2021 Fiscal Year Final Research Report

Exploring new biologically active metabolites using unidentified gene clusters from Basidiomycota genomes

Research Project

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Project/Area Number	19H02891
Research Category	Grant-in-Aid for Scientific Research (B)
Allocation Type	Single-year Grants
Section	一般
Review Section	Basic Section 38040:Bioorganic chemistry-related
Research Institution	Hokkaido University
Principal Investigator	Hideaki Oikawa 北海道大学, 理学研究院, 特任教授 (00185175)
Co-Investigator(Kenkyū-buntansha)	南篤志北海道大学, 理学研究院, 准教授 (40507191) 尾崎太郎北海道大学, 理学研究院, 助教 (40709060)
Project Period (FY)	2019-04-01 – 2022-03-31
Keywords	担子菌 / 天然物 / ゲノム編集 / 生合成 / ゲノムマイニング
Outline of Final Research Achievements	To develop the versatile methodology for genome mining of mushroom metabolites, we examined the production of terpenes 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 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 two biosynthetic gene clusters using multiple 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 produced bioactive terpenoids such as nerve growth- factor synthesis activator erinacine antitumor agent melleolides and other fungal metabolites.
Free Research Field	生物有機化学
Academic Significance and Societal Importance of the Research Achievements	生合成遺伝子の発現による物質生産は、近年実現した新たな物質生産法であり、現在までにゲノム解析株が1600株以上公開されていながら、我々の研究成果が報告されるまで、ほとんど例がなかった。今回担子菌由来の天然物生産におけるスプライシングの問題を解決するため、新たに汎用性の高いHot spot-ノックイン発現法を開発し、その適用により初めて汎用的な生産が可能になった。ここで開発された手法は、担子菌のみならず子嚢菌を含めた糸状菌の代謝産物が生産にも応用出来る点は注目に値する。