title: Development of a sufficient and effective procedure for transformation of an oleaginous yeast, Rhodosporidium toruloides DMKU3-TK16.
abstract: Rhodosporidium toruloides DMKU3-TK16 (TK16), a basidiomycetous yeast isolated in Thailand, can produce a large amount of oil corresponding to approximately 70 % of its dry cell weight. However, lack of a sufficient and efficient transformation method makes further genetic manipulation of this organism difficult. We here developed a new transformation system for R. toruloides using a lithium acetate method with the Sh ble gene as a selective marker under the control of the R. toruloides ATCC 10657 GPD1 promoter. A linear DNA fragment containing the Sh ble gene expression cassette was integrated into the genome, and its integration was confirmed by colony PCR and Southern blot. Then, we further optimized the parameters affecting the transformation efficiency, such as the amount of linear DNA, the growth phase, the incubation time in the transformation mixture, the heat shock treatment temperature, the addition of DMSO and carrier DNA, and the recovery incubation time. With the developed method, the transformation efficiency of approximately 25 transformants/μg DNA was achieved. Compared with the initial trial, transformation efficiency was enhanced 417-fold. We further demonstrated the heterologous production of EGFP in TK16 by microscopic observation and immunoblot analysis, and use the technique to disrupt the endogenous URA3 gene. The newly developed method is thus simple and time saving, making it useful for efficient introduction of an exogenous gene into R. toruloides strains. Accordingly, this new practical approach should facilitate the molecular manipulation, such as target gene introduction and deletion, of TK16 and other R. toruloides strains as a major source of biodiesel.
authors.affiliation: International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.
authors.forename: Yung-Yu
authors.initials: YY
authors.lastname: Tsai
authors.affiliation: International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Ohashi
authors.affiliation: International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.
authors.forename: Takenori
authors.initials: T
authors.lastname: Kanazawa
authors.affiliation: Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand.
authors.forename: Pirapan
authors.initials: P
authors.lastname: Polburee
authors.affiliation: International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Misaki
authors.affiliation: Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand.
authors.forename: Savitree
authors.initials: S
authors.lastname: Limtong
authors.affiliation: International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka, 565-0871, Japan. fujiyama@icb.osaka-u.ac.jp.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001487:Basidiomycota / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D015139:Blotting, Southern; D004352:Drug Resistance, Microbial / Q000187:drug effects; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016681:Genome, Fungal / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008957:Models, Genetic; D009154:Mutation; D009821:Oils / Q000378:metabolism*; D010692:Phleomycins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013696:Temperature; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D009821:Oils; D010692:Phleomycins; C099353:phleomycin resistance protein, Streptoalloteichus hindustanus; D049452:Green Fluorescent Proteins
keywords: Biodiesel; Gene integration; Oleaginous yeast; Rhodosporidium toruloides; Transformation