Discovery of Oleaginous Yeast from Mountain Forest Soil in Thailand.

As an interesting alternative microbial platform for the sustainable synthesis of oleochemical building blocks and biofuels, oleaginous yeasts are increasing in both quantity and diversity. In this study, oleaginous yeast species from northern Thailand were discovered to add to the topology. A total of 127 yeast strains were isolated from 22 forest soil samples collected from mountainous areas. They were identified by an analysis of the D1/D2 domain of the large subunit rRNA (LSU rRNA) gene sequences to be 13 species. The most frequently isolated species were Lipomyces tetrasporus and Lipomyces starkeyi. Based on the cellular lipid content determination, 78 strains of ten yeast species, and two potential new yeast that which accumulated over 20% of dry biomass, were found to be oleaginous yeast strains. Among the oleaginous species detected, Papiliotrema terrestris and Papiliotrema flavescens have never been reported as oleaginous yeast before. In addition, none of the species in the genera Piskurozyma and Hannaella were found to be oleaginous yeast. L. tetrasporus SWU-NGP 2-5 accumulated the highest lipid content of 74.26% dry biomass, whereas Lipomyces mesembrius SWU-NGP 14-6 revealed the highest lipid quantity at 5.20 ± 0.03 g L-1. The fatty acid profiles of the selected oleaginous yeasts varied depending on the strain and suitability for biodiesel production.

Lipid Production from Sugarcane Top Hydrolysate and Crude Glycerol with Rhodosporidiobolus fluvialis using a Two-Stage Batch-Cultivation Strategy with Separate Optimization of Each Stage.

Lipids from oleaginous microorganisms, including oleaginous yeasts, are recognized as feedstock for biodiesel production. A production process development of these organisms is necessary to bring lipid feedstock production up to the industrial scale. This study aimed to enhance lipid production of low-cost substrates, namely sugarcane top and biodiesel-derived crude glycerol, by using a two-stage cultivation process with Rhodosporidiobolus fluvialis DMKU-SP314. In the first stage, sugarcane top hydrolysate was used for cell propagation, and in the second stage, cells were suspended in a crude glycerol solution for lipid production. Optimization for high cell mass production in the first stage, and for high lipid production in the second stage, were performed separately using a one-factor-at-a-time methodology together with response surface methodology. Under optimum conditions in the first stage (sugarcane top hydrolysate broth containing; 43.18 g/L total reducing sugars, 2.58 g/L soy bean powder, 0.94 g/L (NH4)2SO4, 0.39 g/L KH2PO4 and 2.5 g/L MgSO4•7H2O, pH 6, 200 rpm, 28 C and 48 h) and second stage (81.54 g/L crude glycerol, pH 5, 180 rpm, 27 C and 196 h), a high lipid concentration of 15.85 g/L, a high cell mass of 21.07 g/L and a high lipid content of 73.04% dry cell mass were obtained.

Molecular cloning and overexpression of DGA1, an acyl-CoA-dependent diacylglycerol acyltransferase, in the oleaginous yeast Rhodosporidiobolus fluvialis DMKU-RK253.

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.

Meira siamensis sp. nov., a novel anamorphic ustilaginomycetous yeast species isolated from the vetiver grass phylloplane.

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).

Nakazawaea todaengensis f.a., sp. nov., a yeast isolated from a peat swamp forest in Thailand.

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.

Development of a sufficient and effective procedure for transformation of an oleaginous yeast, Rhodosporidium toruloides DMKU3-TK16.

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.

Characterization of oleaginous yeasts accumulating high levels of lipid when cultivated in glycerol and their potential for lipid production from biodiesel-derived crude glycerol.

This study attempted to identify oleaginous yeasts and selected the strain that accumulated the largest quantity of lipid for lipid production from glycerol. Two-step screening of 387 yeast strains revealed 23 oleaginous strains that accumulated quantities of lipid higher than 20 % of their biomass when cultivated in glycerol. These strains were identified to be four ascomycetous yeast species i.e. Candida silvae, Kodamaea ohmeri, Meyerozyma caribbica, and Pichia manshurica, and five basidiomycetous yeast species i.e. Cryptococcus cf. podzolicus, Cryptococcus laurentii, Rhodosporidium fluviale, Rhodotorula taiwanensis, and Sporidiobolus ruineniae. Rhodosporidium fluviale DMKU-RK253 accumulated the highest quantity of lipid equal to 65.2 % of its biomass (3.9 g L(-1) lipid and 6.0 g L(-1) biomass) by shaking flask cultivation in crude glycerol. The main fatty acids in the accumulated lipid of this strain consisted of oleic acid, linoleic acid, and palmitic acid. Therefore, R. fluviale DMKU-RK253 has potential for producing lipid for biodiesel manufacturing using crude glycerol as a feedstock.

Barnettozyma siamensis f.a., sp. nov., a lipid-accumulating ascomycete yeast species.

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 61189(T) = NBRC 109701(T) = CBS 13392(T)).