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Kanna et al. AMB Express 2011, 1:15 http://www.amb-express.com/content/1/1/15

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Figure 1 The structure of the expression cassette in transformation vector pLcbx-1. The deduced b-xylosidase gene was ligated at the indicated restriction site. TrpC; Transient receptor potential hph; hygromycin phosphotransferase

and the hph marker was transformed into A. cellulolyti- cusY-94 using the protoplast-PEG method.

HND05_CDS0018, but the transformation cassette was not inserted at either of this position (data not shown).

We had transformation experiments in A. cellulolyti- cus with protoplast-PEG method using hph as the selec- tion marker, and could obtain transformants. The mean value of transformation efficiency was 0.24 × 106 cells-1. We have isolated a transformed colony with pLcbX-1on a YPSA plate containing 500 g/ml hygromycin. The transformant, YKX1, could grow on PDA plates with 500 g/ml hygromycin, and the presence of hph was confirmed by PCR from genomic DNA of the strain (data not shown). Growth rates were determined by measuring ATP concentration, because growth rates of filamentous fungi cannot be well assessed by measuring optical density. The growth of YKX1 gradually increased relative to that of Y-94 by 3 and 7 days after the start of the cultures, though growth rates were similar on the first day after the start of the cultures (Figure 2).

We measured b-xylosidase activity in YKX1 and Y-94 cultured in medium with cellulose as a sole carbon source, on days 1, 3 and 7. The b-xylosidase activity was markedly higher in YKX1 than in Y-94 (Figure 3). The amount of secreted protein in YKX1 was similar to that in Y-94 on all days (data not shown). To assess bxy3A gene expression in YKX1 and Y-94, we performed real- time PCR (Table 1). bxy3A expression was more than ten-fold higher in YKX1 than in Y-94 when the strains were grown in medium with cellulose. Although the expression of cbh1 was slightly lower on average in YKX1 than in Y-94, FPU values of Y-94 (1.27 ± 0.14 FPU/ml) and YKX (1.13 ± 0.15 FPU/ml) were not signif- icantly different. This fact suggests that the cbh1 gene was not disrupted by homologous recombination. We analyzed the insertion position by the genome walking method (data not shown). The cbh1 was located at

We also measured xylanase activity both in YKX1 and Y-94, and activity was similar in YKX1 and Y-94 on the third day of culture (Table 2). Furthermore, activities of other hydrolyzing enzymes i.e. b-mannanse and b-man- nosidase were also similar in YKX1 and Y-94 (Table 2). We had experiments of xylooligosaccharides hydrolysis of the transformant. After 1 hr incubation, YKX1 cul- tures had a higher xylose yield than did Y-94 cultures (Figure 4). After 48 hr, YKX1 cultures had xylose yield of 60% from xylooligosaccharides.

Discussion In filamentous fungi, b-xylosidase of each species belongs to one of 3 GH families, 3, 43, or 54 (Knob et al. 2010). The putative b-xylosidase gene in A. celluloly- ticus, bxy3A, showed high homology to b-xylosidase of the GH 3 family, and Bxy3A shared a conserved motif with the GH 3 family.

In this study, to increase hemicellulase productivity, the cbh1 promoter was used to drive overexpression of BXY3A. CBH1, which is one of major cellulase, cleaves cellulose at the non-reducing end and produces cello- biose. CBH1 protein accounts for about 60% of all secreted proteins in T. reesei. Therefore, the cbh1 pro- moter is widely used as a promoter for overexpression of homologous or heterologous gene (Keränen and Penttilä 1995). However, expression cassettes containing the cbh1 promoter might integrate at the cbh1 loci by homologous recombination. Actually, it was reported that expression of cbh1 was abolished by homologous recombination, pcbh1-gus in T. reesei PC-3-7 (Rahman et al. 2009). Moreover, the value of cellulase activity of Pcbh1-gus, in which uidA encoding b-glucuronidase is

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