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Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining. IMPORTANCE The recent large influx of genome sequences from basidiomycetes, which are prolific producers of bioactive natural products, may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of basidiomycete genes. For this purpose, we applied the ascomycete Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron prediction, this Aspergillus oryzae system represents a practical method for the production of basidiomycete natural products.

Abscisic acid (ABA) is a well-known phytohormone that regulates abiotic stresses. ABA produced by fungi is also proposed to be a virulence factor of fungal pathogens. Although its biosynthetic pathway in fungi was proposed by a series of feeding experiments, the enzyme catalyzing the reaction from farnesyl diphosphate to α-ionylideneethane remains to be identified. In this work, we identified the novel type of sesquiterpene synthase BcABA3 and its unprecedented three-step reaction mechanism involving two neutral intermediates, β-farnesene and allofarnesene. Database searches showed that BcABA3 has no homology with typical sesquiterpene synthases and that the homologous enzyme genes are found in more than 100 bacteria, suggesting that these enzymes form a new family of sesquiterpene synthases.

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Compacted bentonite, the major mineral being montmorillonite, is a candidate buffer material for geological disposal of high-level radioactive waste. The diffusion behavior of radionuclides in the compacted montmorillonite is an important issue to be clarified for the safety assessment of geological disposal. In this study, one-dimensional, non-steady diffusion experiments using 22 Na at different diffusion temperatures and the measurement of the basal spacings by XRD were conducted for Na-montmorillonite saturated with NaCl solutions of different concentrations. Some basal spacings of the montmorillonite were found to decrease from 1.88 to 1.56 nm as the NaCl concentration increased from 0 to 0.1 M. The apparent self-diffusion coefficients at 298 K obtained in this study slightly increased as the NaCl concentration increased from 0 to 0.5 M, while the activation energies, calculated from the temperature dependences of the diffusion coefficients, were found to be 14, 22, and 17 kJ mol -1 at NaCl concentrations of 0, 0.1, and 0.5 M, respectively. These NaCl concentration dependences of the diffusion coefficients and their activation energies can be explained by assuming three independent diffusion processes (diffusion in pore water, on external surface of montmorillonite, and in interlayer of montmorillonite).

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