Simultaneous bioethanol distillery wastewater treatment and xylanase production by the phyllosphere yeast Pseudozyma antarctica GB-4(0).

Bioethanol production using lignocellulosic biomass generates lignocellulosic bioethanol distillery wastewater (LBDW) that contains a large amount of xylose, making it a potential inexpensive source of xylose for biomaterials production. The main goal of this study was the production of useful enzymes from LBDW during treatment of this wastewater. In this study, we found that xylose strongly induced two yeast strains, Pseudozyma antarctica T-34 and GB-4(0), to produce novel xylanases, PaXynT and PaXynG, respectively. The nucleotide sequence of PaXynT [accession No. DF196774 (GAC73192.1)], obtained from the genome database of strain T-34 using its N-terminal amino acid sequence, was 91% identical to that of PaXynG (accession No. AB901085), and the deduced amino acid sequence is 98% identical. The specific activities of the purified PaXynT and PaXynG were about 52 U/mg. The optimal pH and temperature for both enzymes' activities were 5.2 and 50°C, respectively. They hydrolyzed xylan to xylose and neither had ß-xylosidase (EC 3.2.1.37) activity, indicating that they are endo-ß-xylanases (EC 3.2.1.8). With these results, we expect that PaXyns can be employed in saccharizing lignocellulosic biomass materials for the production of useful products just like other endoxylanases. After 72 h of LBDW fed-batch cultivation using a jar-fermentor, strain GB-4(0) produced 17.3 U/ml (corresponding to about 0.3 g/l) of PaXynG and removed 63% of dissolved organic carbon and 87% of dissolved total phosphorus from LBDW. These results demonstrate the potential of P. antarctica for xylanase production during LBDW treatment.

Purification, characterization, and cloning of the gene for a biodegradable plastic-degrading enzyme from Paraphoma-related fungal strain B47-9.

Paraphoma-related fungal strain B47-9 secreted a biodegradable plastic (BP)-degrading enzyme which amounted to 68 % (w/w) of the total secreted proteins in a culture medium containing emulsified poly(butylene succinate-co-adipate) (PBSA) as sole carbon source. The gene for this enzyme was found to be composed of an open reading frame consisting of 681 nucleotides encoding 227 amino acids and two introns. Southern blot analysis showed that this gene exists as a single copy. The deduced amino acid sequence suggested that this enzyme belongs to the cutinase (E.C.3.1.1.74) family; thus, it was named P araphoma-related fungus cutinase-like enzyme (PCLE). It degraded various types of BP films, such as poly(butylene succinate), PBSA, poly(butylene adipate-co-terephthalate), poly(ε-caprolactone), and poly(DL-lactic acid). It has a molecular mass of 19.7 kDa, and an optimum pH and temperature for degradation of emulsified PBSA of 7.2 and 45 °C, respectively. Ca(2+) ion at a concentration of about 1.0 mM markedly enhanced the degradation of emulsified PBSA.

Treatment of, and Candida utilis biomass production from shochu wastewater; the effects of maintaining a low pH on DOC removal and feeding cultivation on biomass production.

Shochu wastewater (SW; alcoholic distillery wastewater) contains large amounts of organic compounds (25,000 - 60,000 COD mg/L), nitrogen (1,000 - 6,000 T-N mg/L), and phosphorus (500 - 1,000 T-P mg/L). Despite its high nutrient content, SW is highly perishable, which limits its utilization for animal feed and fertilizer. Therefore, SW is mainly treated by methane fermentation. On the other hand, a feed yeast, Candida utilis, can utilize various organic compounds and be utilized as a yeast extract source and animal feed. We previously bred a mutant, C. utilis UNA1, that accumulates a large amount of nitrogen. Here, we investigated the use of C. utilis UNA1 to treat highly concentrated SW. With fed-batch cultivation using a 5-L jar fermenter, controlling pH at 5.0 with H2SO4, 62.9% of DOC, 38.4% of DTN, and 44.5% of DTP were stably removed from non-diluted barley shochu wastewater (BSW), and about 16.7 kg of freeze-dried yeast biomass was obtained. The yeast sludge biomass generated from BSW contains about 60% crude protein. Furthermore, using H2SO4 to control pH increased the sulfur content of wastewater, which increased the methionine composition of yeast sludge biomass.

Evidence for DNA cleavage caused directly by a transfer RNA-targeting toxin.

The killer yeast species Pichiaacaciae produces a heteromeric killer protein, PaT, that causes DNA damage and arrests the cell cycle of sensitive Saccharomyces cerevisiae in the S phase. However, the mechanism by which DNA damage occurs remains elusive. A previous study has indicated that Orf2p, a subunit of PaT, specifically cleaves an anticodon loop of an S. cerevisiae transfer RNA (tRNA(Gln)mcm5s2UUG). This finding raised a question about whether the DNA damage is a result of the tRNA cleavage or whether Orf2p directly associates with and cleaves the genomic DNA of sensitive yeast cells. We showed that Orf2p cleaves genomic DNA in addition to cleaving tRNA in vitro. This DNA cleavage requires the same Orf2p residue as that needed for tRNA cleavage, His299. The expression of Orf2p, in which His299 was substituted to alanine, abolished the cell cycle arrest of the host cell. Moreover, the translation impairment induced by tRNA cleavage enabled Orf2p to enter the nucleus, thereby inducing histone phosphorylation.

Genome-wide screening to study breeding methods to improve the nitrogen accumulation ability of yeast without gene recombinant techniques.

To remove nitrogen efficiently from high-concentration organic wastewater, we studied breeding methods using Saccharomyces cerevisiae as a model yeast with improved nitrogen accumulation ability. By DNA microarray analysis under various nitrogen concentrations with two nitrogen sources (peptone and L-asparagine), we obtained 295 commonly overexpressed (over 2-fold) genes and 283 commonly underexpressed (under one-half) genes under nitrogen-starvation conditions. We speculated that overexpression or underexpression recombination of some of these genes might enhance nitrogen uptake. Because a complete collection of nonessential gene deletion strains had been created, we investigated the nitrogen accumulation profiles of underexpressed gene deletion strains. From 256 nonessential gene deletion strains, three (URE2, SNO1, and AVT3) were selected. Strain SUD2 (ure2Δ::kanMX4) improved by 1.2-fold total nitrogen per cell (TN/OD660) as compared to the parent strain, S288c. Positive selection of methylamine-resistant mutants to obtain URE2 mutants was useful for improving nitrogen accumulation ability without recombinant techniques.

Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica: surface plasmon resonance and atomic force microscopy study.

Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica JCM10317 (PaE) was analyzed by surface plasmon resonance (SPR). The adsorption of PaE and the degradation rate for polyester films were quantitatively monitored by a positive and negative SPR signal shifts, respectively. The decrease in SPR signal and the erosion depth of amorphous poly(L-lactide) (a-PLLA) film measured by atomic force microscopy (AFM) had a linear relationship, and the weight loss was estimated from the AFM data combined with a density of a-PLLA film. Furthermore, SPR sensorgrams for various polyester films showed that degradation rate of poly(ε-caprolactone) and poly(butylene succinate-co-adipate) which contain C6 units was higher than that of other polyesters such as poly(butylene succinate) and a-PLLA. These results suggest that C6 is the preferred chain length as substrates for PaE.

Rapid and simple colorimetric assay for detecting the enzymatic degradation of biodegradable plastic films.

We developed a rapid and simple method for evaluating the degradation of solid biodegradable plastics (BPs). Dye-containing BP films were used as substrates and the release of dye caused by the degradation of BPs was confirmed by a color change in the enzyme solution after a reaction time of 24 h.

Biodegradable plastic-degrading enzyme from Pseudozyma antarctica: cloning, sequencing, and characterization.

Pseudozyma antarctica JCM 10317 exhibits a strong degradation activity for biodegradable plastics (BPs) such as agricultural mulch films composed of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA). An enzyme named PaE was isolated and the gene encoding PaE was cloned from the strain by functional complementation in Saccharomyces cerevisiae. The deduced amino acid sequence of PaE contains 198 amino acids with a predicted molecular weight of 20,362.41. High identity was observed between this sequence and that of cutinase-like enzymes (CLEs) (61-68%); therefore, the gene encoding PaE was named PaCLE1. The specific activity of PaE against emulsified PBSA was 54.8±6.3 U/mg. In addition to emulsified BPs, PaE degraded solid films of PBS, PBSA, poly(ε-caprolactone), and poly(lactic acid).

Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis.

Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca(2+) or Mg(2+) at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 °C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(ε-caprolactone), and poly(lactic acid).

Degradation of biodegradable plastic mulch films in soil environment by phylloplane fungi isolated from gramineous plants.

To improve the biodegradation of biodegradable plastic (BP) mulch films, 1227 fungal strains were isolated from plant surface (phylloplane) and evaluated for BP-degrading ability. Among them, B47-9 a strain isolated from the leaf surface of barley showed the strongest ability to degrade poly-(butylene succinate-co-butylene adipate) (PBSA) and poly-(butylene succinate) (PBS) films. The strain grew on the surface of soil-mounted BP films, produced breaks along the direction of hyphal growth indicated that it secreted a BP-degrading enzyme, and has directly contributing to accelerating the degradation of film. Treatment with the culture filtrate decomposed 91.2 wt%, 23.7 wt%, and 14.6 wt% of PBSA, PBS, and commercially available BP polymer blended mulch film, respectively, on unsterlized soil within 6 days. The PCR-DGGE analysis of the transition of soil microbial community during film degradation revealed that the process was accompanied with drastic changes in the population of soil fungi and Acantamoeba spp., as well as the growth of inoculated strain B47-9. It has a potential for application in the development of an effective method for accelerating degradation of used plastics under actual field conditions.

Specific phase arrest of cell cycle restores cell viability against tRNA cleavage by killer toxin.

Zymocin and PaT are killer toxins that induce cell cycle arrest of sensitive yeast cells in G1 and S phase, respectively. Recent studies have revealed that these two toxins cleave specific tRNAs, indicating that the cell growth impairment is due to the tRNA cleavage. Additionally, we have previously shown that the active domain of colicin D (D-CRD), which also cleaves specific Escherichia coli tRNAs, statically impairs growth when expressed in yeast cells. To verify that phase-specific cell cycle arrest is also induced by the expression of D-CRD, D-CRD and the subunits of zymocin and PaT that have tRNA cleaving activity were expressed in yeast cells and cell cycle status was analyzed. Our results indicate that phase-specific arrest does not commonly occur by tRNA cleavage, and it saves the cell viability. Furthermore, the extent of protein synthesis impairment may determine the phase specificity of cell cycle arrest.

Phyllosphere yeasts rapidly break down biodegradable plastics.

The use of biodegradable plastics can reduce the accumulation of environmentally persistent plastic wastes. The rate of degradation of biodegradable plastics depends on environmental conditions and is highly variable. Techniques for achieving more consistent degradation are needed. However, only a few microorganisms involved in the degradation process have been isolated so far from the environment. Here, we show that Pseudozyma spp. yeasts, which are common in the phyllosphere and are easily isolated from plant surfaces, displayed strong degradation activity on films made from poly-butylene succinate or poly-butylene succinate-co-adipate. Strains of P. antarctica isolated from leaves and husks of paddy rice displayed strong degradation activity on these films at 30°C. The type strain, P. antarctica JCM 10317, and Pseudozyma spp. strains from phyllosphere secreted a biodegradable plastic-degrading enzyme with a molecular mass of about 22 kDa. Reliable source of biodegradable plastic-degrading microorganisms are now in our hands.

Silage produces biofuel for local consumption.

BACKGROUND: In the normal process of bioethanol production, biomass is transported to integrated large factories for degradation to sugar, fermentation, and recovery of ethanol by distillation. Biomass nutrient loss occurs during preservation and degradation. Our aim was to develop a decentralized ethanol production system appropriate for farm or co-operative level production that uses a solid-state fermentation method for producing bio-ethanol from whole crops, provides cattle feed, and produces no wastes. The idea is to incorporate traditional silage methods with simultaneous saccharification and fermentation. Harvested, fresh biomass is ensiled with biomass-degrading enzymes and yeast. Multiple parallel reactions for biomass degradation and ethanol and lactic acid production are induced in solid culture in hermetically sealed containers at a ranch. After fermentation, ethanol is collected on site from the vapor from heated fermented products. RESULTS: The parallel reactions of simultaneous saccharification and fermentation were induced efficiently in the model fermentation system. In a laboratory-scale feasibility study of the process, 250 g of freshly harvested forage rice with 62% moisture was treated with 0.86 filter paper units/g dry matter (DM) of cellulase and 0.32 U/g DM of glucoamylase. After 20 days of incubation at 28°C, 6.4 wt.% of ethanol in fresh matter (equivalent to 169 g/kg DM) was produced. When the 46 wt.% moisture was gathered as vapor from the fermented product, 74% of the produced ethanol was collected. Organic cellular contents (such as the amylase and pronase degradable fractions) were decreased by 63% and organic cell wall (fiber) content by 7% compared to silage prepared from the same material. CONCLUSIONS: We confirmed that efficient ethanol production is induced in nonsterilized whole rice plants in a laboratory-scale solid-state fermentation system. For practical use of the method, further study is needed to scale-up the fermentation volume, develop an efficient ethanol recovery method, and evaluate the fermentation residue as an actual cattle feed.

Ethanol production from ensiled rice straw and whole-crop silage by the simultaneous enzymatic saccharification and fermentation process.

Silage production from rice straw and whole-plant forage paddy rice is increasing in Japan because of decrease in rice consumption. One potential use for this silage is bioethanol production. In this study, we analyzed the effectiveness of three different commercially available cellulases at saccharification of sun-dried rice straw, ensiled rice straw, and rice whole-crop silage (WCS). Furthermore, the ethanol productivity of the simultaneous saccharification and ethanol fermentation process (SSF) from the same plant substrates was analyzed. Among the three kinds of cellulases tested (Novozymes NS50013, Genencor GC220, and Acremonium cellulase), Acremonium cellulase showed the highest ethanol production for the three plant substrates, and the WCS produced the highest ethanol level. Analysis of the enzymatic degradation activity of the cellulases revealed that Acremonium cellulase contained remarkably high glucoamylase and pectinase side activities relative to the other cellulase preparations. The addition of glucoamylase and pectinase to the other two cellulases significantly increased ethanol productivity to levels observed for the Acremonium cellulase preparation, which showed little enhanced performance with the addition of the same enzymes. Finally, we tested whether milling and sterilization had an effect on ethanol production and found that sterilized silage produced higher ethanol levels but that the milling process had no significant effect. These results show that (i) silage made from whole-plant rice can be used for bioethanol production and (ii) the proper selection and combination of commercially available enzymes can make SSF more cost efficient by removing the need for a pre-treatment step.

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica.

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface-active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino-terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene-disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low-temperature tolerance of the yeast.

Mannosylinositol phosphorylceramide is a major sphingolipid component and is required for proper localization of plasma-membrane proteins in Schizosaccharomyces pombe.

In Saccharomyces cerevisiae, three classes of sphingolipids contain myo-inositol--inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)(2)C]. No fission yeast equivalent of Ipt1p, the inositolphosphotransferase that synthesizes M(IP)(2)C from MIPC, has been found in the Schizosaccharomyces pombe genome. Analysis of the sphingolipid composition of wild-type cells confirmed that MIPC is the terminal and most abundant complex sphingolipid in S. pombe. Three proteins (Sur1p, Csg2p and Csh1p) have been shown to be involved in the synthesis of MIPC from IPC in S. cerevisiae. The S. pombe genome has three genes (SPAC2F3.01, SPCC4F11.04c and SPAC17G8.11c) that are homologues of SUR1, termed imt1(+), imt2(+) and imt3(+), respectively. To determine whether these genes function in MIPC synthesis in S. pombe, single and multiple gene disruptants were constructed. Single imt disruptants were found to be viable. MIPC was not detected and IPC levels were increased in the triple disruptant, indicating that the three SUR1 homologues are involved in the synthesis of MIPC. GFP-tagged Imt1p, Imt2p and Imt3p localized to Golgi apparatus membranes. The MIPC-deficient mutant exhibited pleiotropic phenotypes, including defects in cellular and vacuolar morphology, and in localization of ergosterols. MIPC seemed to be required for endocytosis of a plasma-membrane-localized amino acid transporter, because sorting of the transporter from the plasma membrane to the vacuole was severely impaired in the MIPC-deficient mutant grown under nitrogen-limiting conditions. These results suggest that MIPC has multiple functions not only in the maintenance of cell and vacuole morphology but also in vesicular trafficking in fission yeast.

Cellular and transcriptional responses of yeast to the cleavage of cytosolic tRNAs induced by colicin D.

Colicin D is a plasmid-encoded antibacterial protein that specifically cleaves the anticodon loops of four Escherichia coli tRNA(Arg) species. Here, we report that the catalytic domain of colicin D, which is expressed in Saccharomyces cerevisiae, impairs cell growth by cleaving specific tRNAs. DNA microarray analysis revealed that mating-related genes were upregulated, while genes involved in a range of metabolic processes were downregulated, thereby impairing cell growth. The pheromone-signalling pathway was activated only in alpha cells by tRNA cleavage, which was not observed in 'a' cells or diploid cells. On the basis of these results and on the recent identification of two killer toxins that cleave specific tRNAs, the relationship between tRNA depletion and the resultant cellular response is discussed.

Rice exonuclease-1 homologue, OsEXO1, that interacts with DNA polymerase lambda and RPA subunit proteins, is involved in cell proliferation.

Exonuclease 1, a class III member of the RAD2 nuclease family, is a structure-specific nuclease involved in DNA metabolism (replication, repair and recombination). We have identified a homologue to Exonuclease-1 from rice (Oryza sativa L. cv. Nipponbare) and have designated it O. sativa Exonuclease-1 (OsEXO1). The open reading frame of OsEXO1 encodes a predicted product of 836 amino acid residues with a molecular weight of 92 kDa. Two highly conserved nuclease domains (XPG-N and XPG-I) are present in the N-terminal region of the protein. OsEXO1-sGFP fusion protein transiently overexpressed in the onion epidermal cells localized to the nucleus. The transcript of OsEXO1 is highly expressed in meristematic tissues and panicles. Inhibition of cell proliferation by removal of sucrose from the medium or by the addition of cell cycle inhibitors decreased OsEXO1 expression. Functional complementation assays using yeast RAD2 member null mutants demonstrates that OsEXO1 is able to substitute for ScEXO1 and ScRAD27 functions. Yeast two-hybrid analysis shows that OsEXO1 interacted with rice DNA polymerase lambda (OsPol lambda), the 70 kDa subunit b of rice replication protein A (OsRPA70b), and the 32 kDa subunit 1 of rice replication protein A (OsRPA32-1). Irradiation of UV-B induces OsEXO1 expression while hydrogen peroxide treatment represses it. These results suggest that OsEXO1 plays an important role in both cell proliferation and UV-damaged nuclear DNA repair pathway under dark conditions.

Rice shaker potassium channel OsKAT1 confers tolerance to salinity stress on yeast and rice cells.

We screened a rice (Oryza sativa L. 'Nipponbare') full-length cDNA expression library through functional complementation in yeast (Saccharomyces cerevisiae) to find novel cation transporters involved in salt tolerance. We found that expression of a cDNA clone, encoding the rice homolog of Shaker family K(+) channel KAT1 (OsKAT1), suppressed the salt-sensitive phenotype of yeast strain G19 (Deltaena1-4), which lacks a major component of Na(+) efflux. It also suppressed a K(+)-transport-defective phenotype of yeast strain CY162 (Deltatrk1Deltatrk2), suggesting the enhancement of K(+) uptake by OsKAT1. By the expression of OsKAT1, the K(+) contents of salt-stressed G19 cells increased during the exponential growth phase. At the linear phase, however, OsKAT1-expressing G19 cells accumulated less Na(+) than nonexpressing cells, but almost the same K(+). The cellular Na(+) to K(+) ratio of OsKAT1-expressing G19 cells remained lower than nonexpressing cells under saline conditions. Rice cells overexpressing OsKAT1 also showed enhanced salt tolerance and increased cellular K(+) content. These functions of OsKAT1 are likely to be common among Shaker K(+) channels because OsAKT1 and Arabidopsis (Arabidopsis thaliana) KAT1 were able to complement the salt-sensitive phenotype of G19 as well as OsKAT1. The expression of OsKAT1 was restricted to internodes and rachides of wild-type rice, whereas other Shaker family genes were expressed in various organs. These results suggest that OsKAT1 is involved in salt tolerance of rice in cooperation with other K(+) channels by participating in maintenance of cytosolic cation homeostasis during salt stress and thus protects cells from Na(+).

Characterization of the genus Pseudozyma by the formation of glycolipid biosurfactants, mannosylerythritol lipids.

Pseudozyma antarctica is one of the best producers of the glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface-active properties but also versatile biochemical actions. In order to obtain a variety of producers, all the species of the genus were examined for their production of MELs from soybean oil. Pseudozyma fusiformata, P. parantarctica and P. tsukubaensis were newly identified to be MEL producers. Of the strains tested, P. parantarctica gave the best yield of MELs (30 g L(-1)). The obtained yield corresponded to those of P. antarctica, P. aphidis and P. rugulosa, which are known high-level MEL producers. Interestingly, P. parantarctica and P. fusiformata produced mainly 4-O-[(4',6'-di-O-acetyl-2',3'-di-O-alkanoyl)-beta-d-mannopyranosyl]-meso-erythritol (MEL-A), whereas P. tsukubaensis produced mainly 4-O-[(6'-mono-O-acetyl-2',3'-di-O-alkanoyl)-beta-d-mannopyranosyl]-meso-erythritol (MEL-B). Consequently, six of the nine species clearly produced MELs. Based on the MEL production pattern, the nine species seemed to fall into four groups: the first group produces large amounts of MELs; the second produces both MELs and other biosurfactants; the third mainly produces MEL-B; and the fourth is non-MEL-producing. Thus, MEL production may be an important taxonomic index for the Pseudozyma yeasts.