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J Am Chem Soc. 2019 Oct 2;141(39):15519-15523. doi: 10.1021/jacs.9b08935. Epub 2019 Sep 24.

Efficient Reconstitution of Basidiomycota Diterpene Erinacine Gene Cluster in Ascomycota Host Aspergillus oryzae Based on Genomic DNA Sequences.

Author information

1
Department of Chemistry, Faculty of Science , Hokkaido University , Kita-ku, Kita 10 Nishi 8 , Sapporo 060-0810 , Japan.
2
Research Institute of Green Science and Technology , Shizuoka University , Suruga-ku, Shizuoka , 422-8529 , Japan.
3
Graduate School of Integrated Science and Technology , Shizuoka University , Shizuoka , Japan.
4
Graduate School of Science and Technology , Shizuoka University , Shizuoka , Japan.
5
Department of Biotechnology , The University of Tokyo , 1-1-1 Yayoi , Bunkyo-ku , Tokyo 113-8657 , Japan.

Abstract

To develop the versatile methodology for genome mining of mushroom metabolites, we examined the production of bioactive diterpenes erinacines using genomic DNA sequences. In this report, we initially identified high expression loci (hot spots) in Aspergillus oryzae by sequencing the genomic DNAs from highly yielding transformants which were obtained in our previous biosynthetic studies. Genome editing knock-in of all erinacine biosynthetic genes directly to the hot spot showed that A. oryzae correctly spliced more than 90% of the introns in the mushroom genomic DNA gene sequences. Then, we reconstituted the erinacine biosynthetic gene cluster using two rounds of knock-in of the cDNAs and newly developed repeatable genetic engineering by plasmid recycling. At 100% transformation rate, we obtained a transformant that successfully produced erinacine Q and its intermediates. In this study, we elucidated a biosynthetic pathway of erinacines involving functionally unique hydroxylation supported by dehydrogenase EriH and xylose-specific glycosylation by introducing plant genes for supplying UDP-xylose. Our newly developed hot spot knock-in and plasmid recycling allowed us to avoid a time-consuming screening process and to use unlimited introduction of biosynthetic genes due to marker-free genome editing.

PMID:
31535864
DOI:
10.1021/jacs.9b08935

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