TY - JOUR
T1 - Ascomycete Aspergillus oryzae is an efficient expression host for production of basidiomycete terpenes by using genomic DNA sequences
AU - Nagamine, Shota
AU - Liu, Chengwei
AU - Nishishita, Jumpei
AU - Kozaki, Takuto
AU - Sogahata, Kaho
AU - Sato, Yoshiro
AU - Minami, Atsushi
AU - Ozaki, Taro
AU - Schmidt-Dannert, Claudia
AU - Maruyama, Jun ichi
AU - Oikawa, Hideaki
N1 - Publisher Copyright:
© 2019 American Society for Microbiology.
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
KW - Aspergillus oryzae
KW - Basidiomycete fungi
KW - Heterologous expression
KW - Terpene synthase
KW - Terpenes
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U2 - 10.1128/AEM.00409-19
DO - 10.1128/AEM.00409-19
M3 - Article
C2 - 31101615
AN - SCOPUS:85070117029
SN - 0099-2240
VL - 85
JO - Applied and environmental microbiology
JF - Applied and environmental microbiology
IS - 15
M1 - e00409-19
ER -