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Efficient reconstitution of Basidiomycota diterpene erinacine gene cluster in Ascomycota host Aspergillus oryzae based on genomic DNA sequences.

著者 Liu C , Minami A , Ozaki T , Wu J , Kawagishi H , Maruyama JI , Oikawa H
J Am Chem Soc.2019 Sep 19 ; ():.
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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 pre-vious 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 se-quences. Then, we reconstituted the erinacine biosyn-thetic gene cluster using two rounds knock-in of the cDNAs and newly developed repeatable genetic engi-neering by plasmid recycling. At 100% transformation rate, we obtained a transformant that successfully pro-duced erinacine Q and its intermediates. In this study, we elucidated a biosynthetic pathway of erinacines in-volving 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 recy-cling 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 [PubMed - as supplied by publisher]
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