In addition, a mini-CbpA was purified simply by affinity
chromatography, using cellulose as a support. We demonstrate ethanol fermentation from cellulosic NVP-BKM120 cell line materials by a recombinant strain and the synergic effect for hydrolysis by in vivo assembly of minicellulosomes. The Escherichia coli strain used as the host strain for recombinant DNA manipulation in this study was DH5α. Saccharomyces cerevisiae strain YPH499 (Clontech Laboratories Inc.) was used for cellulase expression and fermentation. Saccharomycopsis fibuligera (ATCC 36309), C. thermocellum (ATCC 27405), and C. cellulovorans (ATCC 35296) were used as the source of genomic DNA. Escherichia coli was grown in Luria–Bertani medium (10 g L−1 tryptone, 5 g L−1 yeast extract, 5 g L−1 sodium chloride) containing 50 μg mL−1 ampicillin at 37 °C. Saccharomyces cerevisiae was aerobically cultivated at 30 °C in selection selleck medium [synthetic defined (SD) medium: 20 g L−1 glucose, 6.7 g L−1 yeast–nitrogen base without amino acid (YNB)
and 1.3 g L−1 Trp drop-out amino acid], in reproduction medium (YPD medium: 10 g L−1 yeast extract, 20 g L−1 peptone, 20 g L−1 glucose), and in fermentation medium (CMC medium): 6.7 g L−1 YNB, 1.3 g L−1 Trp drop-out amino acid, 10 g L−1 CMC]. Clostridium thermocellum and C. cellulovorans were grown under strictly anaerobic conditions at 37 °C in round-bottom flasks containing a previously described medium (Sleat et al., 1984; Shoseyov & Doi, 1990). All molecular methods used standard molecular biology techniques (Sambrook et al., 1989). Restriction enzymes and T4 DNA ligase were purchased from Takara (Japan). Genomic DNA of S. cerevisiae, S. fibuligera, C. thermocellum, and C. cellulovorans were isolated using PIK3C2G a genomic DNA purification kit (Promega) according to the manufacturer’s instructions. All oligonucleotide primers
used for plasmid construction are listed in Table 1. The chimeric CelE-doc gene contained the dockerin region of C. cellulovorans EngB attached to the C. thermocellum endoglucanase CelE backbone. The chimeric CelE-doc gene was constructed by a multistep PCR strategy using pairs of overlapping primers: cCelE P1, cCelE P2, cCelE P3, and cCelE P4 (Fig. 1a). The catalytic domain fragment was amplified using C. thermocellum genomic DNA as the template, and primers cCelE P1 and cCelE P2, and corresponded to the catalytic domain of CelE. The dockerin fragment was amplified using C. cellulovorans genomic DNA as the template, and primers cCelE P3 and cCelE p4, and covered the dockerin domain of EngB. Each of the P2 and P3 primers possessed a 10-nucleotide-long 5′ extension complementary to the end of the adjacent fragment of the chimeric CelE-doc gene, which was necessary to fuse the different fragments together.