Takafumi Naganuma

Phylogenetic and biochemical characterization of the oil-producing yeast Lipomyces starkeyi

Lipomyces starkeyi is an oleaginous yeast, and has been classified in four distinct groups, i.e., sensu stricto and custers α, β, and γ. Recently, L. starkeyi clusters α, β, and γ were recognized independent species, Lipomyces mesembrius, Lipomyces doorenjongii, and Lipomyces kockii, respectively. In this study, we investigated phylogenetic relationships within L. starkeyi, including 18 Japanese wild strains, and its related species, based on internal transcribed spacer sequences and evaluated biochemical characters which reflected the phylogenetic tree. Phylogenetic analysis showed that most of Japanese wild strains formed one clade and this clade is more closely related to L. starkeyi s.s. clade including one Japanese wild strain than other clades. Only three Japanese wild strains were genetically distinct from L. starkeyi. Lipomyces mesembrius and L. doorenjongii shared one clade, while L. kockii was genetically distinct from the other three species. Strains in L. starkeyi s.s. clade converted six sugars, d-glucose, d-xylose, l-arabinose, d-galactose, d-mannose, and d-cellobiose to produce high total lipid yields. The Japanese wild strains in subclades B, C, and D converted d-glucose, d-galactose, and d-mannose to produce high total lipid yields. Lipomyces mesembrius was divided into two subclades. Lipomyces mesembrius CBS 7737 converted d-xylose, l-arabinose, d-galactose, and d-cellobiose, while the other L. mesembrius strains did not. Lipomyces doorenjongii converted all the sugars except d-cellobiose. In comparison to L. starkeyi, L. mesembrius, and L. doorenjongii, L. kockii produced higher total lipid yields from d-glucose, d-galactose, and d-mannose. The type of sugar converted depended on the subclade classification elucidated in this study.

An Improved Method for Isolating Yeasts in the Genus Lipomyces and Related Genera from Soil

An improved method for isolating soil yeasts in the genus Lipomyces and related genera was developed on the basis of their physiological properties. Liberation of yeast cells from soil, capture of the yeast cells by a membrane filter, and colony formation on an acidic (pH 3) nitrogen-free agar plate containing 0.1% cycloheximide resulted in success.

Total DNA Preparation Excluding Extracellular Acidic Polysaccharide from Lipomyces Yeasts and its Application to Taxonomic Studies

A preparation method of total DNA from Lipomyces yeasts was improved in order to exclude extracellular acidic polysaccharide thoroughly. The method combined an ultracentrifuge and polyethylene glycol precipitation with the usual method. The total DNAs obtained were analyzed for G+C content and by DNA-DNA hybridization. The results all agreed almost completely with literature data. All the DNA samples prepared using this method were pure enough for these taxonomic analyses and could also be used as templates of PCR for amplification of small subunit ribosomal DNA and the internal transcribed spacer region.

Two novel Lipomycetaceous yeast species, Lipomyces okinawensis sp. nov. and Lipomyces yamanashiensis f.a., sp. nov., isolated from soil in the Okinawa and Yamanashi prefectures, Japan

Four novel Lipomyces strains were isolated from soil collected in the Okinawa and Yamanashi prefectures, Japan. Based on their morphological and biochemical characteristics, along with sequence typing using the D1/D2 domain of the LSU rRNA, internal transcribed spacer (ITS) region including 5.8S rRNA, and translation elongation factor 1 alpha gene (EF-1α), the four strains were shown to represent two novel species of the genus Lipomyces, described as Lipomyces okinawensis sp. nov. (type strain No.3-a(35)T=NBRC 110620T=CBS 14747T) and Lipomyces yamanashiensis f.a., sp. nov. (type strain No.313T=NBRC 110621T=CBS 14748T).