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Construction of xylose- and xylo-oligosaccharide-fermenting Saccharomyces cerevisiae strains is important, because hydrolysates derived from lignocellulosic biomass contain significant amounts of these sugars. We have obtained recombinant S. cerevisiae strain MA-D4 (D-XKXDHXR), expressing xylose reductase, xylitol dehydrogenase and xylulokinase. In the present study, we generated recombinant strain D-XSD/XKXDHXR by transforming MA-D4 with a β-xylosidase gene cloned from the filamentous fungus Trichoderma reesei. The intracellular β-xylosidase-specific activity of D-XSD/XKXDHXR was high, while that of the control strain was under the limit of detection. D-XSD/XKXDHXR produced ethanol, and xylose accumulated in the culture supernatant under fermentation in a medium containing xylo-oligosaccharides as sole carbon source. β-Xylosidase-specific activity in D-XSD/XKXDHXR declined due to xylose both in vivo and in vitro. D-XSD/XKXDHXR converted xylo-oligosaccharides in an enzymatic hydrolysate of eucalyptus to ethanol. These results indicate that D-XSD/XKXDHXR efficiently converted xylo-oligosaccharides to xylose and subsequently to ethanol.

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We have developed a new method to observe the site of big and long spiral callose fiber elongation from a protoplast in liquid culture medium. Protoplasts of embryogenic cells of a conifer, Larix leptolepis, were cultured in the NH 4 NO 3 -free Murashige and Skoog's medium containing 50 mM MgCl 2 and 6% sucrose in a well in a 96-or 24-well culture plates. The protoplasts with elongated callose fibers were selected before fixation with glutaraldehyde, by picking up using a micromanipulator after electric treatment (DC 3 kV/cm) in the medium containing Alexafluor 488 phalloidin. Under laser confocal scanning microscopy (LCSM), two sites in the cell were stained clearly. One site was the nucleus and the other was the plasma membrane from which fibers elongated. Single cell transmission electron microscopy (TEM) was developed for observation of the microstructure at the site of fiber elongation on a single protoplast. A single protoplast which had a fiber was selected using a micromanipulator and transferred to an agarose bead at a low gelling temperature. The cells were fixed with cold glutaraldehyde and processed for TEM analysis. Elongated thin fibrils with vesicle-like structures could be observed by TEM.

We developed a method to measure mechanical properties of single fibers of callose in liquid protoplast cultures of Larix leptolepis and Betula platyphylla, which were formed in media containing 50 mM of MgCl 2 or 100 mM of CaCl 2 , respectively. Tensile test was performed using two micromanipulators loading micropipettes under an inverted microscope. Spring constant of the pipette used was first calibrated and calculated from using a microbalance. The callose fiber was wired between the two micropipettes. The Young's modulus of single fibers for Larix and Betula was 7-9 kPa (1.4-1.9 x 10 4 N/m 2 ) though the diameters of the fiber varied from 10 μm for Larix and 22-26 μm for Betula. No big difference was found between experiments with and without medium containing high concentrations of salts. Tensile strength at break was 1.1-1.8 kPa (2.3-3.6 x 10 3 N/m 2 ). The values are compared to other materials including cellulose containing plant cell wall, cell membranes, and amorphous callose. The value of the Young's modulus observed was discussed.