Production Of Mannosylerythritol Lipids Research Articles

Growth Behavior of Selected Ustilaginaceae Fungi Used for Mannosylerythritol Lipid (MEL) Biosurfactant Production - Evaluation of a Defined Culture Medium.

Fungi of the Ustilaginaceae family are a promising source for many biotechnologically relevant products. Among these, mannosylerythritol lipid (MEL) biosurfactants have drawn a special interested over the last decades due to their manifold application possibilities. Nevertheless, there is still a knowledge gap regarding process engineering of MEL production. As an example, no reports on the use of a chemically defined culture medium have been published yet, although such a defined medium might be beneficial for scaling-up the production process toward industrial scale. Our aim therefore was to find a mineral medium that allows fast biomass growth and does not negatively affect the successive MEL production from plant oils. The results showed comparable growth performance between the newly evaluated mineral medium and the established yeast extract medium for all seven investigated Ustilaginaceae species. Final biomass concentrations and specific growth rates of 0.16-0.25 h–1 were similar for the two media. Oxygen demand was generally higher in the mineral medium than in the yeast extract medium. It was shown that high concentrations of vitamins and trace elements were necessary to support the growth. Increasing starting concentrations of the media by a factor of 10 resulted in proportionally increasing final biomass concentrations and up to 2.3-times higher maximum growth rates for all species. However, it could also lead to oxygen limitation and stagnant growth rates when too high medium concentrations were used, which was observed for Ustilago siamensis and Moesziomyces aphidis. Successive MEL production from rapeseed oil was effectively shown for 4 out of 7 organisms when the mineral medium was used for cell growth, and it was even enhanced for two organisms, M. aphidis and Pseudozyma hubeiensis pro tem., as compared to the established yeast extract medium. Conversion of rapeseed oil into MEL was generally improved when higher biomass concentrations were achieved during the initial growth phase, indicating a positive relationship between biomass concentration and MEL production. Overall, this is the first report on the use of a chemically defined mineral medium for the cell growth of Ustilaginaceae fungi and successive MEL production from rapeseed oil, as an alternative to the commonly employed yeast extract medium.

Production of Mannosylerythritol Lipids (MELs) to be Used as Antimicrobial Agents Against S. aureus ATCC 6538

Antimicrobial resistance (AMR) is a current major health issue, both for the high rates of resistance observed in bacteria that cause common infections and for the complexity of the consequences of AMR. Pathogens like Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Mycobacterium tuberculosis among others are clear examples of antibiotic-resistant threats. Biosurfactants have recently emerged as a potential new generation of anti-adhesive and anti-biofilm agents; mannosylerythritol lipids (MELs) are biosurfactants produced by a range of fungi. A range of structural variants of MELs can be formed and the proportion of each isomer in the fermentation depends on the yeast used, the carbon substrate used for growth and the duration of the fermentation. In order to allow assessment of the possible functions of MELs as antimicrobial molecules, small quantities of MEL were produced by controlled fermentation. Fermentations of the yeast Pseudozyma aphidis using rapeseed oil as a carbon source yielded up to 165 gMELs/kgSubstrate. The MELs formed by this strain was a mixture of MEL-A, MEL-B, MEL-C and MEL-D. The MELs produced were tested against S. aureus ATCC 6538 on pre-formed biofilm and on co-incubation biofilm experiments on silicone discs; showing a disruption of biomass, reduction of the biofilm metabolic activity and a bacteriostatic/bactericidal effect confirmed by a release of oxygen uptake p_{{{text{O}}_{2} }}, the reduction of citrate synthase activity and scanning electron microscopy. The results show that MELs are promising antimicrobial molecules for biomedical technological applications that could be studied in detail in large-scale systems and in conjunction with animal tissue models.

High-Quality Draft Genome Sequence and Annotation of the Basidiomycete Yeast Sporisorium graminicola CBS10092, a Producer of Mannosylerythritol Lipids.

The basidiomycete Sporisorium graminicola (formally Pseudozyma graminicola) strain CBS10092 was originally isolated from an herbaceous plant in Russia. It is a known producer of mannosylerythritol lipids (MELs), the main component being MEL-C. Here, we present the 19.9-Mb draft genome sequence, which comprises 6,602 genes, including those encoding the MEL biosynthetic pathway.

Elucidation of substrate specificities of decorating enzymes involved in mannosylerythritol lipid production by cross-species complementation

Mannosylerythritol lipids (MELs) are surface active molecules produced by many basidiomycetous fungi. MELs consist of a mannosylerythritol disaccharide, which is acylated with short and medium chain fatty acids at the mannosyl moiety. A gene cluster composed of five genes is required for MEL biosynthesis. Here we show that the plant pathogenic fungus Ustilago hordei secretes these glycolipids under nitrogen starvation conditions. In contrast to MELs produced by the closely related fungus Ustilago maydis those secreted by U. hordei are mostly mono-acetylated and contain a different mixture of acyl groups. Cross-species complementation between these fungi revealed that these differences result from different catalytic activities of the acetyltransferase Mat1 and the acyltransferases Mac1 and Mac2. U. maydis mat1 mutants expressing the homologous mat1 gene from U. hordei produced mostly mono-acetylated variants and lack di-acetylated MELs normally produced by U. maydis. Furthermore, we determined that the acyltransferase Mac1 acylates the mannosylerythritol moiety at position C2 while Mac2 acylates C3. The identification of decorating enzymes with different substrate specificities will allow the tailor-made production of novel subsets of MELs.

Cover Image, Volume 94, Issue 4

The cover image is based on the Research Article Production of mannosylerythritol lipids from lignocellulose hydrolysates: tolerance thresholds of Moesziomyces antarcticus to inhibitors by Marisa V. Santos et al., DOI: 10.1002/jctb.5853. Image Credit: Marisa V. Santos. Thanks to FCT grant CRUISE (PTDC/AAG‐TEC/0696/2014). image

Production and characterization of a new glycolipid, mannosylerythritol lipid, from waste cooking oil biotransformation by Pseudozyma aphidis ZJUDM34.

Mannosylerythritol lipids (MELs) are glycolipids possessing unique biosurfactant properties. However, the prices of substrates currently used for MEL formation caused its unsustainable commercial development. Waste cooking oil poses significant ecological and economical problems. Thus, the production of MELs from used waste cooking oil using the biotransformation route is one of the better alternatives to utilize it efficiently and economically. This work aims at the production of MELs using waste cooking oil instead of soybean oil and evaluating the major characteristics and compositions of MELs. The titers reached 61.50 g/L by the optimization of culture medium, higher than the counterpart (10.25 ± 0.32 g/L) of the nonoptimized medium. MELs exhibited good surface activity and better performance in contrast to MELs grown on soybean oil. The water phase behavior of MEL‐A was also evaluated. The process showed higher productivity of MELs with better surface activity and application stability than the conventional process using soybean oil. The findings of this study imply that the use of inexpensive fermentation substrates associated with straightforward downstream processing is expected to have a great impact on the economy of MEL production.

Production of mannosylerythritol lipids from lignocellulose hydrolysates: tolerance thresholds of Moesziomyces antarcticus to inhibitors

AbstractBACKGROUNDMoesziomyces antarcticus is an efficient producer of mannosylerythritol lipids (MEL), a biosurfactant with a wide range of potential applications. The use of lignocellulosic biomass can contribute to sustainable MEL production. While lignocellulosic sugars (e.g. D‐glucose and D‐xylose) can be converted to MEL, the required pretreatment of lignocellulosic biomass releases by‐products that are potentially inhibitory for yeasts. A design of experiment (DoE) was performed to evaluate the effect of furfural, acetate and formate on M. antarcticus and their capacity to produce MEL from lignocellulose hydrolysates.RESULTSFurfural presented a higher inhibitory effect on MEL production than the two dissociated weak acids. The DoE was developed for 7‐days D‐glucose cultures with inhibitors up to 0.7 g L−1 furfural, 2.0 g L−1 acetate and 1.7 g L−1 formate. The model equations relate D‐glucose consumption rate and the production of cell biomass, lipids and MEL with the concentration of inhibitors. For example, MEL titre is reduced by 25% when 0.08 g L−1 furfural, 0.29 g L−1 acetate and 0.25 g L−1 formate were used. The model was validated in D‐glucose and used to study MEL production in D‐glucose and D‐glucose/D‐xylose mixtures. The use of D‐xylose showed a positive effect on MEL production in the presence of inhibitors since similar MEL titres were attained with (0.08 g L−1 furfural, 0.29 g L−1 acetate and 0.30 g L−1 formate) or without inhibitors when using a D‐glucose/D‐xylose mixture.CONCLUSIONThis study provides insight to the conditions required by M. antarcticus for MEL production from lignocellulosic hydrolysates and points towards further process and strain development requirements. © 2018 Society of Chemical Industry

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.

Selective production of deacetylated mannosylerythritol lipid, MEL-D, by acetyltransferase disruption mutant of Pseudozyma hubeiensis

Mannosylerythritol lipids (MELs) are produced by several smut fungi of the Ustilaginaceae family; they are promising microbial biosurfactants and have excellent surface-active and self-assembling properties. Pseudozyma hubeiensis is a candidate for abundant MEL production and produces large amounts of 4-O-[(4'-mono-O-acetyl-2',3'-di-O-alkanoyl)-β-d-mannopyranosyl]-meso-erythritol (MEL-C). An acetyltransferase disruption mutant of P.hubeiensis, SY62-MM36, was obtained to selectively produce deacetylated 4-O-[(2',3'-di-O-alkanoyl)-β-d-mannopyranosyl]-meso-erythritol (MEL-D), and the structures of the products were determined. Lower mobility of major spots of the mutant on silica gel thin-layer chromatography verified its more hydrophilic nature than that of wild-type MEL-A, B, and C. Structural analyses confirmed the product to be MEL-D, which comprises acyl chains of caproic acid (C6:0), capric acid (C10:0), and lauric acid (C12:0). The critical micelle concentration (CMC) and the surface tension (γCMC) of the MEL-D were 2.0×10-5M and 29.7mN/m, respectively. SY62-MM36 also produced a minor product that was estimated as triacylated MEL-D. The triacylated MEL-D had a CMC of 3.5×10-5M and a γCMC of 29.6mN/m. In water, MEL-D formed a lamella liquid crystal phase over a broad range of concentrations. By fed-batch cultivation, the mutant produced 91.6±6.3g/L of MEL-D for 7 days.

A novel approach for the production and purification of mannosylerythritol lipids (MEL) by Pseudozyma tsukubaensis using cassava wastewater as substrate

P. tsukubaensis is a yeast-like microorganism that synthesized the biosurfactant mannosylerythritol lipids-B (MEL-B). Production cost can be one of the drawbacks of biosurfactants production. Therefore the development of efficient and cost effective purification strategies and the use of by-products in the culture medium could serve as important strategies to reduce overall process cost. The aim of this work was to evaluate the production of MEL using cassava wastewater, a hydrophilic medium composed of a low-cost substrate which is a by-product of cassava processing, followed by foam fractionation and ultrafiltration of MEL. Cassava wastewater proved to be a feasible culture medium for P. tsukubaensis and MEL-B production as the yield (1.26 g L−1) was similar to that reported by others using water-soluble carbon sources (up to 2 g L−1). Interestingly ultrafiltration with 100 kDa MWCO membranes (using 20 mL centrifugal devices) led to the purification of MEL-B in one step since ≈ 80% of MEL was recovered, while more than 95% of proteins were found in the permeate. The scale up of the ultrafiltration (up to 500 mL) using a cross flow filtration unit led to very similar results. Overall the ultrafiltration led to a threefold increase in MEL purity in terms of protein (at both scales). The chemical characterization by NMR confirmed the production of MEL-B homologue and also the production of a second stereoisomer ≈9%, while the CG-MS and MALDI-TOFMS analysis confirmed the main fatty acids within the structure of MEL-B (C8:0 and 12:0 and C8:0 and C14:1). Therefore, the process developed here was found to be a good alternative to the conventional production of MEL which uses synthetic culture medium, solvent extraction (ethyl acetate) and column chromatography (silica) for its purification.

Characterization, enhancement and modelling of mannosylerythritol lipid production by fungal endophyte Ceriporia lacerate CHZJU.

The glycolipid biosurfactants mannosylerythritol lipids (MELs) attract great attention for their biodegradability, super emulsifying properties and versatile bioactivities. In this study, the MEL deriving from Ceriporia lacerate CHZJU was identified as MEL-A, and its critical micelle concentration and emulsifying activities were assessed. To examine the production of MELs from Ceriporia lacerate, a Plackett-Burman design and response surface methodology were used to optimize the culture nutrients. The optimal medium contains 1g/L yeast extract, 1.5g/L (NH4)2SO4, 0.5g/L KH2PO4, 0.04g/L CaCl2, 119.6mL/L soybean oil and 0.297g/L MnSO4. Subsequent verification revealed that the yield of MELs was 129.64±5.67g/L. Furthermore, an unstructured kinetic model was developed for mycelial growth, MEL production and substrate utilization. This work provides insight into Ceriporia lacerate CHZJU, a predominant fungus producing MEL-A. Optimization using response surface methodology enhanced the mannosylerythritol lipid recovery. Importantly, we developed fermentation kinetic modelling for mannosylerythritol lipid production.

The transcriptomic profile of Pseudozyma aphidis during production of mannosylerythritol lipids.

The basidiomycetous fungus Pseudozyma aphidis is able to convert vegetable oils to abundant amounts of the biosurfactant mannosylerythritol lipid (MEL) with a unique product pattern of MEL-A, MEL-B, MEL-C, and MEL-D. To investigate the metabolism of MEL production, we analyzed the transcriptome of P. aphidis DSM 70725 under MEL-inducing and non-inducing conditions using deep sequencing. Following manual curation of the previously described in silico gene models based on RNA-Seq data, we were able to generate an experimentally verified gene annotation containing 6347 genes. Using this database, our expression analysis revealed that only four of the five cluster genes required for MEL synthesis were clearly induced by the presence of soybean oil. The acetyltransferase encoding gene PaGMAT1 was expressed on a much lower level, which may explain the secretion of MEL with different degrees of acetylation in P. aphidis. In parallel to MEL synthesis, microscopic observations showed morphological changes accompanied by expression of genes responsible for cell development, indicative of a coregulation between MEL synthesis and cell morphology. In addition a set of transcription factors was identified which may be responsible for regulation of MEL synthesis and cell development. The upregulation of genes required for nitrogen metabolism and other assimilation processes indicate additional metabolic pathways required under the MEL-inducing conditions used. We also searched for a conserved gene cluster for cellobiose lipids (CL) but only found seven genes with limited homology distributed over the genome. However, we detected characteristic TLC spots in fermentations using P. aphidis DSM 70725, indicative of CL secretion.

Mannosylerythritol lipids: production and applications.

Mannosylerythritol lipids (MELs) are a glycolipid class of biosurfactants produced by a variety yeast and fungal strains that exhibit excellent interfacial and biochemical properties. MEL-producing fungi were identified using an efficient screening method for the glycolipid production and taxonomical classification on the basis of ribosomal RNA sequences. MEL production is limited primarily to the genus Pseudozyma, with significant variability among the MEL structures produced by each species. Outside of Pseudozyma, one recently isolated strain, Ustilago scitaminea, has been shown to exhibit abundant MEL-B production from sugarcane juice. Structural analyses of these compounds suggest a role for MELs in numerous cosmetic applications. MELs act as effective topical moisturizers and can repair damaged hair. Furthermore, these compounds have been shown to exhibit both protective and healing activities, to activate fibroblasts and papilla cells, and to act as natural antioxidants. In this review, we provide a brief summary of MEL research over the past few decades, focusing on the identification of MEL-producing fungi, the structural characterization of MELs, the use of alternative compounds as a primary carbon source, and the use of these compounds in cosmetic applications.

Direct xylan conversion into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma antarctica PYCC 5048T

Mannosylerythritol lipids (MEL) are glycolipid biosurfactants, produced by Pseudozyma spp., with increasing commercial interest. While MEL can be produced from d-glucose and d-xylose, the direct conversion of the respective lignocellulosic polysaccharides, cellulose and xylan, was not reported yet. The ability of Pseudozyma antarctica PYCC 5048T and Pseudozyma aphidis PYCC 5535T to use cellulose (Avicel®) and xylan (beechwood) as carbon and energy source has been assessed along with their capacity of producing cellulolytic and hemicellulolytic enzymes, toward a consolidated bioprocess (CBP) for MEL production. The yeasts assessed were neither able to grow in medium containing Avicel® nor produce cellulolytic enzymes under the conditions tested. On contrary, both yeasts were able to efficiently grow in xylan, but MEL production was only detected in P. antarctica PYCC 5048T cultures. MEL titers reached 1.3g/l after 10 days in batch cultures with 40g/l xylan, and 2.0g/l in fed-batch cultures with xylan feeding (additional 40g/l) at day 4. High levels of xylanase activities were detected in xylan cultures, reaching 47–62U/ml (31–32U/mg) at 50°C, and still exhibiting more than 10U/ml under physiological temperature (28°C). Total β-xylosidase activities, displayed mainly as wall-bounded and extracellular activity, accounted for 0.154 and 0.176U/ml in P. antarctica PYCC 5048T and P. aphidis PYCC 5535T cultures, respectively. The present results demonstrate the potential of Pseudozyma spp. for using directly a fraction of lignocellulosic biomass, xylan, and combining in the same bioprocess the production of xylanolytic enzymes with MEL production.

Conversion of cellulosic materials into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma spp. under SHF and SSF processes.

BackgroundMannosylerythritol lipids (MEL) are glycolipids with unique biosurfactant properties and are produced by Pseudozyma spp. from different substrates, preferably vegetable oils, but also sugars, glycerol or hydrocarbons. However, solvent intensive downstream processing and the relatively high prices of raw materials currently used for MEL production are drawbacks in its sustainable commercial deployment. The present work aims to demonstrate MEL production from cellulosic materials and investigate the requirements and consequences of combining commercial cellulolytic enzymes and Pseudozyma spp. under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes.ResultsMEL was produced from cellulosic substrates, Avicel® as reference (>99% cellulose) and hydrothermally pretreated wheat straw, using commercial cellulolytic enzymes (Celluclast 1.5 L® and Novozyme 188®) and Pseudozyma antarctica PYCC 5048T or Pseudozyma aphidis PYCC 5535T. The strategies included SHF, SSF and fed-batch SSF with pre-hydrolysis. While SSF was isothermal at 28°C, in SHF and fed-batch SSF, yeast fermentation was preceded by an enzymatic (pre-)hydrolysis step at 50°C for 48 h. Pseudozyma antarctica showed the highest MEL yields from both cellulosic substrates, reaching titres of 4.0 and 1.4 g/l by SHF of Avicel® and wheat straw (40 g/l glucan), respectively, using enzymes at low dosage (3.6 and 8.5 FPU/gglucan at 28°C and 50°C, respectively) with prior dialysis. Higher MEL titres were obtained by fed-batch SSF with pre-hydrolysis, reaching 4.5 and 2.5 g/l from Avicel® and wheat straw (80 g/l glucan), respectively.ConclusionsThis work reports for the first time MEL production from cellulosic materials. The process was successfully performed through SHF, SSF or Fed-batch SSF, requiring, for maximal performance, dialysed commercial cellulolytic enzymes. The use of inexpensive lignocellulosic substrates associated to straightforward downstream processing from sugary broths is expected to have a great impact in the economy of MEL production for the biosurfactant market, inasmuch as low enzyme dosage is sufficient for good systems performance.

Production of glycolipid biosurfactants, mannosylerythritol lipids, from pentoses and d-glucose/d-xylose mixtures by Pseudozyma yeast strains

The pentose-assimilating capacity of yeasts from the genus Pseudozyma, P. antarctica PYCC 5048T, P. aphidis PYCC 5535T and P. rugulosa PYCC 5537T, was exploited towards the production of mannosylerythritol lipids (MEL), a glycolipid with biosurfactant properties. The three strains tested were able to grow on d-xylose and l-arabinose with similar maximum specific growth rates to those estimated on d-glucose (around 0.2h−1). The highest MEL titres (4.8–5.4g/l) and yields (0.11–0.14g/g) from d-xylose were found in P. antarctica PYCC 5048T, which presented similar values to those estimated on d-glucose and on d-xylose/d-glucose mixtures. P. rugulosa PYCC 5537T showed a pattern of sugar conversion into MEL similar to P. antarctica, but at 40% lower titres. P. aphidis PYCC 5535T presented lower MEL titres from d-xylose (1.2g/l) than from d-glucose (3.4g/l). Nitrate supply increased sugar consumption rate and, when accompanied by d-glucose or d-xylose feeding, additional biomass production. In this case, sugar was completely consumed before sugar feeding at day 7, but not when feeding is performed at day 4. Higher MEL titres where obtained for the later condition reaching 7.3g/l and 5.8g/l, in fed-batch cultures with glucose and xylose, respectively.

Peroxisomes contribute to biosynthesis of extracellular glycolipids in fungi.

Many microorganisms secrete surface-active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis-targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross-talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell.

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.

Production of mannosylerythritol lipids and their application in cosmetics

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants abundantly produced by different basidiomycetous yeasts such as Pseudozyma, and show not only excellent interfacial properties but also versatile biochemical actions. These features of MELs make their application in new technology areas possible. Recently, the structural and functional variety of MELs was considerably expanded by advanced microbial screening methods. Different types of MELs bearing different hydrophilic and hydrophobic parts have been reported. The genes responsible for MEL biosynthesis were identified, and their genetic study is now in progress, aiming to control the chemical structure. The excellent properties leading to practical cosmetic ingredients, i.e., moisturization of dry skin, repair of damaged hair, activation of fibroblast and papilla cells and antioxidant and protective effects in skin cells, have been demonstrated on the yeast glycolipid biosurfactants. In this review, the current status of research and development on MELs, particularly the commercial application in cosmetics, is described.

Accumulation of cellobiose lipids under nitrogen-limiting conditions by two ustilaginomycetous yeasts,Pseudozyma aphidisandPseudozyma hubeiensis

Some basidiomycetous yeast strains extracellularly produce cellobiose lipids (CLs), glycolipid biosurfactants which have strong fungicidal activity. The representative CL producer Ustilago maydis produces CLs together with the other glycolipids, mannosylerythritol lipids (MELs); the preference of the two glycolipids is affected considerably by the nitrogen source. To develop new CL producers, 12 MEL producers were cultured under the nitrogen-limited conditions. Pseudozyma aphidis and Pseudozyma.hubeiensis were characterized as new CL producers. CL production was induced on three strains, P.aphidis, Pseudozyma graminicola, and P.hubeiensis under these conditions. The putative homologous genes of U.maydis cyp1, which encodes a P450 monooxygenase, essential for CL biosynthesis, were partially amplified from their genomic DNA. The nucleotide sequences of the gene fragments from P.hubeiensis and P.aphidis shared identities with U.maydis cyp1 of 99% and 78%, respectively. Furthermore, all of the deduced translation products are tightly clustered in the phylogenic tree of the monooxygenase. These results suggest that the genes involved with CL biosynthesis must be widely distributed in the basidiomycetous fungi as well as the MEL biosynthesis genes, and thus, the genus Pseudozyma has great potential as a biosurfactant producer.