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

Page 5 of 8

Figure 2 The amount of ATP as the indicator of fungal growth at 1, 3, and 7 days. Closed column; Y-94, Open column; YKX1. Standard deviations are calculated from triplicate experiments.

ligated between the cbh1 promoter and the cbh1 termi- nator, was half of that in wild type (Rahman et al. 2009). In this study, FPU activity of YKX1 was similar to that of Y-94. In most transformation experiments in filamen- tous fungi, a terminator is added downstream of the open reading frame. We did not ligated the terminator of cbh1 at downstream of bxy3A. Therefore, the expres- sion cassette was less likely to induce a double cross- over. However, it is possible that a single crossover could have occurred at cbh1 gene. Although cbh1 gene of YKX1 was slightly lower than that of Y-94, expression cassette did not disrupt the original cbh1 gene.

b-xylosidase gene in the integrated transformation cassette was expressed and the produced enzyme func- tioned. Xylosidase activity in YKX1 was higher than that in Y-94 grown in PD medium (data not shown), while activity of YKX1 was much higher than that of Y-94 in medium with 4%Solcaflock (Figure 3). Another potential problem with using the cbh1 promoter was a possible titration effect; the cellulase genes may have bound

most of the transcription factor, xyr1, in T. reesei (Stricker et al. 2006). In A. niger, xlnR which is an ortholog of xyr1, induces expression of the cellulase gene (Stricker et al. 2008). In both cases, expression from the endogenous cbh1 gene might decrease by an additional cbh1 promoter due to competition for the transcription factor. However, we found that FPU activ- ity of YKX1 was similar to Y-94, which suggests titration effect did not occur in YKX1.

We were also concerned that a titration effect might decrease expression from the endogenous b-xylosidase gene because xyr1 induces bxl1 and xlnR induces xlnD in T. reesei and A. niger, respectively (Stricker et al. 2006,, Stricker et al. 2008). It was difficult to determine whether the endogenous b-xylosidase was influenced by a titration effect because YKX1 was transformed with the open reading frame from the endogenous b-xylosi- dase gene. However, expression of bxy3A driven by the cbh1 promoter in YKX1 was higher than expression of the endogenous bxy3A in the parental strain (Table 1).

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