Isolation and characterization of Lipomyces starkeyi mutants with greatly increased lipid productivity following UV irradiation.

The oleaginous yeast Lipomyces starkeyi is an intriguing lipid producer that can produce triacylglycerol (TAG), a feedstock for biodiesel production. We previously reported that the L. starkeyi mutant E15 with high levels of TAG production compared with the wild-type was efficiently obtained using Percoll density gradient centrifugation. However, considering its use for biodiesel production, it is necessary to further improve the lipid productivity of the mutant. In this study, we aimed to obtain mutants with better lipid productivity than E15, evaluate its lipid productivity, and analyze lipid synthesis-related gene expression in the wild-type and mutant strains. The mutants E15-11, E15-15, and E15-25 exhibiting higher lipid productivity than E15 were efficiently isolated from cells exposed to ultraviolet light using Percoll density gradient centrifugation. They exhibited approximately 4.5-fold higher lipid productivity than the wild-type on day 3. The obtained mutants did not exhibit significantly different fatty acid profiles than the wild-type and E15 mutant strains. E15-11, E15-15, and E15-25 exhibited higher expression of acyl-CoA synthesis- and Kennedy pathway-related genes than the wild-type and E15 mutant strains. Activation of the pentose phosphate pathway, which supplies NADPH, was also observed. These results suggested that the increased expression of acyl-CoA synthesis- and Kennedy pathway-related genes plays a vital role in lipid productivity in the oleaginous yeast L. starkeyi.

Fourteen novel lipomycetaceous yeast species isolated from soil in Japan and transfer of Dipodascopsis anomala to the genus Babjevia based on ascospore production phenotype.

Fourteen novel lipomycetaceous yeasts species were isolated from soil samples collected from the Hokkaido, Chiba and Okinawa prefectures of Japan. Phylogenetic analyses of the D1/D2 domains of the large subunit rRNAs and translation elongation factor 1 alpha genes (TEF1-α) revealed that five strains of two species from the soil in Furano-shi, Hokkaido were related to Dipodascopsis anomala and 29 strains representing 12 species from soils in Kamogawa-shi, Chiba and Iriomote Island, Okinawa were in the Myxozyma clade. The two species of Dipodascopsis form globose or ellipsoid ascospores in their sac-like ascus and pseudohyphae. Furthermore, these species produce ascospores in their pseudohyphae and do not produce an acicular ascus, which is common among the three species including D. anomala. Therefore, we propose transferring D. anomala to the genus Babjevia and amending Babjevia. Two novel species were described and included in the genus Babjevia: Babjevia hyphoforaminiformans sp. nov. (holotype NBRC 111233; MycoBank no. MB 829051) and Babjevia hyphasca sp. nov. (holotype NBRC 112965; MycoBank no. MB 829053). The 12 species in the Myxozyma clade produce neither ascospores nor pseudohyphae and have different characteristics in assimilating several carbon sources from each other. Thus, we propose that the novel species of Lipomyces be classified as forma asexualis (f.a.). From Kamogawa-shi, Chiba (19 strains representing five species): Lipomyces melibiosiraffinosiphilus f.a., sp. nov. (holotype NBRC 111411; MycoBank no. MB 829034), Lipomyces kiyosumicus f.a., sp. nov. (holotype NBRC 111424; MycoBank no. MB 829035), Lipomyces chibensis f.a., sp. nov. (holotype NBRC 111413; MycoBank no. MB 829036), Lipomyces kamogawensis f.a., sp. nov. (holotype NBRC 112967; MycoBank no. MB 829037), Lipomyces amatsuensis f.a., sp. nov. (holotype NBRC 111420; MycoBank no. MB 829041). From Iriomote island, Okinawa (10 strains representing seven species): Lipomyces taketomicus f.a., sp. nov. (holotype NBRC 112966; MycoBank no. MB 829042), Lipomyces yaeyamensis f.a., sp. nov. (holotype NBRC 110433; MycoBank no. MB 829050), Lipomyces iriomotensis f.a., sp. nov. (holotype NBRC 110436; MycoBank no. MB 829045), Lipomyces haiminakanus f.a., sp. nov. (holotype NBRC 110435; MycoBank no. MB 829046), Lipomyces komiensis f.a., sp. nov. (holotype NBRC 110440; MycoBank no. MB 829047), Lipomyces nakamensis f.a., sp. nov. (holotype NBRC 110434; MycoBank no. MB 829048), Lipomyces sakishimensis f.a., sp. nov. (holotype NBRC 110439; MycoBank no. MB 829049).

Highly selective isolation and characterization of Lipomyces starkeyi mutants with increased production of triacylglycerol.

The oleaginous yeast Lipomyces starkeyi is an attractive organism for the industrial production of lipids; however, the amount of lipid produced by wild-type L. starkeyi is insufficient. The study aims to obtain L. starkeyi mutants that rapidly accumulate large amounts of triacylglycerol (TAG). Mutagenized yeast cells at the early stages of cultivation were subjected to Percoll density gradient centrifugation; cells with increased production of TAG were expected to be enriched in the resultant upper fraction because of their lower density. Among 120 candidates from the upper fractions, five mutants were isolated that accumulated higher amounts of TAG. Moreover, when omitting cells with mucoid colony morphology, 11 objective mutants from 11 candidates from the upper fraction were effectively (100%) isolated. Of total 16 mutants obtained, detailed characterization of five mutants was performed to reveal that five mutants achieved about 1.5-2.0 times TAG concentration (4.7-6.0 g/L) as compared with the wild-type strain (3.6 g/L) at day 5. Among these five mutants, strain E15 was the best for industrial use because only strain E15 showed significantly higher TAG concentration as well as significantly higher degree of lipid to glucose and biomass to glucose yields than the wild-type strain. Thus, Percoll density gradient centrifugation is an effective method to isolate mutant cells that rapidly accumulate large amounts of TAG. It is expected that by repeating this procedure as part of a yeast-breeding program, L. starkeyi mutants suitable for industrial lipid production can be easily and effectively obtained.

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).

Potential biodegradation of crude petroleum oil by newly isolated halotolerant microbial strains from polluted Red Sea area.

Two microbial isolates from oil polluted Red Sea water in Egypt, designated as RS-Y1 and RS-F3, were found capable of degrading Belayim mix (BX) crude oil. Strains RS-Y1 and RS-F3 were assigned to the genera Lipomyces tetrasporus and Paecilomyces variotii based on their morphological and physiological characteristics. Both isolates were compared for the biodegradation of crude petroleum-oil hydrocarbons in basal salt medium supplemented with 5% (w/v) of BX-crude oil. Gas chromatography profile showed that the biodegradation of total petroleum hydrocarbons (TPHs) inoculated with L. tetrasporus (68.3%) and P. variotii (58.15%) along with their consortium (66%) significantly reduced TPHs levels as compared to the control after 30days. L. tetrasporus (44.5%) was more effective than P. variotii strain (32.89%) in reducing the unresolved complex mixtures (UCM) content from the medium. Both isolates exhibited a strong growth over a wide range of salinity (5-45g/L NaCl).

ETHANOL PRODUCTION FROM STARCH BY YEASTS ISOLATED FROM CROPS AND DAIRY PRODUCTS.

The aim of this work was to study the ability ofyeasts isolated from crops and dairy products to convert starch to ethanol. The isolated yeasts were screened for their ability to hydrolyze starch. Six most active strains were identified as Lipomyces mesembrius spp. 5.4, 5.5 and 6.4, Shwanniomyces vanrijiae var yarowii F33, Torulaspora sp. F7 and Candida sp. S26. The selected yeasts produced low levels of ethanol from starch under aerobic conditions - 0.006-0.129 g/l (0.3-0.87 % of theoretical yield) and microaerobic conditions - 0. 089-0.35 g/l (1.61-6.07 % of theoretical yield). These amylolytic yeast strains will be studied as the potential candidates for the cocultivation with efficient ethanol producers which do not possess the ability to directly hydrolyze starch.

Lipomyces chichibuensis sp. nov., isolated in Japan, and reidentification of the type strains of Lipomyces kononenkoae and Lipomyces spencermartinsiae.

We isolated two strains of a novel Lipomyces species from soil collected in Chichibu forest, Saitama prefecture, Japan. Based on their morphological and biochemical characteristics, along with multilocus sequence typing using the D1/D2 domain of the large-subunit (LSU) rRNA gene, the internal transcribed spacer (ITS) region and the translation elongation factor 1 alpha gene (EF-1α), the two strains were shown to represent a novel species of the genus Lipomyces, described as Lipomyces chichibuensis sp. nov. (type strain CB08-2(T) = NBRC 109582(T) = CBS 12929(T); Mycobank no. MB808164). In addition, we reidentified the type strains of Lipomyces kononenkoae and Lipomyces spencermartinsiae maintained in culture collections based on phenotypic characters and/or DNA-DNA hybridization to ensure correct future identification of species of the genus Lipomyces. The correct type strains of L. kononenkoae and L. spencermartinsiae are NBRC 107661(T) ( = CBS 2514(T)) and NBRC 10376(T) ( = CBS 5608(T)), respectively.

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, ß, and γ. Recently, L. starkeyi clusters α, ß, 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.