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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.

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Strain DMKU-PS11(1)T was isolated from peat in a swamp forest in Thailand. DNA sequence analysis showed that it belonged to a novel species that was most closely related to Nakazawaea laoshanensis. However, it differed from the type strain of N. laoshanensis (NRRL Y-63634T) by 2.3 % nucleotide substitutions in the D1/D2 region of the large subunit (LSU) rRNA gene, 1.0 % nucleotide substitutions in the small subunit (SSU) rRNA gene and 8.0 % nucleotide substitutions in the internal transcribed spacer (ITS) region. The phylogenetic analyses based on the combined sequences of the SSU and the D1/D2 region and that of the SSU sequences alone confirmed the placement of the novel species in the Nakazawaea clade and its close affinity with N. laoshanensis. Hence, the species Nakazawaea todaengensis f.a., sp. nov. is proposed. The type strain is DMKU-PS11(1)T (=CBS 14555T=TBRC 6559T). The MycoBank number for Nakazawaea todaengensis f.a., sp. nov. is MB 819513.

Two strains, DMKU-LV83 and DMKU-LV85, of a novel yeast species were isolated from the phylloplane of vetiver grass collected in Thailand by plating of leaf washings. Analysis of the sequences of the D1/D2 region of the large subunit (LSU) rRNA gene showed that the two strains represent a single novel species and most closely related to Meira miltonrushii. However, the novel species differed from the type strain of M. miltonrushii (MCA 3882T) by 5.5 % nucleotide substitutions in the D1/D2 region and 8.9 % nucleotide substitutions in the ITS region. The phylogenetic analysis based on the D1/D2 region of the LSU rRNA gene confirmed the placement of the novel species in the Meira clade and its close affinity with M. miltonrushii. Therefore, the species Meira siamensis sp. nov. is proposed. The type strain is DMKU-LV83T (=CBS 12860T=BCC 61180T).

Two strains, DMKU-UbN24(1)T and DMKU-CPN24(1), of a novel yeast species were obtained from soil and palm oil fruit, respectively, collected in Thailand by an enrichment isolation technique using a nitrogen-limited medium containing glycerol as the sole source of carbon. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics and sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, the two strains were found to represent a novel species of the genus Barnettozyma although the formation of ascospores was not observed. The novel species was related most closely to the type strain of Candida montana but differed by 5.4 % nucleotide substitutions in the D1/D2 region of the LSU rRNA gene and by 10.3–10.5 % nucleotide substitutions in the ITS region. The name Barnettozyma siamensis f.a., sp. nov. is proposed. The type strain is DMKU-UbN24(1)T ( = BCC 61189T = NBRC 109701T = CBS 13392T).

The original publication of this paper contained an error in the part of "Materials and methods". In the text part of transformation, transformation mixture (5% PEG-4000, 100 mM lithium acetate, 10 mM Tris-HCl, 1 mM EDTA at 4.9) should be corrected as transformation mixture (35% PEG-4000, 100 mM lithium acetate, 10 mM Tris-HCl at pH 4.9, 1 mM EDTA).

Triacylglycerol (TAG) is a major component of lipid storage in yeast. The acyl CoA: diacylgycerol acyltransferase (DGAT) that catalyzes the final and rate-limiting step in the production of TAG is rather interesting. Consequently, cloning and analysis of the gene-encoding TAG synthase, diacylglycerol acyltransferase gene (DGA1), of the oleaginous yeast Rhodosporidiobolus fluvialis DMKU-RK253 were undertaken. Analysis of the deduced amino acid sequence of DGA1 from R. fluvialis DMKU-RK253 (RfDGA1) showed similarity with the acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) from other organisms. The cDNA of RfDGA1 was cloned into the yeast expression vector pYES2 and heterologously overexpressed in Saccharomyces cerevisiae. One of the transformants showed a 1.6-fold increase in lipid content compared with the wild-type strain harbouring the pYES2 empty vector. Furthermore, DGA1 overexpression in R. fluvialis DMKU-RK253 resulted in a 2.5-fold increase in lipid content when compared with the wild-type strain, and no significant differences in fatty acid composition were observed between RfDGA1-overexpressed and wild-type strains. Taken together, our results supported our hypothesis that the RfDGA1 is a genetic factor that can be used for the development of a strain with improved lipid accumulation capabilities.