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 - Funding Information:
This work was financially supported by Japan Society for the Promotion of Science grants-in-aid JP15H01835 to H.O., JP16H03277 and JP16H06446 to A.M., and JP18K14342 to C.L.
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.
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.
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 -