Updated component analysis method for naturally occurring sophorolipids from Starmerella bombicola.

Sophorolipids (SLs) are promising glycolipid biosurfactants as they are easily produced and functional. SLs from microorganisms are comprised of mixtures of multiple derivatives that have different structures and properties, including well-known acidic and lactonic SL (ASLs and LSLs, respectively). In this study, we established a method for analyzing all SL derivatives in the products of Starmerella bombicola, a typical SL-producing yeast. Detailed component analyses of S. bombicola products were carried out using reversed-phase high-performance liquid chromatography and mass spectrometry. Methanol was used as the eluent as it is a good solvent for all SL derivatives. With this approach, it was possible to not only quantify the ratio of the main components of ASL, LSL, and SL glycerides but also confirm trace components such as SL mono-glyceride and bola-form SL (sophorose at both ends); notably, this is the first time these components have been isolated and identified successfully in naturally occurring SLs. In addition, our results revealed a novel SL derivative in which a fatty acid is bonded in series to the ASL, which had not been reported previously. Using the present analysis method, it was possible to easily track compositional changes in the SL components during culture. Our results showed that LSL and ASL are produced initially and that SL glycerides accumulate from the middle stage during the fermentation process. KEY POINTS: • An easy and detailed component analysis method for sophorolipids (SLs) is introduced. • Multiple SL derivatives were identified different from known SLs. • A novel hydrophobic acidic SL was isolated and characterized.

Characterization of a KU70-disrupted strain of the mannosylerythritol lipid-producing yeast Pseudozyma tsukubaensis constructed by a marker recycling system.

The basidiomycetous yeast Pseudozyma tsukubaensis is known as an industrial mannosylerythritol lipid producer. In this study, the PtURA5 marker gene was deleted by homologous recombination. Using the PtURA5-deleted mutant as a host strain, we obtained a derivative disrupted for the PtKU70 gene, a putative ortholog of the KU70 gene encoding a protein involved in the non-homologous end-joining pathway of DNA repair. Subsequently, the introduced PtURA5 gene was re-deleted by marker recycling. These results demonstrated that the PtURA5 gene can be used as a recyclable marker gene. Although the frequency of homologous recombination has been shown to be increased by KU70 disruption in other fungi, the PtKU70-disrupted strain of P. tsukubaensis did not demonstrate an elevated frequency of homologous recombination. Furthermore, the PtKU70-disrupted strain did not show increased susceptibility to bleomycin. These results suggested that the function of this KU70 ortholog in P. tsukubaensis is distinct from that in other fungi.

Xylobiose treatment strengthens intestinal barrier function by regulating claudin 2 and heat shock protein 27 expression in human Caco-2 cells.

BACKGROUND: Xylobiose, a non-digestible disaccharide, largely contributes to the beneficial physiological effects of xylooligosaccharides. However, there is insufficient evidence to assess the direct effect of xylobiose on intestinal barrier function. Here, we investigated the intestinal barrier function in human intestinal Caco-2 cells treated with xylobiose. RESULTS: In total, 283 genes were upregulated and 256 genes were downregulated in xylobiose-treated Caco-2 cells relative to the controls. We focused on genes related to intestinal barrier function, such as tight junction (TJ) and heat shock protein (HSP). Xylobiose decreased the expression of the TJ gene Claudin 2 (CLDN2) and increased the expression of the cytoprotective HSP genes HSPB1 and HSPA1A, which encode HSP27 and HSP70, respectively. Immunoblot analysis confirmed that xylobiose suppressed CLDN2 expression and enhanced HSP27 and HSP70 expression. A quantitative reverse transcription-PCR and promoter assays indicated that xylobiose post-transcriptionally regulated CLDN2 and HSPB1 levels. Additionally, selective inhibition of phosphatidyl-3-inositol kinase (PI3K) inhibited xylobiose-mediated CLDN2 expression, whereas HSP27 expression induced by xylobiose was sensitive to the inhibition of PI3K, mitogen-activated protein kinase kinase and Src. CONCLUSION: The results of the present study reveal that xylobiose suppresses CLDN2 and increases HSP27 expression in intestinal Caco-2 cells via post-transcriptional regulation, potentially strengthening intestinal barrier integrity; however, these effects seem to occur via different signaling pathways. Our findings may help to assess the physiological role of xylobiose. © 2023 Society of Chemical Industry.

Crystal structure of reducing-end xylose-releasing exoxylanase in subfamily 7 of glycoside hydrolase family 30.

TcXyn30A from Talaromyces cellulolyticus, which belongs to subfamily 7 of the glycoside hydrolase family 30 (GH30-7), releases xylose from the reducing end of xylan and xylooligosaccharides (XOSs), the so-called reducing-end xylose-releasing exoxylanase (ReX). In this study, the crystal structures of TcXyn30A with and without xylose at subsite +1 (the binding site of the xylose residue at the reducing end) were determined. This is the first report on the structure of ReX in the family GH30-7. TcXyn30A forms a dimer. The complex structure of TcXyn30A with xylose revealed that subsite +1 is located at the dimer interface. TcXyn30A recognizes xylose at subsite +1 composed of amino acid residues from each monomer and blocks substrate binding to subsite +2 by dimer formation. Thus, the dimeric conformation is responsible for ReX activity. The structural comparison between TcXyn30A and the homologous enzyme indicated that subsite -2 is composed of assembled three stacked Trp residues, Trp49, Trp333, and Trp334, allowing TcXyn30A to accommodate xylan and any branched XOSs decorated with a substitution such as α-1,2-linked 4-O-methyl-d-glucuronic acid or α-1,2- and/or -1,3-linked L-arabinofuranose. These findings provide an insight into the structural determinants for ReX activity of TcXyn30A.

Self-Assembling Properties and Recovery Effects on Damaged Skin Cells of Chemically Synthesized Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs), which are one of the representative sugar-based biosurfactants (BSs) produced by microorganisms, have attracted much attention in various fields in the sustainable development goals (SDGs) era. However, they are inseparable mixtures with respect to the chain length of the fatty acids. In this study, self-assembling properties and structure-activity relationship (SAR) studies of recovery effects on damaged skin cells using chemically synthesized MELs were investigated. It was revealed, for the first time, that synthetic and homogeneous MELs exhibited significant self-assembling properties to form droplets or giant vesicles. In addition, a small difference in the length of the fatty acid chains of the MELs significantly affected their recovery effects on the damaged skin cells. MELs with medium or longer length alkyl chains exhibited much higher recovery effects than that of C18-ceramide NP.

Synthesis of Mannosylerythritol Lipid Analogues and their Self-Assembling Properties, Recovery Effects on Damaged Skin Cells, and Antibacterial Activity.

Synthesis of three types of purpose-designed mannosylerythritol lipid (MEL)-D analogues with decanoyl groups, ß-GlcEL-D, α-GlcEL-D, and α-MEL-D, was accomplished utilizing our boron-mediated aglycon delivery (BMAD) methods. Their self-assembling properties, recovery effects on damaged skin cells, and antibacterial activity were evaluated. It was revealed, for the first time, that α-GlcEL-D and α-MEL-D only generated giant vesicles, indicating that slight differences in the steric configuration of an erythritol moiety and fatty acyl chains affect the ability to form vesicles. Analogue α-MEL-D exhibited significant recovery effects on damaged skin cells. Furthermore, α-MEL-D exhibited antibacterial activity as high as that for MEL-D, indicating that α-MEL-D is a promising artificial sugar-based material candidate for enhancing the barrier function of the stratum corneum, superior to a known cosmetic ingredient, and possesses antibacterial activity.

Evaluating haloarchaeal culture media for ultrahigh-molecular-weight polyhydroxyalkanoate biosynthesis by Haloferax mediterranei.

A series of culture media for haloarchaea were evaluated to optimize the production of ultrahigh-molecular-weight (UHMW) poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei. Cells of H. mediterranei grew (> 1 g/L of dry cell weight) and accumulated PHBV upon flask cultivation in 10 medium types with neutral pH and NaCl concentration > 100 g/L. Molecular weight and compositional analysis revealed that the number-average molecular weight (Mn) of PHBV produced with six selected types of media ranged from 0.8 to 3.5 × 106 g/mol and the 3-hydroxyvalerate (3HV) composition ranged from 8 to 36 mol%. Cultivation in two NBRC media, 1214 and 1380, resulted in the production of PHBV with an Mn of more than 3.0 × 106 g/mol and a weight-average molecular weight of more than 5.0 × 106 g/mol, indicating the production of UHMW-PHBV. These culture media contained small amount of complex nutrients like yeast extract and casamino acids, suggesting that H. mediterranei likely produced UHMW-PHBV on poor nutrient condition. Haloferax mediterranei grown in NBRC medium 1380 produced PHBV with the highest 3HV composition. A solvent-cast film of UHMW-PHBV with 26.4 mol% 3HV produced from 1-L flask cultivation with NBRC medium 1380 was found to be flexible and semi-transparent. Thermal analysis of the UHMW-PHBV cast film revealed melting and glass-transition temperatures of 90.5 °C and - 2.7 °C, respectively. KEY POINTS: • Haloarchaeal culture media were evaluated to produce UHMW-PHBV by H. mediterranei. • UHMW-PHBV with varied molecular weight was produced dependent on culture media. • Semi-transparent film could be made from UHMW-PHBV with 26.4 mol% 3HV.

A putative transporter gene PtMMF1-deleted strain produces mono-acylated mannosylerythritol lipids in Pseudozyma tsukubaensis.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by various yeasts. Mmf1, a putative transporter of MELs, is conserved in the MEL biosynthesis gene clusters of diverse MEL producers, including the genera Ustilago, Pseudozyma, Moesziomyces, and Sporisorium. To clarify the function of Mmf1, we generated the gene-deleted strain of P. tsukubaensis ΔPtMMF1 and evaluated its MEL production. Using thin-layer chromatography analyses, we detected most MELs produced by ΔPtMMF1 in the culture supernatant. The spot size of diacylated MEL-B (the only product of the parental strain) was significantly smaller for strain ΔPtMMF1 than for the parental strain, and a mono-acylated MEL-D spot was detected. In addition, an unknown glycolipid was detected in the sample extracted from strain ΔPtMMF1. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that the unknown glycolipid was a novel MEL homologue, mono-acylated MEL-B. KEY POINTS: • P. tsukubaensis is able to secrete MELs without PtMMF1p. • Strain ΔPtMMF1 mainly produced mono-acylated MELs.

Construction of a Pseudozyma antarctica strain without foreign DNA sequences (self-cloning strain) for high yield production of a biodegradable plastic-degrading enzyme.

The basidiomycetous yeast Pseudozyma antarctica GB-4(0) esterase (PaE) is a promising candidate for accelerating degradation of used biodegradable plastics (BPs). To increase safety and reduce costs associated with the use of PaE, we constructed a self-cloning strain with high-PaE productivity. A Lys12 gene (PaLYS12)-deleted lysine auxotroph strain GB4-(0)-L1 was obtained from GB-4(0) by ultraviolet mutagenesis and nystatin enrichment. Subsequently, the PaE gene (PaCLE1) expression cassette consisting of GB-4(0)-derived PaCLE1, under the control of a xylose-inducible xylanase promoter with PaLYS12, was randomly introduced into the GB4-(0)-L1 genome. A PaE high-producing strain, PGB474, was selected from among the transformants by high throughput double-screening based on its ability to degrade emulsified polybutylene succinate-co-adipate. Quantitative PCR revealed that four copies of the PaE gene expression cassette were introduced into the PGB474 genome. PGB474 produced 2.0 g/L of PaE by xylose-fed-batch cultivation using a 3-L jar fermentor for 72 h.

Tailor-made mannosylerythritol lipids: current state and perspectives.

Mannosylerythritol lipids (MELs) are a type of glycolipid biosurfactant produced by basidiomycetous yeasts, most notably those belonging to the genera Pseudozyma and Ustilago. Mannosylerythritol lipids are environmentally friendly and possess many unique functions, such as gene delivery, bio-activation, and human skin repair, and thus have potential applications in cosmetic, pharmaceutical, agriculture, food, and environmental industries. However, MELs will require overcoming same issues related to the commercialization, e.g., expansion of the structure and function variety and cost reduction. In the past decade, various studies have attempted to tailor production of targeted MELs in order to expand the utility of these biosurfactants. Moreover, the rapid development of genomic sequencing techniques will enhance our ability to modify MEL producers. In this review, we focus on current research into the tailored production of MELs, including conventional and advanced approaches.

Biosynthesis of mono-acylated mannosylerythritol lipid in an acyltransferase gene-disrupted mutant of Pseudozyma tsukubaensis.

The basidiomycetous yeast genus Pseudozyma produce large amounts of mannosylerythritol lipids (MELs), which are biosurfactants. A few Pseudozyma strains produce mono-acylated MEL as a minor compound using excess glucose as the sole carbon source. Mono-acylated MEL shows higher hydrophilicity than di-acylated MEL and has great potential for aqueous applications. Recently, the gene cluster involved in the MEL biosynthesis pathway was identified in yeast. Here, we generated an acyltransferase (PtMAC2) deletion strain of P. tsukubaensis 1E5 with uracil auxotrophy as a selectable marker. A PtURA5-mutant with a frameshift mutation in PtURA5 was generated as a uracil auxotroph of strain 1E5 by ultraviolet irradiation on plate medium containing 5-fluoro-orotic acid (5-FOA). In the mutant, PtMAC2 was replaced with a PtURA5 cassette containing the 5' untranslated region (UTR) (2000 bp) and 3' UTR (2000 bp) of PtMAC2 by homologous recombination, yielding strain ΔPtMAC2. Based on TLC and NMR analysis, we found that ΔPtMAC2 accumulates MEL acylated at the C-2' position of the mannose moiety. These results indicate that PtMAC2p catalyzes acylation at the C-3' position of the mannose of MEL.

Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment.

The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single-stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice-derived P. antarctica strain GB-4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine-5'-phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB-4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR-amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil-auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 µg DNA and a gene-targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley & Sons, Ltd.

Enhanced production of a diastereomer type of mannosylerythritol lipid-B by the basidiomycetous yeast Pseudozyma tsukubaensis expressing lipase genes from Pseudozyma antarctica.

Basidiomycetous yeasts in the genus Pseudozyma are known to produce extracellular glycolipids called mannosylerythritol lipids (MELs). Pseudozyma tsukubaensis produces a large amount of MEL-B using olive oil as the sole carbon source (> 70 g/L production). The MEL-B produced by P. tsukubaensis is a diastereomer type of MEL-B, which consists of 4-O-ß-D-mannopyranosyl-(2R,3S)-erythritol as a sugar moiety, in contrast to the conventional type of MELs produced by P. antarctica, which contain 4-O-ß-D mannopyranosyl-(2S,3R)-erythritol. In this study, we attempted to increase the production of the diastereomer type of MEL-B in P. tsukubaensis 1E5 by introducing the genes encoding two lipases, PaLIPAp (PaLIPA) and PaLIPBp (PaLIPB) from P. antarctica T-34. Strain 1E5 expressing PaLIPA exhibited higher lipase activity than the strain possessing an empty vector, which was used as a negative control. Strains of 1E5 expressing PaLIPA or PaLIPB showed 1.9- and 1.6-fold higher MEL-B production than the negative control strain, respectively, and oil consumption was also accelerated by the introduction of these lipase genes. MEL-B production was estimated using time course analysis in the recombinant strains. Strain 1E5 expressing PaLIPA produced 37.0 ± 1.2 g/L of MEL-B within 4 days of cultivation, whereas the strain expressing an empty vector produced 22.1 ± 7.5 g/L in this time. Overexpression of PaLIPA increased MEL-B production by P. tsukubaensis strain 1E5 from olive oil as carbon source by more than 1.7-fold.

High-level recombinant protein production by the basidiomycetous yeast Pseudozyma antarctica under a xylose-inducible xylanase promoter.

Yeast host-vector systems are useful tools for the production of recombinant proteins. Here, we report the construction of a new high-level expression plasmid pPAX1-neo for the basidiomycetous yeast, Pseudozyma antarctica. pPAX1-neo harbours a xylose-inducible expression cassette under control of the xylanase promoter and terminator of P. antarctica T-34, a selection cassette of neomycin/G418 with an Escherichia coli neomycin resistance gene under control of the homocitrate synthase promoter of strain T-34, and an autonomously replicating sequence fragment of Ustilago maydis (UARS). Biodegradable plastic (BP)-degrading enzymes of P. antarctica JCM10317 (PaE) and Paraphoma-related fungal strain B47-9 (PCLE) were used as reporter proteins and inserted into pPAX1-neo, resulting in pPAX1-neo::PaCLE1 and pPAX1-neo::PCLE, respectively. Homologous and heterologous BP-degrading enzyme production of transformants of P. antarctica T-34 were detected on agar plates containing xylose and emulsified BP. Recombinant PaE were also produced by transformants of other Pseudozyma strains including Pseudozyma aphidis, Pseudozyma rugulosa, and Pseudozyma tsukubaensis. To improve the stability of transformed genes in cells, the UARS fragment was removed from linearized pPAX1-neo::PaCLE1 and integrated into the chromosome of the P. antarctica strain, GB-4(0), which was selected as a PaE producer in xylose media. Two transformants, GB-4(0)-X14 and X49, had an 11-fold higher activity compared with the wild type strain in xylose-containing liquid media. By xylose fed-batch cultivation using a 3-L jar fermentor, GB-4(0)-X14 produced 73.5 U mL(-1) of PaE, which is 13.4-fold higher than that of the wild type strain GB-4(0), which produced 5.5 U mL(-1) of PaE.

Selective formation of mannosyl-L-arabitol lipid by Pseudozyma tsukubaensis JCM16987.

To develop a structural homolog of mannosylerythritol lipids (MELs), Pseudozyma tsukubaensis JCM16987 (known to be a specific producer of the diastereomer type of mono-acetylated MEL (MEL-B)) was cultivated in medium containing 4 % (w/v) olive oil as the primary carbon source and 4 % L-arabitol as the supplemental sugar alcohol. Based on thin-layer chromatography (TLC), the glycolipid extract showed two major spots corresponding to MEL-B and an unknown glycolipid (GL1). Based on high-performance liquid chromatography after acid hydrolysis, GL1 from the L-arabitol culture showed two primary peaks identical to mannose and arabitol using the sugar analysis column, and one peak identical to L-arabitol was detected using the chiral resolution column. Based on NMR analysis, GL1 was identified as mono-acetylated mannosyl-L-arabitol lipid (MLAL-B) consisting of mannose, with L-arabitol as the sugar moiety. The observed critical micelle concentration (CMC) and surface tension at the CMC (γCMC) of MLAL-B were 1.2 × 10(-5) M and 32.8 mN/m, which were significantly higher than MEL-B (CMC = 3.1 × 10(-6) M and γcmc = 26.1 mN/m). Furthermore, based on a water-penetration scan, MLAL-B efficiently formed lamellar phase (Lα) and myelins at a broad concentration range. Thus, the present glycolipid showed higher hydrophilicity and/or water solubility and increased our understanding of environmentally advanced biosurfactants.

Isolation and characterization of bacterial strains with the ability to utilize high concentrations of levulinic acid, a platform chemical from inedible biomass.

Nineteen levulinic acid (LA)-utilizing bacteria were isolated from environmental samples. Following examination of the use of 80 g/L LA by some isolated strains, Brevibacterium epidermidis LA39-2 consumed 62.6 g/L LA following 8 days incubation. The strain also utilized both 90 and 100 g/L LA, with consumption ratio of 84.3 and 53.3%, respectively, after 10 days incubation.

Selective production of two diastereomers of disaccharide sugar alcohol, mannosylerythritol by Pseudozyma yeasts.

Mannosylerythritol (ME) is the hydrophilic backbone of mannosylerythritol lipids as the most promising biosurfactants produced by different Pseudozyma yeasts, and has been receiving attention as a new sugar alcohol. Different Pseudozyma yeasts were examined for the sugar alcohol production using glucose as the sole carbon source. P. hubeiensis KM-59 highly produced a conventional type of ME, i.e., 4-O-ß-D-mannopyranosyl-D-erythritol (4-ME). Interestingly, P. tsukubaensis KM-160 produced a diastereomer of 4-ME, i.e., 1-O-ß-D-mannopyranosyl-D-erythritol (1-ME). In shake flask culture with 200 g/l of glucose, strain KM-59 produced 4-ME at a yield of 33.2 g/l (2.2 g/l/day of the productivity), while strain KM-160 produced 1-ME at 30.0 g/l (2.0 g/l/day). Moreover, the two strains were found to produce ME from glycerol; the maximum yields of 4-ME and 1-ME from 200 g/l of glycerol were 16.1 g/l (1.1 g/l/day) and 15.8 g/l (1.1 g/l/day), respectively. The production of 1-ME as the new diastereomer was further investigated in fed batch culture using a 5-l jar-fermenter. Compared to the flask culture, strain KM-160 gave three times higher productivity of 1-ME at 38.0 g/l (6.3 g/l/day) from glucose and at 31.1 g/l (3.5 g/l/day) from glycerol, respectively. This is the first report on the selective production of two diastereomers of ME, and should thus facilitate the functional development and application of the disaccharide sugar alcohol in the food and relative industries.

Mannosylerythritol lipids secreted by phyllosphere yeast Pseudozyma antarctica is associated with its filamentous growth and propagation on plant surfaces.

The biological function of mannosylerythritol lipids (MELs) towards their producer, Pseudozyma antarctica, on plant surfaces was investigated. MEL-producing wild-type strain and its MEL production-defective mutant strain (ΔPaEMT1) were compared in terms of their phenotypic traits on the surface of plastic plates, onion peels, and fresh leaves of rice and wheat. While wild-type cells adhering on plastic surfaces and onion peels changed morphologically from single cells to elongated ones for a short period of about 4 h and 1 day, respectively, ΔPaEMT1 cells did not. Microscopic observation of both strains grown on plant leaf surfaces verified that the wild type colonized a significantly bigger area than that of ΔPaEMT1. However, when MELs were exogenously added to the mutant cells on plant surfaces, their colonized area became enlarged. High-performance liquid chromatography analysis revealed a secretion of higher amount of MELs in the cell suspension incubated with wheat leaf cuttings compared to that in the suspension without cuttings. Transcriptional analysis by real-time reverse transcriptase PCR verified that the expression of erythritol/mannose transferase gene and MELs transporter gene of P. antarctica increased in the cells inoculated onto wheat leaves at 4, 6, and 8 days of incubation, indicating a potential of P. antarctica to produce MELs on the leaves. These findings demonstrate that MELs produced by P. antarctica on plant surfaces could be expected to play a significant role in fungal morphological development and propagation on plant surfaces.

Production of D-arabitol from raw glycerol by Candida quercitrusa.

To promote the effective use of raw glycerol (a by-product of biodiesel production), 110 yeast strains that produce D-arabitol from glycerol were isolated from environmental samples. Among them, strain 17-2A was an effective D-arabitol producer in the presence of 250 g/l glycerol and was identified as Candida quercitrusa based on morphological, physicochemical, and phylogenetic analyses. C. quercitrusa type strain NBRC1022 produced the greatest quantity of D-arabitol (41.7 g/l) when the ability to produce D-arabitol from raw glycerol was compared among C. quercitrusa 17-2A and its phylogenetically related strains in flask culture. Under optimized culture conditions, strain NBRC1022 produced D-arabitol at a concentration of 58.2 g/l after a 7-day cultivation in 250 g/l glycerol, 6 g/l yeast extract, and 2 g/l CaCl2. The culture conditions were further investigated with raw glycerol using a jar fermenter; the concentration of D-arabitol reached 67.1 g/l after 7 days and 85.1 g/l after 10 days, respectively, which corresponded to 0.40 g/g of glycerol. To our knowledge, the present D-arabitol yield from glycerol is higher than reported previously using microbial production.

Biosurfactant-producing yeasts widely inhabit various vegetables and fruits.

The isolation of biosurfactant-producing yeasts from food materials was accomplished. By a combination of a new drop collapse method and thin-layer chromatography, 48 strains were selected as glycolipid biosurfactant producers from 347 strains, which were randomly isolated from various vegetables and fruits. Of the producers, 69% were obtained from vegetables of the Brassica family. Of the 48 producers, 15 strains gave relatively high yields of mannosylerythritol lipids (MELs), and were identified as Pseudozyma yeasts. These strains produced MELs from olive oil at yields ranging from 8.5 to 24.3 g/L. The best yield coefficient reached 0.49 g/g as to the carbon sources added. Accordingly, MEL producers were isolated at high efficiency from various vegetables and fruits, indicating that biosurfactant producers are widely present in foods. The present results should facilitate their application in the food and related industries.