Monday, July 27, 2009
P74

Agrobacterium tumefaciens-mediated transformation as a tool for genomics in an industrially important fungus

Masato Yamada1, Kazuki Yawata1, Seiji Hashimoto1, and Makoto Shimosaka2. (1) Department of Biotechnology, Faculty of Engineering, TOYAMA Prefectural University, 5180, Kurokawa, Imizu, Toyama, 939-0398, Japan, (2) Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1, Tokida, Ueda, Nagano, 386-8567, Japan

  Coleophoma empetri F-11899 is a filamentous fungus used for a production of micafungin, MycaminTM, which has been prescribed to treat deep seated mycoses. Notwithstanding its industrial importance, no genetic information on C. empetri F-11899 is so far available. Agrobacterium tumefaciens-mediated transformation (ATMT) was attempted to make genetic manipulation possible in this strain, and an optimum condition of ATMT was developed for cell density of bacteria and time period of co-cultivation.  Using the established ATMT method, hygromycin B resistant gene was successfully transferred into genome of C. empetri F-11899 and stably maintained even after a serial passage. C. empetri F-11899 produces an acylated cyclic hexapeptide, which is the parent compound of micafungin and structurally belongs to echinocandin family. Although a mechanism for a production of echinocandins is presumed to be non-ribosomal protein synthesis (NRPS), their biosynthetic genes have not been clarified yet. To elucidate a precise mechanism leading to a production of the parent compound of micafungin, a mutant library of C. empetri F-11899 was constructed by ATMT, in which the binary plasmid used was deigned to make plasmid rescue possible. The hygromycin B resistant transformants unable to produce the parent compound were selected from the library, and the nucleotide sequences of genome adjacent to the inserted T-DNAs were determined.

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