The mini-CbpA carried a CBD, a hydrophilic domain, and two VX809 cohesin domains with a C-terminal FLAG tag from the pADHα vector (Fig. 2). The expressed mini-CbpA was secreted by means of the α-mating factor of the pADHα vector. The CBD of CbpA from C. cellulovorans was used as a cellulose-binding module (Murashima et al., 2002). Because the mini-CbpA was designed to contain the CBD at its N terminus, purification of the nondegraded mini-CbpA was achieved in a single step, as shown by electrophoretic analysis using 10% SDS-PAGE. The calculated molecular mass of the mini-CbpA

was 58.2 kDa (57 208 Da mini-CbpA plus 1012 Da FLAG tag residues). After purification of the culture supernatant by the cellulose purification method (Shpigel et al., 1999), a homogeneous band was observed by SDS-PAGE analysis (Fig. 4). The mini-CbpA presented an apparent check details molecular mass of 58.2 kDa, which was in good agreement with the calculated

molecular mass. We have tested native-PAGE and CMCase zymogram to confirm the assembly of minicellulosome in the medium (Fig. 5). This shifted halo band confirmed that mini-CbpA and chimeric CelE had been assembled into minicellulosomes in vivo. We have previously demonstrated direct fermentation of CMC to ethanol using the S. cerevisiae strain transformed with an expression plasmid containing endoglucanase CelE and β-glucosidase Bgl1. As the wild-type S. cerevisiae was unable to hydrolyze cellulose to glucose, this suggested that CMC was hydrolyzed to glucose by sequential reactions of CelE and Bgl1. In this study, CMC utilization by cells expressing mini-CbpA, chimeric CelE, of and Bgl1 was compared with that of cells expressing chimeric CelE and Bgl1 (Fig. 6). Figure 6 shows the time course of CMC fermentation by the recombinant strain in CMC medium at 30 °C. The level of ethanol production was consistently higher for cells expressing mini-CbpA,

chimeric CelE, and Bgl1. These results indicate that the scaffolding protein could function and that dockerin-fused enzymes on the scaffolding protein had synergistic activity in CMC degradation. Similar synergistic activity on cellulosic substrates by assembly of minicellulosomes has been reported (Murashima et al., 2002). The highest ethanol concentration was approximately 3.45 g L−1 from CMC after 16 h of fermentation. No ethanol was produced by the S. cerevisiae strain transformed with the pADHα plasmid as the control. The results demonstrated the feasibility of using cellulosic material medium for use in fermentation, and the synergic effect of minicellulosomes. We generated a recombinant yeast strain with minicellulosome-assembling ability by transforming genes into a S. cerevisiae strain. The fermentation performance of the recombinant strain using cellulosic substrates was improved.