Identification of a gene cluster for biosynthesis of mannosylerythritol lipids in the basidiomycetous fungus Ustilago maydis

Many microorganisms produce surface-active substances that enhance the availability of water-insoluble substrates. Although many of these biosurfactants have interesting potential applications, very little is known about their biosynthesis. The basidiomycetous fungus Ustilago maydis secretes large amounts of mannosylerythritol lipids (MELs) under conditions of nitrogen starvation. We recently described a putative glycosyltransferase, Emt1, which is essential for MEL biosynthesis and whose expression is strongly induced by nitrogen limitation. We used DNA microarray analysis to identify additional genes involved in MEL biosynthesis. Here we show that emt1 is part of a gene cluster which comprises five open reading frames. Three of the newly identified proteins, Mac1, Mac2, and Mat1, contain short sequence motifs characteristic for acyl- and acetyltransferases. Mutational analysis revealed that Mac1 and Mac2 are essential for MEL production, which suggests that they are involved in the acylation of mannosylerythritol. Deletion of mat1 resulted in the secretion of completely deacetylated MELs, as determined by mass spectrometry. We overexpressed Mat1 in Escherichia coli and demonstrated that this enzyme acts as an acetyl coenzyme A-dependent acetyltransferase. Remarkably, Mat1 displays relaxed regioselectivity and is able to acetylate mannosylerythritol at both the C-4 and C-6 hydroxyl groups. Based on these results, we propose a biosynthesis pathway for the generation of mannosylerythritol lipids in U. maydis.     

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.     

Production of Glycolipid Biosurfactants,Mannosylerythritol Lipids,Using Sucrose by Fungal and Yeast Strains,and Their Interfacial Properties

Glycolipid biosurfactants, mannosylerythritol lipids (MELs), were produced from glucose and sucrose without vegetable oils. Pseudozyma antarctica JCM 10317, Ustilago maydis NBRC 5346, U. scitaminea NBRC 32730, and P. siamensis CBS 9960 produced mainly MEL-A, MEL-A, MEL-B, and MEL-C respectively. The sucrose-derived MELs showed excellent interfacial properties: low critical micelle concentration as well as that of oil-derived MELs.     

Formation and analysis of mannosylerythritol lipids secreted by Pseudozyma aphidis

Pseudozyma aphidis DSM 70725 was found to be a novel producer of mannosylerythritol lipids (MELs). The MELs were quantified by HPLC. Glucose as carbon source for precultivation supported growth well. By contrast, at concentrations >30 g l^–1 in preculture, subsequent MEL formation in the main culture with soybean oil as sole carbon source was reduced. The type of substrate supply considerably influenced MEL formation. High concentrations of soybean oil (80 ml l^–1) at init favored the production process when compared to a stepwise (20 ml l^–1) addition. Mannose or erythritol were suitable second carbon sources that enhanced the MEL yield with soybean oil as preferred primary substrate. After 10 days, a maximum yield of 75 g l^–1 was attained during shake-flask cultivation. Biofuel (rapeseed oil methyl ester) also resulted in high yields of MEL, but glucose reduced the MEL yield. Analysis by GC-MS showed that all fatty acids contained in MEL and derived from soybean oil or related methyl ester were degraded by C2-units to differing extents. The surface (water/air) and interfacial (water/hexadecane) tension of the MELs produced from different carbon sources were reduced to a minimum of 26.2 mN m^–1 and 1 mN m^–1, respectively.     

Fed-batch bioreactor production of mannosylerythritol lipids secreted by Pseudozyma aphidis

Two strains of Pseudozyma aphidis, DSM 70725 and DSM 14930, were used for the bioreactor production of mannosylerythritol lipids (MELs). Foam formation interfered substantially with the cultivation process. Soybean oil was simultaneously employed as both carbon source and anti-foam agent. Primary MEL formation occurred after nitrate limitation. After a first short time-period of nitrate limitation and further nitrate addition, MELs were secreted in spite of nitrate excess. The sedimentation of MEL-enriched beads indicated enhanced product formation. Maximum yield, productivity and yield coefficient of 165 g l⁻¹, 13.9 g l⁻¹ day⁻¹ and 0.92 g g⁻¹ were achieved using strain DSM 14930 with additional substrate-feeding (glucose, sodium nitrate, yeast extract) and a foam-controlled soybean oil supply.     

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.     

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.     

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.     

Mannosylerythritol lipids: a review

Mannosylerythritol lipids (MELs) are surface active compounds that belong to the glycolipid class of biosurfactants (BSs). MELs are produced by Pseudozyma sp. as a major component while Ustilago sp. produces them as a minor component. Although MELs have been known for over five decades, they recently regained attention due to their environmental compatibility, mild production conditions, structural diversity, self-assembling properties and versatile biochemical functions. In this review, the MEL producing microorganisms, the production conditions, their applications, their diverse structures and self-assembling properties are discussed. The biosynthetic pathways and the regulatory mechanisms involved in the production of MEL are also explained here.     

Genetic analysis of biosurfactant production in Ustilago maydis

The dimorphic basidiomycete Ustilago maydis produces large amounts of surface-active compounds under conditions of nitrogen starvation. These biosurfactants consist of derivatives of two classes of amphipathic glycolipids. Ustilagic acids are cellobiose lipids in which the disaccharide is O-glycosidically linked to 15,16-dihydroxyhexadecanoic acid. Ustilipids are mannosylerythritol lipids derived from acylated beta-d-mannopyranosyl-d-erythritol. Whereas the chemical structure of these biosurfactants has been determined, the genetic basis for their biosynthesis and regulation is largely unknown. Here we report the first identification of two genes, emt1 and cyp1, that are essential for the production of fungal extracellular glycolipids. emt1 is required for mannosylerythritol lipid production and codes for a protein with similarity to prokaryotic glycosyltransferases involved in the biosynthesis of macrolide antibiotics. We suggest that Emt1 catalyzes the synthesis of mannosyl-d-erythritol by transfer of GDP-mannose. Deletion of the gene cyp1 resulted in complete loss of ustilagic acid production. Cyp1 encodes a cytochrome P450 monooxygenase which is highly related to a family of plant fatty acid hydroxylases. Therefore we assume that Cyp1 is directly involved in the biosynthesis of the unusual 15,16-dihydroxyhexadecanoic acid. We could show that mannosylerythritol lipid production is responsible for hemolytic activity on blood agar, whereas ustilagic acid secretion is required for long-range pheromone recognition. The mutants described here allow for the first time a genetic analysis of glycolipid production in fungi.     

Characterization of new glycolipid biosurfactants,tri-acylated mannosylerythritol lipids,produced by <Emphasis Type="Italic">Pseudozyma</Emphasis> yeasts

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by Pseudozyma yeasts. They show not only the excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from soybean oil by P. antarctica and P. rugulosa, some new extracellular glycolipids (more hydrophobic than the previously reported di-acylated MELs) were found in the culture medium. The most hydrophobic one was identified as 1-O-alka(e)noyl-4-O-[(4′,6′-di-O-acetyl-2′,3′-di-O-alka(e)noyl)-β-d-mannopyranosyl]-d-erythritol, namely tri-acylated MEL. Others were tri-acylated MELs bearing only one acetyl group. The tri-acylated MEL could be prepared by the lipase-catalyzed esterification of a di-acylated MEL with oleic acid implying that the new glycolipids are synthesized from di-acylated MELs in the culture medium containing the residual fatty acids.     

A basidiomycetous yeast, Pseudozyma crassa, produces novel diastereomers of conventional mannosylerythritol lipids as glycolipid biosurfactants

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These compounds show not only excellent surface-active properties, but also versatile biochemical actions. During a survey of new MEL producers, we found that a basidiomycetous yeast, Pseudozyma crassa, extracellularly produces three glycolipids. When glucose and oleic acid were used as the carbon source, the total amount of glycolipids reached approximately 4.6 g/L in the culture medium. The structures of these glycolipids were similar to those of well-known MEL-A, -B, and -C, respectively. Very interestingly, in all the present glycolipids, the configuration of the erythritol moiety was entirely opposite to that of conventional MELs. The present glycolipids were identified to have the carbohydrate structure of 4-O-β-d-mannopyranosyl-(2R,3S)-erythritol, stereochemically different from 4-O-β-d-mannopyranosyl-(2S,3R)-erythritol of conventional MELs. Furthermore, these new glycolipids possessed both short-chain acids (C₂ or C₄) and long-chain acids (C₁₄, C₁₆, or C₁₈) on the mannose moiety. The major component of the present glycolipids clearly showed different interfacial and biological properties, compared to conventional MELs comprising two medium-chain acids on the mannose moiety. Accordingly, the novel MEL diastereomers produced by P. crassa should provide us with different glycolipid functions, and facilitate a broad range of applications of MELs.     

Physiological differences in the formation of the glycolipid biosurfactants,mannosylerythritol lipids,between <Emphasis Type="Italic">Pseudozyma antarctica</Emphasis> and <Emphasis Type="Italic">Pseudozyma aphidis</Emphasis>

Vegetable oil is the usual carbon source for the production of biosurfactants (BS), mannosylerythritol lipids (MEL). To simplify the procedures of BS production and recovery, we investigated the extracellular production of MEL from water-soluble carbon sources instead of vegetable oils by using two representative yeast strains. The formation of extracellular MEL from glucose was confirmed by thin layer chromatography (TLC) and HPLC analysis. On glucose cultivation, pure MEL were easily prepared by only solvent extraction of the culture medium, different from the case of soybean oil cultivation. The fatty acid profile of the major MEL produced from glucose was similar to that produced from soybean oil based on GC–MS analysis. The resting cells of Pseudozyma antarctica T-34 produced MEL by feeding of glucose only and gave a yield of 12 g l⁻¹. In contrast, Pseudozyma aphidis ATCC 32657 gave no MEL from glucose. Moreover, the extracellular lipase activities were detected at high levels during the cultivation regardless of the carbon sources. These results indicate that all the biosynthesis pathways for MEL in P. antarctica T-34 should constitutively function. In conclusion, P. antarctica T-34 thus has potential for BS production from glucose.     

A basidiomycetous yeast, Pseudozyma tsukubaensis, efficiently produces a novel glycolipid biosurfactant. The identification of a new diastereomer of mannosylerythritol lipid-B

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These compounds show not only excellent surface-active properties but also versatile biochemical activities. In the course of MEL production by Pseudozyma tsukubaensis, we found an unusual MEL that had a different carbohydrate structure from that of conventional MELs. The carbohydrate structure was identified as 1-O-beta-D-mannopyranosyl-D-erythritol, and the MEL was confirmed to be 1-O-beta-(2',3'-di-O-alka(e)noyl-6'-O-acetyl-D-mannopyranosyl)-D-erythritol. Interestingly, the configuration of the erythritol moiety in the present MEL was opposite to that of the known MEL-B, 4-O-beta-(2',3'-di-O-alka(e)noyl-6'-O-acetyl-D-mannopyranosyl)-D-erythritol, and to that of all MELs hitherto reported. The present MEL should thus provide different interfacial and biochemical properties compared to conventional MELs.     

Structural characterization and surface-active properties of a new glycolipid biosurfactant,mono-acylated mannosylerythritol lipid,produced from glucose by <Emphasis Type="Italic">Pseudozyma antarctica</Emphasis>

Mannosylerythritol lipids (MELs), which are glycolipid biosurfactants produced by Pseudozyma yeasts, show not only excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from glucose as the sole carbon source, P. antarctica was found to produce unknown glycolipids more hydrophilic than conventional “di-acylated MELs,” which have two fatty acyl esters on the mannose moiety. Based on a detailed characterization, the most hydrophilic one was identified as 4-O-(3′-O-alka(e)noyl-β-d-mannopyranosyl)-d-erythritol namely, “mono-acylated MEL.” The mono-acylated MEL reduced the surface tension of water to 33.8 mN/m at a critical micelle concentration (CMC) of 3.6 × 10⁻⁴ M, and its hydrophilic–lipophilic balance was tentatively calculated to be 12.15. The observed CMC was 100-fold higher than that of the MELs hitherto reported. Interestingly, of the yeast strains of the genus Pseudozyma, only P. antarctica and P. parantarctica gave the mono-acylated MEL from glucose, despite a great diversity of di-acylated MEL producers in the genus. These strains produced MELs including the mono-acylated one at a rate of 20–25%. From these results, the new MEL is likely to have great potential for use in oil-in-water-type emulsifiers and washing detergents because of its higher water solubility compared to conventional MELs and will thus contribute to facilitating a broad range of applications for the environmentally advanced surfactants.     

Discovery of Pseudozyma rugulosa NBRC 10877 as a novel producer of the glycolipid biosurfactants, mannosylerythritol lipids, based on rDNA sequence

The search for a novel producer of glycolipid biosurfactants, mannosylerythritol lipids (MEL) was undertaken based on the analysis of ribosomal DNA sequences on the yeast strains of the genus Pseudozyma. Pseudozyma rugulosa NBRC 10877 was found to produce a large amount of glycolipids from soybean oil. Fluorescence microscopic observation also demonstrated that the strain significantly accumulates polar lipids in the cells. The structure of the glycolipids produced by the strain was analyzed by ¹H and ¹³C nuclear magnetic resonance and gas chromatography–mass spectrometry methods, and was determined to be the same as MEL produced by Pseudozyma antarctica, a well-known MEL producer. The major fatty acids of the present MEL consisted of C8 and C10 acids. Based on high performance liquid chromatography, the composition of the produced MEL was as follows: MEL-A (68%), MEL-B (12%), and MEL-C (20%). To enhance the production of MEL by the novel strain, factors affecting the production, such as carbon and nitrogen sources, were further examined. Soybean oil and sodium nitrate were the best carbon and nitrogen sources, respectively. The supplementation of a MEL precursor, such as erythritol, drastically enhanced the production yield from soybean oil at a rate of 70 to 90%. Under the optimal conditions in a shake culture, a maximum yield, productivity, and yield coefficient (on a weight basis to soybean oil supplied) of 142 g l⁻¹, 5.0 g l⁻¹ day⁻¹, and 0.5 g g⁻¹ were achieved by intermittent feeding of soybean oil and erythritol using the yeast.     

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

Applied Microbiology and Biotechnology - The basidiomycetous yeast genus Pseudozyma produce large amounts of mannosylerythritol lipids (MELs), which are biosurfactants. A few Pseudozyma strains...     

Effects of biosurfactants produced by Candida antarctica on the biodegradation of petroleum compounds

The effects of biosurfactants on the biodegradation of petroleum compounds were investigated. Candida antarctica T-34 could produce extracellular biosurfactant mannosylerythritol lipids (MELs) when it was cultured in vegetable oil. In addition, in our previous study, it was found that this strain could also produce a new type of biosurfactant while it grew on n-undecane (C₁₁H₂₄), and the biosurfactant was named as BS-UC. In flask culture of Candida antarctica, the addition of BS-UC could improve the biodegradation rate of some n-alkanes (e.g. 90.2% for n-decane, 90.2% for n-undecane, 89.0% for dodecane), a mixture of n-alkanes (82.3%) and kerosene (72.5%). By comparing the effects of the biosurfactants BS-UC and MEL and chemical surfactants on the biodegradation of crude oil, it was found that biosurfactants could be used to enhance the degradation of petroleum compounds instead of chemical surfactants. In a laboratory scale immobilized bioreactor, the addition of biosurfactant improved not only the emulsification of kerosene in simulated wastewater but also its biodegradation rate. The highest degradation rate of kerosene by addition of MEL and BS-UC reached 87 and 90% at 15 h, respectively. The results showed that the biosurfactant BS-UC was highly promising for work on biodegradation of hydrophobic contaminants.