title: Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for Thraustochytrids.
abstract: A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo(r)), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo(r) marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo(r) mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C(20:3n-6)) and eicosatetraenoic acid (C(20:4n-3)), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C(20:4n-6)) and eicosapentaenoic acid (C(20:5n-3)), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Keishi
authors.initials: K
authors.lastname: Sakaguchi
authors.forename: Takanori
authors.initials: T
authors.lastname: Matsuda
authors.forename: Takumi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Jun-Ichiro
authors.initials: J
authors.lastname: Ohara
authors.forename: Rie
authors.initials: R
authors.lastname: Hamaguchi
authors.forename: Eriko
authors.initials: E
authors.lastname: Abe
authors.forename: Naoki
authors.initials: N
authors.lastname: Nagano
authors.forename: Masahiro
authors.initials: M
authors.lastname: Hayashi
authors.forename: Mayumi
authors.initials: M
authors.lastname: Ueda
authors.forename: Daiske
authors.initials: D
authors.lastname: Honda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Okita
authors.forename: Yousuke
authors.initials: Y
authors.lastname: Taoka
authors.forename: Shinichi
authors.initials: S
authors.lastname: Sugimoto
authors.forename: Nozomu
authors.initials: N
authors.lastname: Okino
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D044943:Fatty Acid Desaturases / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D011995:Recombination, Genetic; D012641:Selection, Genetic; D058009:Stramenopiles / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D000890:Anti-Infective Agents; D012337:RNA, Ribosomal, 18S; D049452:Green Fluorescent Proteins; D044943:Fatty Acid Desaturases