To make possible genetic manipulation in F. velutipes, we constructed the pFvT plasmid containing the hygromycin phosphotransferase gene (hph) under the control of the

tryptophan synthetase gene (trpl) promoter, and developed an easy transformation method for F. velutipes by the REMI method (Maehara et al., 2010a). Here, we focused on the promoter of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene because many tools such as promoters and selection markers are desirable for effective metabolic pathway engineering of F. velutipes Fv-1. The gpd promoters are the most frequently used constitutive promoters in basidiomycetes. GPD constitutes up to 5% of the soluble protein in Saccharomyces cerevisiae and other higher eukaryotic organisms (Piechaczyk et al., 1984; Punt et al., 1990), and gpd mRNA accounts for 2-5% of the poly (A)+ RNA in yeast (Holland & Holland, 1978).

In this section, we described that construction of new plasmids having the hph gene from Escherichia coli as a selection marker, which regulated by the gpd promoter and the potency of the gpd promoter from F. velutipes were evaluated.

First we constructed three vectors, pFvG, pFvTgh, and pFvGgh, by modification of pFvT. The pFvT vector possessed a trp1 promoter and terminator regulating the expression of the constructed genes, and the hph gene as selection marker (Fig. 8A, Maehara et al., 2010a). Vectors pFvG (Fig. 8B) and pFvGgh (Fig. 8D) contained the gpd promoter and the terminator of F. velutipes (Kuo et al., 2004) located upstream and downstream of a multiple cloning site (MCS), and both pFvTgh (Fig. 8C) and pFvGgh (Fig. 8D) contained the gpd promoter and the terminator located upstream and downstream of the hph gene (Maehara et al., 2010b).

To determine the potency of the gpd promoter, we compared transformation efficiency by the gpd promoter with that by the trp1 promoter. Gene integrations were performed by the REMI method. Protoplasts were prepared from mycelia of the F. velutipes Fv-1 strain, and then plasmids were transformed into the protoplasts with PstI (25 U). As shown in Table 2, about 10 transformants (10.7 to 12.3) were obtained by the transformation of pFvT and of pFvG, which contain the hph gene controlled by the trp1 promoter. In contrast, as for the results of the transformation of pFvTgh and pFvGgh, the numbers of transformants were significantly increased and about 24.7 to 33.3 transformants were obtained, suggesting that the activity of the gpd promoter was higher than that of the trp1 promoter in F. velutipes Fv-1. There is a difference of about 500-bp in the length of pFvT and pFvG, or pFvTgh and pFvGgh, but no significant difference in the number of transformants obtained by pFvT and by pFvG, and by pFvTgh and pFvGgh was not observed. It might suggest, that the difference of the sizes of these plasmids was not affected on transformation efficiency.

To compare the activity of the gpd and the trp1 promoter, the expression levels of the hph gene in each transformant were examined by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from each set of three transformants and equal amounts of RNAs from each set of three clones were mixed and used as template for RT — PCR. As shown in Fig. 10A, the intensities of the bands of the pFvTgh and pFvGgh transformants were stronger than that of the pFvT and pFvG transformants (upper panel), suggesting that the expression level of the hph gene in the pFvTgh and pFvGgh transformants was higher than that in the pFvT and pFvG transformants. The results were corresponded to the transformation efficiency presented in Table 2, and strongly suggest that the gpd promoter is functional in the heterologous gene expression system in F. velutipes Fv-1 to improve the expression level of the target gene.

Finally, in order to determine whether the plasmid vector was integrated into the genomic DNA by the REMI method, the genomic DNAs of 10 randomly selected pFvGgh transformants were analyzed by Southern blot using the digoxigenin-labeled hph gene as a probe (Fig. 10B). Hybridization signals were detected in all the transformants, and multiple

Ftrp-p-hph Fgpd-p-hph

Fig. 10. Analysis of the transformants obtained by REMI method hybridization signals were also detected in some transformants. There was no signal from the genomic DNA of wild-type Fv-1 as a negative control (data not shown). These results indicate that at least a single hph gene was introduced into all the transformants, and the hph gene is thought to exist as a multicopy in the genomic DNAs of many transformants (Fig. 10B, lanes 3, 4, 5, 6, 8, 9 and 11). The same size bands were detected between 2,027 and 3,530- bp in four transformants (Fig. 10B, lanes 3, 4, 8 and 9). These bands might represent about 2,700-bp of the full-length gpd promoter-hph-gpd terminator region. A 6.9-kb DNA fragment, corresponding to the size of the pFvGgh plasmid, was observed in the genome of only one clone (Fig. 10B, lane 6), indicating that the full length of the plasmid was successfully introduced into the transformant. Consequently, we estimate the probability of integration of full-length pFvGgh vector by the REMI method to be approximately 10%. In our previous study, the probability of integration of the full-length vector was 30% so that the frequency of REMI events of Fv-1 was 10-30% (Maehara et al., 2010a). This value seems to be the comparable level in the case of model mushroom, Coprinus cinereus (8-56%) (Granado et al., 1997).

In conclusion, we demonstrated that the gpd promoter from F. velutipes Fv-1 would be a useful in the transformation system of the strain. The transformation efficiency was about 3 times improved by the use of the gpd promoter. The vectors constructed in this study will be available to improve the genetic engineering of F. velutipes Fv-1 for ethanol fermentation from pentose.

2. Conclusion

In spite of CBP is gaining recognition of a low-cost biomass processing as it involves enzyme production, completely no enzyme process which does not add the saccharification enzymes have not been established. In this study, we demonstrated that F. velutipes can highly convert biomass to ethanol using only small amount of saccharification enzyme even in the quite high concentration of biomass such as 30% w/ v. These results suggest F. velutipes has favorable properties for CBP. Generally, artificial cultivation of mushrooms in polypropylene bottles is performed under the condition of water content 70 to 80%. The condition must be most suitable condition to cultivate the mushrooms. Therefore, F. velutipes will be especially effective in situations that CBP performed under the high concentration of biomass. We believe that this point would be advantage of F. velutipes compared with the other microorganisms engineered for CBP and even for fungus which is possible to ferment the both pentose and hexose. In the future, we would like to develop a novel bioethanol production process by using F. velutipes.

3. Acknowledgment

This work was financially supported by a grant-in-aid (Development of Biomass Utilization Technologies for Revitalizing Rural Areas) from the Ministry of Agriculture, Forestry, and Fisheries of Japan.