, the forward primer with engineered SpeΙ site (5’-GCACTAGTATGGTCTACACCACG) and the reverse primer with engineered KpnΙ site (5’-GCGGT ACCTCAATTAGAATCAGGC) were designed based on sequence from the A. cellulolyticus genome sequence information (unpublished data) using Augustus 2.2 soft- ware The promoter from the cellobiohydrolase Ι (cbh1) gene (GenBank Accession number; E39854) was amplified with the forward primer with an XhoΙ site (5’-GCCTCGA- GAAGCTTGGAAGCT) and the reverse primer with a SpeΙ site (5’-TACCATGGCTGCACTAGTGTGTC- GATTGCTT). The amplified fragment of the cbh1 pro- moter was connected to b-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 DH5a 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 trea- ted with in 10 mM KH2PO4, 0.8 M NaCl, and 0.2% Yatalase (Takara Bio) to prepare protoplasts. The proto- plasts 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 g) was added to the protoplast sus- pension, then 50 l of Solution B (40% PEG4000, 10 mM Tris-HCl, 10 mM CaCl2) was added to the proto- plast suspension, and the suspension was incubated on ice for 30 min and RT for 15 min. Then, 8.5 ml of solu- tion A was added to the cell suspension to dilute Solu- tion 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 g hygromycin was piled on to the YPSA plate. A single colony was iso- lated 3 days after the addition of PD medium. To con- firm 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 incu- bated with the substrate at 50°C for 60 min. DNS
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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 b-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 Ger- many). 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 b-xylosidase: 5’-TTCCCGGTTAG GGTTTGATG-3’ (forward) and 5’-GGGACACCATT- CACCGAGTT-3’ (reverse), for cellobiohydrolase I: 5’-ACTGCCTCCTTCAGCAAACAC-3’ (forward) and 5’-GGCGTAGTCGTCCCACAAA-3’ (reverse), for b-actin (the internal control): 5’-CAACTGGGACGA- CATGGAGA-3’ (forward) and 5’-GTTGGACTTGG GGTTGATGG-3’ (reverse).
Nucleotide sequence accession number The DDBJ accession number of bxy3A sequence is AB613265.
Results A putative b-xylosidase gene, bxy3A, was identified in
cellulolyticus genome sequence information using the
nidulans xlnD sequence as the query for a homology
search. The size of the gene was 2.4-Kb and it encoded 798 amino acids without introns. The amino acid sequence deduced from bxy3A was aligned with other b-xylosidase sequences (data not shown). This protein had 74% identity to T. reesei Bxl1 (Margolles-Clark et al. 1996) and 61% identity to A. nidulans XlnD (Perez- Gonzalez et al. 1998).
The Bxy3A expression construct is shown in Figure 1. The cbh1 promoter was used to drive expression of the deduced bxy3A open reading frame. The hph gene cod- ing region driven by the transient receptor potential (trp) C promoter was used as the selection marker. The plasmid, pLcbX-1, with the Bxy3A expression construct