Japanese sake making using wild yeasts isolated from natural environments.

Saccharomyces cerevisiae is one of the most important microorganisms for the food industry, including Japanese sake, beer, wine, bread, and other products. For sake making, Kyokai sake yeast strains are considered one of the best sake yeast strains because these strains possess fermentation properties that are suitable for the quality of sake required. In recent years, the momentum for the development of unique sake, which is distinct from conventional sake, has grown, and there is now a demand to develop unique sake yeasts that have different sake making properties than Kyokai sake yeast strains. In this minireview, we focus on "wild yeasts," which inhabit natural environments, and introduce basic research on the wild yeasts for sake making, such as their genetic and sake fermentation aspects. Finally, we also discuss the molecular breeding of wild yeast strains for sake fermentation and the possibility for sake making using wild yeasts.

Features and application potential of microbial urethanases.

Urethanase (EC 3.5.1.75) can reduce ethyl carbamate (EC), a group 2A carcinogen found in foods and liquor. However, it is not yet commercially available. Urethanase has been detected as an intracellular enzyme from yeast, filamentous fungi, and bacteria. Based on the most recent progress in the sequence analysis of this enzyme, it was observed that amidase-type enzyme can degrade EC. All five enzymes had highly conserved sequences of amidase signature family, and their molecular masses were in the range of 52-62 kDa. The enzymes of Candida parapsilosis and Aspergillus oryzae formed a homotetramer, and that of Rhodococcus equi strain TB-60 existed as a monomer. Most urethanases exhibited amidase activity, and those of C. parapsilosis and A. oryzae also demonstrated high activity against acrylamide, which is a group 2A carcinogen. It was recently reported that urease and esterase also exhibited urethanase activity. Although research on the enzymatic degradation of EC has been very limited, recently some sequences of EC-degrading enzyme have been elucidated, and it is anticipated that new enzymes would be developed and applied into practical use. KEY POINTS: • Recently, some urethanase sequences have been elucidated • The amino acid residues that formed the catalytic triad were conserved • Urethanase shows amidase activity and can also degrade acrylamide.

Cutinase-like biodegradable plastic-degrading enzymes from phylloplane yeasts have cutinase activity.

Phylloplane yeast genera Pseudozyma and Cryptococcus secrete biodegradable plastic (BP)-degrading enzymes, termed cutinase-like enzymes (CLEs). Although CLEs contain highly conserved catalytic sites, the whole protein exhibits ≤30% amino acid sequence homology with cutinase. In this study, we analyzed whether CLEs exhibit cutinase activity. Seventeen Cryptococcus magnus strains, which degrade BP at 15 °C, were isolated from leaves and identified the DNA sequence of the CLE in one of the strains. Cutin was prepared from tomato leaves and treated with CLEs from 3 Cryptococcus species (C. magnus, Cryptococcus flavus, and Cryptococcus laurentii) and Pseudozyma antarctia (PaE). A typical cutin monomer, 10,16-dihydroxyhexadecanoic acid, was detected in extracts of the reaction solution via gas chromatography-mass spectrometry, showing that cutin was indeed degraded by CLEs. In addition to the aforementioned monomer, separation analysis via thin-layer chromatography detected high-molecular-weight products resulting from the breakdown of cutin by PaE, indicating that PaE acts as an endo-type enzyme.

Effect of light on carotenoid and lipid production in the oleaginous yeast Rhodosporidium toruloides.

The oleaginous yeast Rhodosporodium toruloides is receiving widespread attention as an alternative energy source for biofuels due to its unicellular nature, high growth rate and because it can be fermented on a large-scale. In this study, R. toruloides was cultured under both light and dark conditions in order to understand the light response involved in lipid and carotenoid biosynthesis. Our results from phenotype and gene expression analysis showed that R. toruloides responded to light by producing darker pigmentation with an associated increase in carotenoid production. Whilst there was no observable difference in lipid production, slight changes in the fatty acid composition were recorded. Furthermore, a two-step response was found in three genes (GGPSI, CAR1, and CAR2) under light conditions and the expression of the gene encoding the photoreceptor CRY1 was similarly affected.

Amycolatopsis oliviviridis sp. nov., a novel polylactic acid-bioplastic-degrading actinomycete isolated from paddy soil.

A novel bioplastic-degrading actinomycete, strain SCM_MK2-4T, was isolated from paddy soil in Thailand. The 16S rRNA gene sequence showed that strain SCM_MK2-4T belonged to the genus Amycolatopsis, with the highest sequence similarity to Amycolatopsisazurea JCM 3275T (99.4 %), and was phylogenetically clustered with this strain along with Amycolatopsislurida JCM 3141T (99.3 %), A. japonica DSM 44213T (99.2 %), A. decaplanina DSM 44594T (99.0 %), A. roodepoortensis M29T (98.9 %), A. keratiniphilasubsp. nogabecina DSM 44586T (98.8 %), A. keratiniphilasubsp. keratiniphila DSM 44409T (98.5 %), A. orientalis DSM 40040T (98.4 %) and A. regifaucium GY080T (98.3 %). A combination of DNA-DNA hybridization results ranging from 42.8±3.2 to 66.2±1.4 % with the type strains of A. azurea and A. lurida and some different phenotypic characteristics indicated that the strain could be distinguished from its closest phylogenetic neighbours. Whole-cell hydrolysates of the strain were shown to contain meso-diaminopimelic acid, arabinose, galactose, glucose, ribose, mannose, rhamnose and xylose. The predominant menaquinone was MK-9(H4). The major cellular fatty acid profile consisted of iso-C15 : 0, iso-C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2OH) and C16 : 0. The polar lipid composition of the strain consisted of phosphatidyl-N-methylethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylglycerol, aminophospholipids, an unidentified phospholipid and two unidentified glycolipids. The G+C content of the genomic DNA was 68.2 mol%. On the basis of phylogenetic analyses, DNA-DNA hybridization experimentation and the phenotypic characteristics, it was concluded that strain SCM_MK2-4T represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis oliviviridis sp. nov. is proposed. The type strain is SCM_MK2-4T (=TBRC 7186T=JCM 32134T).

Effective enhancement of polylactic acid-degrading enzyme production by Amycolatopsis sp. strain SCM_MK2-4 using statistical and one-factor-at-a-time approaches.

This study aims to find the optimal medium and conditions for polylactic acid (PLA)-degrading enzyme production by Amycolatopsis sp. SCM_MK2-4. Screening of the most effective components in the enzyme production medium by Plackett-Burman design revealed that the silk cocoon and PLA film were the most significant variables enhancing the PLA-degrading enzyme production. After an response surface methodology, a maximum amount of PLA-degrading enzyme activity at 0.74 U mL-1 was predicted and successfully validated at 95% after 0.39% (w/v) silk cocoon and 1.62% (w/v) PLA film were applied to the basal medium. The optimal initial pH value, temperature, and inoculum size were evaluated by a method considering one-factor-at-a-time. The values were recorded at an initial pH in the range of 7.5-9.0, a temperature of 30-32°C, and an inoculum size of 4-10%. The highest activity of approximately 0.95 U mL-1 was achieved after 4 days of cultivation using the optimized medium and under optimized conditions in a shake flask. Upscaling to the use of a 3-L stirred tank fermenter was found to be successful with a PLA-degrading activity of 5.53 U mL-1; which represents a 51-fold increase in the activity compared with that obtained from the nonoptimized medium and conditions in the shake flask.

Isolation and screening of biopolymer-degrading microorganisms from northern Thailand.

Forty agricultural soils were collected from Chiang Mai and Lampang provinces in northern Thailand. Bacteria, actinomycetes and fungi were isolated and screened for their ability to degrade polylactic acid (PLA), polycaprolactone (PCL) and poly(butylene succinate) (PBS) by the agar diffusion method. Sixty-seven actinomycetes, seven bacteria and five fungal isolates were obtained. The majority of actinomycetes were Streptomyces based on morphological characteristic, chemotaxonomy and 16S rRNA gene data. Seventy-nine microorganisms were isolated from 40 soil samples. Twenty-six isolates showed PLA-degradation (32.9 %), 44 isolates showed PBS-degradation (55.7 %) and 58 isolates showed PCL-degradation (73.4 %). Interestingly, 16 isolates (20.2 %) could degrade all three types of bioplastics used in this study. The Amycolatopsis sp. strain SCM_MK2-4 showed the highest enzyme activity for both PLA and PCL, 0.046 and 0.023 U/mL, respectively. Moreover, this strain produced protease, esterase and lipase on agar plates. Approximately, 36.7 % of the PLA film was degraded by Amycolatopsis sp. SCM_MK2-4 after 7 days of cultivation at 30 °C in culture broth.

Constant enthalpy change value during pyrophosphate hydrolysis within the physiological limits of NaCl.

A decrease in water activity was thought to result in smaller enthalpy change values during PPi hydrolysis, indicating the importance of solvation for the reaction. However, the physiological significance of this phenomenon is unknown. Here, we combined biochemistry and calorimetry to solve this problem using NaCl, a physiologically occurring water activity-reducing reagent. The pyrophosphatase activities of extremely halophilic Haloarcula japonica, which can grow at ∼4 M NaCl, and non-halophilic Escherichia coli and Saccharomyces cerevisiae were maximal at 2.0 and 0.1 M NaCl, respectively. Thus, halophilic and non-halophilic pyrophosphatases exhibit distinct maximal activities at different NaCl concentration ranges. Upon calorimetry, the same exothermic enthalpy change of -35 kJ/mol was obtained for the halophile and non-halophiles at 1.5-4.0 and 0.1-2.0 M NaCl, respectively. These results show that solvation changes caused by up to 4.0 M NaCl (water activity of ∼0.84) do not affect the enthalpy change in PPi hydrolysis. It has been postulated that PPi is an ATP analog, having a so-called high energy phosphate bond, and that the hydrolysis of both compounds is enthalpically driven. Therefore, our results indicate that the hydrolysis of high energy phosphate compounds, which are responsible for biological energy conversion, is enthalpically driven within the physiological limits of NaCl.

Heterologous production of horseradish peroxidase C1a by the basidiomycete yeast Cryptococcus sp. S-2 using codon and signal optimizations.

In the present study, we attempted to improve the production of recombinant horseradish peroxidase C1a (HRP-C1a; a heme-binding protein) by Cryptococcus sp. S-2. Both native and codon-optimized HRP-C1a genes were expressed under the control of a high-level expression promoter. When the HRP-C1a gene with native codons was expressed, poly(A) tails tended to be added within the coding region, producing truncated messenger RNAs (mRNAs) that lacked the 3' ends. Codon optimization prevented polyadenylation within the coding region and increased both the mRNA and protein levels of active HRP-C1a. To improve secretion of the recombinant protein, we tested five types of N-terminal signal peptide (NTP). These included the native HRP-C1a NTP (C1a-NTP), short and long xylanase secretion signals (X1-NTP and X2-NTP), cutinase signal (C-NTP), and amylase signal (A-NTP), with and without a C-terminal propeptide (CTP). X2-NTP without CTP resulted in the highest HRP-C1a secretion into the culture medium. HRP-C1a secretion was further increased by using xylose fed-batch fermentation. The production of HRP-C1a in this study was 2.7 and 15 times higher than the production reported in previous studies that used insect cell and Pichia expression systems, respectively.

Modified COLD-PCR for detection of minor microorganisms in wine samples during the fermentation.

The detection of low-abundant microorganism is difficult when in a sample in which a specific microorganism represents an overwhelming majority using polymerase chain reaction (PCR)-based methods. A modified CO-amplification at Lower Denaturation temperature PCR (mCOLD-PCR) method was developed to detect low-abundant microorganisms using a double-strand RNA probe to inhibit the amplification of the sequence of a major microorganism. Combining the mCOLD-PCR and downstream application (e.g., denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS)), low-abundant microorganisms were detected more efficiently, even when a specific microorganism represents an overwhelming majority of the sample. We demonstrated that mCOLD-PCR-DGGE enabled us to detect Schizosaccharomyces pombe in a model sample coexisting with 10,000 times as many Saccharomyces cerevisiae. When mCOLD-PCR-DGGE was applied in the microbiota analysis of a fermenting white wine, Candida sp. and Cladosporium sp., which were not detected by conventional PCR, were detected. According to the NGS analysis after mCOLD-PCR of a fermenting red wine, the detection ratio of Saccharomyces was decreased dramatically, and the detection ratios of other microorganisms and the numbers of genera detected were increased compared with the conventional PCR. Thus, the application of mCOLD-PCR will reveal comprehensive microbiota of fermented foods, beverages, and so on.

Adenosine kinase-deficient mutant of Saccharomyces cerevisiae accumulates S-adenosylmethionine because of an enhanced methionine biosynthesis pathway.

To isolate an S-adenosylmethionine (SAM)-accumulating yeast strain and to develop a more efficient method of producing SAM, we screened methionine-resistant strains using the yeast deletion library of budding yeast and isolated 123 strains. The SAM content in 81 of the 123 strains was higher than that in the parental strain BY4742. We identified ADO1 encoding adenosine kinase as one of the factors participating in high SAM accumulation. The X∆ado1 strain that was constructed from the X2180-1A strain (MAT a, ATCC 26786) could accumulate approximately 30-fold (18 mg/g dry cell weight) more SAM than the X2180-1A strain in yeast extract peptone dextrose medium. Furthermore, we attempted to identify the molecular basis underlying the differences in SAM accumulation between X∆ado1 and X2180-1A strains. DNA microarray analysis revealed that the genes involved in the methionine biosynthesis pathway, phosphate metabolism, and hexose transport were mainly overexpressed in the X∆ado1 strain compared with the X2180-1A strain. We also determined the levels of various metabolites involved in the methionine biosynthesis pathway and found increased content of SAM, tetrahydrofolate (THF), inorganic phosphate, polyphosphoric acid, and S-adenosylhomocysteine in the X∆ado1 strain. In contrast, the content of 5-methyl-THF, homocysteine, glutathione, and adenosine was decreased. These results indicated that the ∆ado1 strain could accumulate SAM because of preferential activation of the methionine biosynthesis pathway.

Modified Cre-loxP recombination in Aspergillus oryzae by direct introduction of Cre recombinase for marker gene rescue.

Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination system has been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the transformant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced into Aspergillus oryzae protoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as a carrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced into A. oryzae protoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flanked by loxP sites. By using this method, we readily constructed a marker gene-rescued strain lacking ligD to optimize homologous recombination. Furthermore, we succeeded in integrative recombination at a loxP site in A. oryzae. Thus, we developed a simple method to use the Cre-loxP recombination system in A. oryzae by direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme.

Construction of a new recombinant protein expression system in the basidiomycetous yeast Cryptococcus sp. strain S-2 and enhancement of the production of a cutinase-like enzyme.

Yeast host-vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host-vector system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host-vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5 times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately 15 times greater than that of the wild type.

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.

Decolorization and treatment of Kokuto-shochu distillery wastewater by the combination treatment involving biodecolorization and biotreatment by Penicillium oxalicum d, physical decolorization by ozonation and treatment by activated sludge.

Kokuto-shochu is a traditional Japanese distilled liquor made from brown sugar. Kokuto-shochu distillery wastewater (KDW) contains high concentrations of organic compounds and brown pigments (called molasses pigments) which are hardly decolorized by general biological wastewater treatment. A fungus, Penicillium oxalicum d, which we isolated in a previous study, decolorizes 47% of the color from KDW without the addition of any nutrients. P. oxalicum d decolorizes KDW by absorbing the pigments into its mycelia. Here we describe a KDW treatment system that combines biodecolorization and biotreatment by P. oxalicum d with treatment by activated sludge and physical decolorization by ozonation. Adding HClO to suppress bacterial growth and replacing fresh seed sludge at regular intervals helped to maintain the dominance and decolorization ability of P. oxalicum d. In a laboratory-scale demonstration, 48 cycles (12 days) achieved a decolorization ratio of 90% and removed more than 97% of dissolved organic carbon (DOC), dissolved total nitrogen (DTN) and dissolved total phosphorus (DTP). A major feature of our system is that it uses only 6% of the water used in an activated sludge-ozonation system.

New urethanase from the yeast Candida parapsilosis.

Urethanase (EC 3.5.1.75) is an effective enzyme for removing ethyl carbamate (EC) present in alcoholic beverages. However, urethanase is not well studied and has not yet been developed for practical use. In this study, we report a new urethanase (CPUTNase) from the yeast Candida parapsilosis. Because C. parapsilosis can assimilate EC as its sole nitrogen source, the enzyme was extracted from yeast cells and purified using ion-exchange chromatography. The CPUTNase was estimated as a homotetramer comprising four units of a 61.7 kDa protein. In a 20% ethanol solution, CPUTNase had 73% activity compared with a solution without ethanol. Residual activity after 18 h indicated that CPUTNase was stable in 0%-40% ethanol solutions. The optimum temperature of CPUTNase was 43°C. This enzyme showed urethanase activity at pH 5.5-10.0 and exhibited its highest activity at pH 10. The gene of CPUTNase was identified, and a recombinant enzyme was expressed in the yeast Saccharomyces cerevisiae. Characteristics of recombinant CPUTNase were identical to the native enzyme. The putative amino acid sequence indicated that CPUTNase was an amidase family protein. Further, substrate specificity supported this sequence analysis because CPUTNase showed higher activities toward amide compounds. These results suggest that amidase could be a candidate for urethanase. We discovered a new enzyme and investigated its enzymatic characteristics, sequence, and recombinant CPUTNase expression. These results contribute to a further understanding of urethanase.

Aspergillus oryzae acetamidase catalyzes degradation of ethyl carbamate.

Urethanase (EC 3.5.1.75) catalyzes the hydrolysis of ethyl carbamate (EC) to ethanol, carbon dioxide, and ammonia. From our recent study, we expected that an acetamidase encoded by amdS of Aspergillus oryzae may catalyze the degradation of EC because it is homologous with a Candida parapsilosis urethanase (CPUTNase) recently identified. Urethanase is a prospective candidate to reduce EC in alcoholic beverages, but knowledge of this enzyme is very limited. Recombinant AmdS was expressed to study its enzymatic properties. Purified AmdS was identified as a homo-tetramer consisting of four 60 kDa units and exhibited urethanase activity. In a 20% ethanol solution, AmdS had 65% activity compared with a solution without ethanol. Residual activity after 18 h indicated that AmdS was stable in 0%-40% ethanol solutions. The optimum temperature of AmdS was 40 °C. This enzyme showed urethanase activity at pH 6.4-9.6 and exhibited its highest activity at pH 9.6. The Km value of AmdS for EC was 8.2 mM, similar to the Km value (7.6 mM) of CPUTNase. AmdS showed activity not only for EC and acetamide but also other amide compounds. In this study, we investigated the enzymatic properties of AmdS that was identified as acetamidase and showed that an amidase can be an enzymatic candidate that degrades EC.

Crystal structure and enhanced activity of a cutinase-like enzyme from Cryptococcus sp. strain S-2.

The structural and enzymatic characteristics of a cutinase-like enzyme (CLE) from Cryptococcus sp. strain S-2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 A resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long-chain substrates, as well as the preferred specificity of FS cutinase for short-chain substrates. In addition, site-directed mutagenesis was performed to increase the hydrolysis activity on long-chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long-chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity.

Evaluation of Candida easanensis JK8 ß-glucosidase with potentially hydrolyse non-volatile glycosides of wine aroma precursors.

Important 'floral' aromas naturally occur in grapes predominantly as flavourless glycoconjugate precursors. Since these aroma compounds can be released by hydrolysis, different glycosidase enzymes can potentially contribute different aromas to wines. In this paper, the effects of crude and purified Candida easanensis JK8 ß-glucosidases on wine aroma precursors of Muscat of Alexandria grape powder were investigated by GC/MS combined with stir bar sorptive extraction (SBSE). A total of 19 bound volatile compounds were identified, including phenols, terpenes, aldehyde, ester and alcohols. The concentrations of terpenes especially nerol and geraniol, and ß-Damascenone, a C13-norisoprenoid, contributes flowery and slightly fruity aromas were significantly increased in enzyme treated. These results suggest the potential application of this yeast ß-glucosidase as an aroma-enhancing enzyme in winemaking.

Screening for Lipomyces strains with high ability to accumulate lipids from renewable resources.

The yeast Lipomyces accumulates triacylglycerols (TAGs) as intracellular fat globules, and these TAGs can be used as source materials for biodiesel production. In this study, we aimed to use this yeast to produce lipids from renewable resources. Using plate culture and micrograph methods, strains with a high lipid-accumulation ability were screened from 15,408 types of systems combining renewable resources, strains, and culture temperatures. The lipid-accumulation ability of the strains was estimated from the fat globule volume, which was calculated using a micrograph. The reliability of this method was examined, and strains with a high lipid-accumulation ability were identified for each renewable resource. Seventy-seven Lipomyces strains (7 deposit, 68 wild-type, 2 mutants) with a high lipid-accumulation ability were selected. A few strains possessed the ability to accumulate large amounts of TAGs from more than four different renewable resources. We found that strains with a high lipid-accumulation ability could efficiently convert consumed carbon sources into TAGs, which could be easily recovered from the fat globules of these strains through physical disruption.