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Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences

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Title: Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences
Authors: Nagamine, Shota Browse this author
Liu, Chengwei Browse this author
Nishishita, Jumpei Browse this author
Kozaki, Takuto Browse this author
Sogahata, Kaho Browse this author
Sato, Yoshiro Browse this author
Minami, Atsushi Browse this author
Ozaki, Taro Browse this author
Schmidt-Dannert, Claudia Browse this author
Maruyama, Jun-ichi Browse this author
Oikawa, Hideaki Browse this author →KAKEN DB
Keywords: Aspergillus oryzae
basidiomycete fungi
heterologous expression
terpene synthase
Issue Date: Aug-2019
Publisher: American Society for Microbiology
Journal Title: Applied and Environmental Microbiology
Volume: 85
Issue: 15
Start Page: e00409-19
Publisher DOI: 10.1128/AEM.00409-19
Abstract: 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.
Type: article (author version)
Appears in Collections:理学院・理学研究院 (Graduate School of Science / Faculty of Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 及川 英秋

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