Rikiya Endoh

Selection of oleaginous yeasts with high lipid productivity for practical biodiesel production.

The lipid-accumulating ability of 500 yeast strains isolated in Japan was evaluated. Primary screening revealed that 31 strains were identified as potential lipid producers, from which 12 strains were cultivated in a medium containing 3% glucose. It was found that JCM 24511 accumulated the highest lipid content, up to 61.53%, while JCM 24512 grew the fastest. They were tentatively identified as Cryptococcus sp. and Cryptococcus musci, respectively. The maximum lipid concentration of 1.49g/L was achieved by JCM 24512. Similarly, JCM 24511 also achieved a high lipid production of 1.37g/L. High lipid productivity is the most important characteristic of oleaginous yeasts from the viewpoint of practical production. Among the strains tested here, JCM 24512 had the best lipid productivity, 0.37g/L/day. The results show that the isolated yeasts could be promising candidates for biodiesel production.

Cryptococcus terricola is a promising oleaginous yeast for biodiesel production from starch through consolidated bioprocessing

Starch is considered a potential feedstock for biofuel production, particularly in light of the large-scale landfilling of food waste and other starchy materials worldwide. Lipid accumulation by oleaginous yeast is a promising method for biodiesel production from starch. However, most oleaginous yeasts are grown on monosaccharides or oligosaccharides because they cannot directly utilize starch. We therefore investigated the starch-assimilation ability of 1,200 yeasts. We found that Cryptococcus terricola could be used for fuel production through consolidated bioprocessing. C. terricola JCM 24523 exhibited the highest lipid content of 61.96% on medium with 5% starch at 10 days. Fatty acid methyl ester analysis showed that this strain produced high proportions of C16:0 and C18 fatty acids when grown on starch, which are ideal for use in biodiesel. Considering the yield and cost, lipids derived from starch using C. terricola would be a promising alternative source for biodiesel production.

Taxonomic richness of yeasts in Japan within subtropical and cool temperate areas.

Background An understanding of the role of yeasts in the environment has been uncertain because estimates of population size and diversity have often been based on species identifications that were determined from a limited number of phenotypic characteristics. DNA-based species identification has now become widely used, allowing an accurate assessment of species in different habitats. However, there are still problems in classification because some genera are polyphyletic. Consequently, the identification of yeasts and measurement of their diversity at the genus level remains difficult, as does assignment of genera to higher taxonomic ranks. Methodology/Principal Findings A total of 1021 yeast strains was isolated from soil samples and plant materials collected from Japan’s subtropical Iriomote Island and the cool temperate Rishiri Island. Based on sequence analyses of the D1/D2 domain of the LSU rRNA gene, these 1021 strains were tentatively classified into 183 species, with apparent new species accounting for approximately half of the total species isolated (60 and 46, Iriomote and Rishiri, respectively). The yeast species composition was statistically different between the two sites with only 15 species in common. Rarefaction curves of respective sources/areas gave distinctive patterns when the threshold of sequence identity became broader, indicating that the yeast diversity was distinct at the different taxonomic levels compared. Conclusions/Significance Our isolation study of yeasts in Japan has enabled us to expand the inventory of species diversity because a large number of new species was observed in the sampling areas. Further, we propose use of a particular diversity threshold as an “indicator” to recognize species, genera and higher taxonomic ranks.

Draft Genome Sequence of Raffaelea quercivora JCM 11526, a Japanese Oak Wilt Pathogen Associated with the Platypodid Beetle, Platypus quercivorus

ABSTRACT The Japanese oak wilt pathogen Raffaelea quercivora and the platypodid beetle, Platypus quercivorus, cause serious mass mortality of Quercus spp. in Japan. Here, we present the first draft genome sequence of R. quercivora JCM 11526 to increase our understanding of the mechanism of pathogenicity and symbiosis with the ambrosia beetle.

Multiple losses of photosynthesis and convergent reductive genome evolution in the colourless green algae Prototheca.

Autotrophic eukaryotes have evolved by the endosymbiotic uptake of photosynthetic organisms. Interestingly, many algae and plants have secondarily lost the photosynthetic activity despite its great advantages. Prototheca and Helicosporidium are non-photosynthetic green algae possessing colourless plastids. The plastid genomes of Prototheca wickerhamii and Helicosporidium sp. are highly reduced owing to the elimination of genes related to photosynthesis. To gain further insight into the reductive genome evolution during the shift from a photosynthetic to a heterotrophic lifestyle, we sequenced the plastid and nuclear genomes of two Prototheca species, P. cutis JCM 15793 and P. stagnora JCM 9641, and performed comparative genome analyses among trebouxiophytes. Our phylogenetic analyses using plastid- and nucleus-encoded proteins strongly suggest that independent losses of photosynthesis have occurred at least three times in the clade of Prototheca and Helicosporidium. Conserved gene content among these non-photosynthetic lineages suggests that the plastid and nuclear genomes have convergently eliminated a similar set of photosynthesis-related genes. Other than the photosynthetic genes, significant gene loss and gain were not observed in Prototheca compared to its closest photosynthetic relative Auxenochlorella. Although it remains unclear why loss of photosynthesis occurred in Prototheca, the mixotrophic capability of trebouxiophytes likely made it possible to eliminate photosynthesis.

Occultifur plantarum f.a., sp. nov., a novel cystobasidiomycetous yeast species

Nine strains representing a single anamorphic novel yeast species in dispersed tropical and subtropical habitats were isolated from sugarcane leaf tissue (DMKU-SE24, DMKU-SE45T, DMKU-SE129 and DMKU-SE134) and corn leaf tissue (DMKU-CE36) in Thailand, phylloplane and rhizoplane of sugarcane in Brazil (IMUFRJ 52018 and IMUFRJ 52019), bromeliad leaf tissue in Brazil (IMUFRJ 51954) and plant leaf in Japan (IPM31-24). These strains showed identical or only 1 nt substitution in the sequences of the D1/D2 region of the LSU rRNA gene and 0-5 nt substitutions in the internal transcribed spacer (ITS) region. Phylogenetic analysis based on the combined sequences of the ITS and the D1/D2 regions showed that the eight of these strains represented a single species in the genus Occultifur that was distinct from related species. Occultifur kilbournensis was the most closely related species, but with 0.9-1.2 % nucleotide substitutions in the D1/D2 region of the LSU rRNA gene, and 2.4-2.6 % nucleotide substitutions in the ITS region. They are therefore considered to represent a novel species of the genus Occultifur although the formation of basidia was not observed. The name Occultifur plantarum f.a., sp. nov. is proposed. The type strain is DMKU-SE45T (=CBS 14554T=TBRC 6561T).

Allodekkera sacchari, gen. nov., sp. nov., a novel yeast species in the Saccharomycetales isolated from a sugar factory in Thailand.

Three yeast isolates, G5-5(5)T, G5-9(3) and G5-9(4), were obtained from the sugar cane juice and waste from sugar production plant (Korach Industry Co., Ltd) in Korach province, Thailand. They were found to belong to the same species based on DNA sequence identity of the small subunit ribosomal RNA gene (SSU) and the D1/D2 region of the large subunit rRNA gene (LSU D1/D2). A blastn search of the GenBank database revealed they had 93 % nucleotide sequence identity to Dekkera bruxellensis for the SSU (1742 bp), but their LSU D1/D2 sequence (572 bp) showed less than 90 % identity to all available sequences in the database. Phylogenetic analyses with neighbour-joining and maximum-parsimony methods using the aligned LSU D1/D2 and SSU sequences (a total of 2072 positions after removal of gaps) inferred that the three isolates were separated from all known taxa in the Saccharomycetales, and that the neighbouring taxa were species of Dekkera/Brettanomyces. Physiological and biochemical characters revealed distinct differences between the three isolates and Dekkera/Brettanomyces species, including the ability to assimilate several carbon sources and inability to ferment glucose. Thus, isolates G5-5(5)T, G5-9(3) and G5-9(4) should be assigned to a novel taxon, for which the name Allodekkera sacchari gen. nov., sp. nov. is proposed. The type strain of the type species is G5-5(5)T (=CBS 14167T=JCM 18455T=TISTR 5950T), with MycoBank number MB815477 (for the genus) and MB817751 (for the species). Two additional strains of the species are G5-9(3) (=JCM 18456) and G5-9(4) (=JCM 18457).

Novel urease-negative basidiomycetous yeast, Trichosporon heliocopridis sp. nov., associated with dung beetles (Heliocopris bucephalus Fabricius) in the north of Thailand.

Ninety-six yeast isolates associated with dung beetles (Heliocopris bucephalus Fabricius) were examined based on a culture-dependent method. A comparison of the colony morphology and PCR-fingerprints obtained by (GTG)5 microsatellite-primed PCR indicated that 84 of these isolates belonged to one group. Five strains (DD1-1T, DD2-33, DD4-11, DD5-15 and DD6-1) were selected as the representatives of this main group, where each of the five selected strains had been derived from a different dung beetle collected in northern Thailand. A comparison of the D1/D2 domain sequence of the large subunit rRNA gene (LSU D1/D2) and the internal transcribed spacer (ITS) sequences revealed that these five strains were the same and were related to the genus Trichosporon. Phylogenetic analysis based on the LSU D1/D2 plus ITS sequences placed this group within the Trichosporon brassicae clade, but it was clearly separated from any known species. In addition, physiological tests showed that this group had the unusual property of the inability to hydrolyse urea, which was distinctly different from the related taxon. Therefore a novel yeast species named Trichosporon heliocopridis sp. nov. (ex-type strain DD1-1T = TISTR 5946T = JCM 30786T = CBS 14168T) is proposed. The MycoBank number is MB812098.

Gut bacterial and fungal communities in ground-dwelling beetles are associated with host food habit and habitat

Beetles (Coleoptera) have the highest species diversity among all orders, and they have diverse food habits. Gut microbes may have contributed to this diversification of food habits. Here, we identified the pattern of the relationship between ground-dwelling beetles and their gut microbial communities (bacteria and fungi) in the field. We collected 46 beetle species of five families from secondary deciduous forests and grasslands in Japan and extracted microbial DNA from whole guts for amplicon sequencing. The gut bacterial and fungal communities differed among all habitats and all food habits of their hosts (carnivores, herbivores, omnivores, and scavengers) except for the fungal communities between carnivores and scavengers. Specifically, the abundant bacterial group varied among food habits: Xanthomonadaceae were abundant in scavengers, whereas Enterobacteriaceae were abundant in carnivores and herbivores. Phylogenetically closely related beetles had phylogenetically similar communities of Enterobacteriaceae, suggesting that the community structure of this family is related to the evolutionary change in beetle ecology. One of the fungal groups, Yarrowia species, which has been reported to have a symbiotic relationship with silphid beetles, was also detected from various carnivorous beetles. Our results suggest that the symbiotic relationships between ground-dwelling beetles and these microbes are widespread.

Lipid production via simultaneous conversion of glucose and xylose by a novel yeast, Cystobasidium iriomotense

The yeast strains IPM32-16, ISM28-8sT, and IPM46-17, isolated from plant and soil samples from Iriomote Island, Japan, were explored in terms of lipid production during growth in a mixture of glucose and xylose. Phylogenetically, the strains were most closely related to Cystobasidium slooffiae, based on the sequences of the ITS regions and the D1/D2 domain of the LSU rRNA gene. The strains were oleaginous, accumulating lipids to levels > 20% dry cell weight. Moreover, kinetic analysis of the sugar-to-lipid conversion of a 1:1 glucose/xylose mixture showed that the strains consumed the two sugars simultaneously. IPM46-17 attained the highest lipid content (33%), mostly C16 and C18 fatty acids. Thus, the yeasts efficiently converted lignocellulosic sugars to lipids, aiding in biofuel production (which benefits the environment, promotes rural jobs, and strengthens fuel security). The strains constituted a novel species of Cystobasidium, for which we propose the name Cystobasidium iriomotense (type strain ISM28-8sT = JCM 24594T = CBS 15015T).