1.2 Development of a gene transfer system for the mycelia of F. velutipes

As shown in above, we found the edible mushroom F. velutipes Fv-1 strain to be an efficient ethanol producer, and, we demonstrated its preferable properties of ethanol fermentation from various sugars (Mizuno et al., 2009b), whole crop sorghums and rice straw (Mizuno et al., 2009a). However, the strain can only slightly convert pentoses, which account for approximately 20-30% of plant cell walls, into ethanol (Mizuno et al., 2009a). Therefore, genetic engineering of the pentose metabolism is necessary to make possible the ethanol fermentation from pentose. Furthermore, more efficient (low cost) conversion of biomass to ethanol could be expected if saccharification ability was strengthened by expressing cellulases. A transformation method of F. velutipes by the electroporation protocol for basidiospores has been reported (Kuo et al., 2004), but it requires a long period to produce basidiospores because it must go through fruiting body formation, and cannot eliminate the risk of contamination in the process of spore harvest. Since screening of many transformants is needed for improvement of the metabolic pathway by genetic engineering, the development of a simpler transformation method is desired to obtain high numbers of transformants.

Therefore, an adequate condition for protoplast preparation from mycelia of F. velutipes Fv-1 strain was investigated, and simpler a transformation protocol for this fungus was developed by the calcium-PEG method and the restriction enzyme-mediated-integration (REMI) method.

First, we constructed a pFvT vector for transformation of the F. velutipes Fv-1 strain (Fig. 8A). The vector possessed a F. velutipes tryptophan synthetase gene promoter and terminator (GenBank no. AB028647) to regulate expression of the constructed genes, and the hygromycin phosphotransferase gene (hph) from Escherichia coli as selection marker. The hph gene was obtained from pCAMBIA1201 vector (CAMBIA; http://www. cambia. org/).

Next, conditions to prepare protoplast from the mycelia of F. velutipes were optimized by modifying a method for Phanerochaete sordida (Yamagishi et al., 2007). The F. velutipes Fv-1

MCS, multiple cloning sites; Ftrp-p, trpl promoter from F. velutipes; Ftrp-t, trpl terminator from F. velutipes; Fgpd-p, gpd promoter from F. velutipes; Fgpd-t, gpd terminator from F. velutipes; hph, hygromycin B phosphotransferase gene; ampr, ampicilin resistance gene; Eori, pUC19 ori. (Reproduced from Maehara et al., 2010b)

Fig. 8. Structures of the plasmids used in this study

strain was grown in PCMY (1% polypeptone, 0.2% casamino acid, 1% malt extract, and 0.4% yeast extract) medium at 25°C for 3 d. Then the mycelia were collected and incubated in enzyme solution [1.5% cellulase Onozuka-RS (Yakult Pharmaceutical, Tokyo) and 1.5% lysis enzyme (Sigma, St. Louis, MO) in 0.75 M MgOsm (0.75 M MgSO4, 20 mM MES, pH 6.3)] at 30°C for 5 h. The protoplasts were filtered through Miracloth (Cosmo Bio, Tokyo), washed at twice with 1 M SorbOsm (1.0 M sorbitol, 10 mM MES, pH 6.3), and suspended in SorbOsm plus 40 mM CaCl2 solution to a final concentration of approximately 108 protoplasts ml-1. Genetic transformation was investigated using the pFvT vector and the protoplasts prepared as described above. The transformation procedures for Lentinus edodes (Sato et al., 1998) and Schizophyllum commune (Van Peer et al., 2009) were modified for the transformation of F. velutipes Fv-1. In the course of the transformation process, the effect of the structure of the plasmid DNA on transformation was evaluated using circular and linear pFvT plasmids. Approximately 6-fold transformants were obtained when the plasmid DNA was linearized (Table 2).

Because the REMI method is a popular transformation tool for fungi (Hirano et al., 2000; Maier & Schafer, 1999; Riggle & Kumamoto, 1998; Sato et al., 1998), we evaluated the effect of REMI on the transformation for F. velutipes Fv-1. The F. velutipes Fv-1 strain was transformed by pFvT with a restriction enzyme, BglI, Kpnl, or Pstl. The addition of the restriction enzymes increased the number of transformants by about 1.6- to 5.8-fold (Table 2). The suggests that the addition of restriction enzymes enhanced the transformation efficiency of F. velutipes. Therefore, to find the optimum enzyme concentration for REMI, we

performed transformation using circular pFvT plasmid with the presence of various concentrations of PstI (Fig. 9). As for the results, the number of transformants obtained was affected by the amount of restriction enzyme. The efficiency was significantly increased by the addition of PstI at 25 units, by it gradually decreased when the PstI amount was over 25 units, suggesting that the optimal value for transformation mediated by the PstI is 25 units. In conclusion, we found a simple transformation procedure for the mycelia of F. velutipes Fv — 1 strain by the calcium-PEG method combined with REMI. The transformation method of F. velutipes Fv-1 strain does not require a process of spore formation, because the mycelia could be used as starting material. Moreover, a high efficiency of transformation was obtained by the adoption of REMI.