Production Of Mannosylerythritol Lipids Research Articles

Past, present and future of glycolipids from Ustilaginaceae – A review on cellobiose lipids and mannosylerythritol lipids

AbstractThe glycolipids cellobiose lipids (CL) and mannosylerythritol lipids (MEL) are biosurfactants mainly synthesized by microorganisms of the Ustilaginaceae family. They have a large structural diversity, varying in their sugar moieties and the attached fatty acids, resulting in a prospectively broad range of applications. This literature review provides a detailed overview of known microbial producers of CL and MEL, and their respective metabolic pathways that result in different molecular structures. Further, current advances in the aerobic fermentative synthesis of the glycolipids and their purification methods are illustrated. All influencing factors identified to date with regard to the fermentation are highlighted in detail: For CL synthesis usually hydrophilic carbon sources are used as substrate, whereas hydrophobic carbon sources are usually metabolized to MEL. Nitrogen limitation was described as a major trigger for glycolipid synthesis and an acidic pH range was favored for increased CL production. An overview of applied fermentation parameters in recent publications (e.g., substrate‐concentrations, feeding approaches) demonstrates the future potential of CL and MEL production optimization. Foaming during fermentation is either combated or exploited by foam fractionation as the first purification step. The current purification processes focus on solvent extractions and chromatography in the laboratory scale and a need for development was identified for future scale‐up. Finally, environmental hotspots during CL and MEL production are presented and future optimization potentials are highlighted.

Promising Application, Efficient Production, and Genetic Basis of Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs) are a class of glycolipids that have been receiving increasing attention in recent years due to their diverse biological activities. MELs are produced by certain fungi and display a range of bioactivities, making them attractive candidates for various applications in medicine, agriculture, and biotechnology. Despite their remarkable qualities, industrial-scale production of MELs remains a challenge for fungal strains. Excellent fungal strains and fermentation processes are essential for the efficient production of MELs, so efforts have been made to improve the fermentation yield by screening high-yielding strains, optimizing fermentation conditions, and improving product purification processes. The availability of the genome sequence is pivotal for elucidating the genetic basis of fungal MEL biosynthesis. This review aims to shed light on the applications of MELs and provide insights into the genetic basis for efficient MEL production. Additionally, this review offers new perspectives on optimizing MEL production, contributing to the advancement of sustainable biosurfactant technologies.

A review on the upstream production and downstream purification of mannosylerythritol lipids.

Biosurfactants are natural compounds with remarkable surface-active properties that may offer an eco-friendly alternative to conventional surfactants. Among them, mannosylerythritol lipids (MELs) stand out as an intriguing example of a glycolipid biosurfactant. MELs have been used in a variety of sectors for various applications, and are currently commercially produced. Industrially, they are used in the pharmaceutical, cosmetic, food, and agricultural industries, based on their ability to reduce surface tension and enhance emulsification. However, despite their utility, their production is comparatively limited industrially. From a bioprocessing standpoint, two areas of interest to improve the production process are upstream production and downstream (separation and purification) product recovery. The former has seen a significant amount of research, with researchers investigating several production factors: the microbial species or strain employed, the producing media composition, and the production strategy implemented. Improvement and optimization of these are key to scale-up the production of MELs. On the other hand, the latter has seen comparatively limited work presented in the literature. For the most part traditional separation techniques have been employed. This systematic review presents the production and purification methodologies used by researchers by comprehensively analyzing the current state-of-the-art with regards the production, separation, and purification of MELs. By doing so, the review presents different possible approaches, and highlights some potential areas for future work by identifying opportunities for the commercialization of MELs.

Substrates of Opposite Polarities and Downstream Processing for Efficient Production of the Biosurfactant Mannosylerythritol Lipids from Moesziomyces spp.

Biosurfactants can replace fossil-driven surfactants with positive environmental impacts, owing to their low eco-toxicity and high biodegradability. However, their large-scale production and application are restricted by high production costs. Such costs can be reduced using renewable raw materials and facilitated downstream processing. Here, a novel strategy for mannosylerythritol lipid (MEL) production explores the combination of hydrophilic and hydrophobic carbon sources sideways with a novel downstream processing strategy, based on nanofiltration technology. Co-substrate MEL production by Moesziomyces antarcticus was threefold higher than using D-glucose with low levels of residual lipids. The use of waste frying oil instead of soybean oil (SBO) in co-substrate strategy resulted in similar MEL production. Moesziomyces antarcticus cultivations, using 3.9 M of total carbon in substrates, yields 7.3, 18.1, and 20.1 g/L of MEL, and 2.1, 10.0, and 5.1 g/L of residual lipids, for D-glucose, SBO, and a combination of D-Glucose and SBO, respectively. Such approach makes it possible to reduce the amount of oil used, offset by the equivalent molar increase in D-glucose, improving sustainability and decreasing residual unconsumed oil substrates, facilitating downstream processing. Moesziomyces spp. also produces lipases that broken down the oil and, thus, residual unconsumed oils are in the form of free fatty-acids or monoacylglycerol, which are smaller molecules than MEL. Therefore, nanofiltration of ethyl acetate extracts from co-substrate-based culture broths allows to improve MEL purity (ratio of MEL per total MEL and residual lipids) from 66 to 93% using 3-diavolumes.

Enhanced fermentation of biosurfactant mannosylerythritol lipids on the pilot scale under efficient foam control with addition of soybean oil

Biosurfactant mannosylerythritol lipids (MELs) are green alternatives to chemically synthesized surfactants. However, these biosurfactants typically have a high cost-of-production limiting their adoption for industrial applications. In this study, an optimized fermentation process at pilot scale to furnish MELs with Pseudozyma aphidis DSM 70725 was developed. This involved the optimization of the levels of Fe2+, Fe3+ to improve the fermentation efficacy and the use of soybean oil to reduce the foam generated during fermentative production of MELs. The MELs titer, production rate and the conversion ratio of the carbon source was enhanced to 61 g/L, 0.255 g/L/h and 0.68 g/g, respectively. These optimizations resulted in approximately 100% improvement over regular fermentation processes and are amenable to the large-scale fermentation of MELs employing soybean oil as an antifoam agent to overcome foaming issues.

Production of Mannosylerythritol Lipids Using Oils from Oleaginous Microalgae: Two Sequential Microorganism Culture Approach.

Mannosylerythritol lipids (MELs) are biosurfactants with excellent biochemical properties and a wide range of potential applications. However, most of the studies focusing on MELs high titre production have been relying in the use of vegetable oils with impact on the sustainability and process economy. Herein, we report for the first time MELs production using oils produced from microalgae. The bio-oil was extracted from Neochloris oleoabundans and evaluated for their use as sole carbon source or in a co-substrate strategy, using as an additional carbon source D-glucose, on Moesziomyces spp. cultures to support cell growth and induce the production of MELs. Both Moesziomyces antarcticus and M. aphidis were able to grow and produce MELs using algae-derived bio-oils as a carbon source. Using a medium containing as carbon sources 40 g/L of D-glucose and 20 g/L of bio-oils, Moesziomyces antarcticus and M. aphidis produced 12.47 ± 0.28 and 5.72 ± 2.32 g/L of MELs, respectively. Interestingly, there are no significant differences in productivity when using oils from microalgae or vegetable oils as carbon sources. The MELs productivities achieved were 1.78 ± 0.04 and 1.99 ± 0.12 g/L/h, respectively, for M. antarcticus fed with algae-derived or vegetable oils. These results open new perspectives for the production of MELs in systems combining different microorganisms.

Towards Mannosylerythritol Lipids (MELs) for Bioremediation: Effects of NaCl on M. antarcticus Physiology and Biosurfactant and Lipid Production; Ecotoxicity of MELs

Mannosylerythritol lipids (MELs) are a group of biosurfactants with a wide range of potential applications, due to their excellent tensioactive properties, biocompatibility, and biodegradability. One of the envisioned uses for MELs is in bioremediation in marine and terrestrial environments. However, knowledge regarding their ecotoxicity is limited. The current costs of production for this biosurfactant are too high to make it competitive in the surfactant market. In an effort to facilitate the use of MELs for marine bioremediation purposes, their production using sea water in medium formulation was explored. Cells were exposed to different levels of NaCl during fermentation, and the effects of increased salinity on the cells and their performance was monitored. In addition, cells were briefly exposed to an osmotic shock, by introducing pure NaCl into the broth, to measure their physiological response. Although the overall effect of NaCl in the medium was negative, cells produced more lipases in these stress conditions. Furthermore, the changes triggered by the osmotic shock caused changes in the cell surface and affected their hydrophobicity, reducing the levels of MELs adsorbed to the cells, which in turn led to an increase in formation of MEL-rich beads. Marine-level salinity (3.5%) was found to be sufficient to enable production of MELs in unsterile conditions and inhibited an introduced bacterial contaminant. Finally, the toxicity levels of MELs to a model marine organism and plant model were lower than other biosurfactants and that of a commercial chemical dispersant used for bioremediation.

Efficient production of mannosylerythritol lipids by a marine yeast Moesziomyces aphidis XM01 and their application as self-assembly nanomicelles

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants because of their excellent physicochemical properties, high environmental compatibility, and various biological functions. In this study, a mangrove yeast strain Moesziomyces aphidis XM01 was identified and used for efficient extracellular MEL production. The MEL titer reached 64.5 ± 0.7 g/L at flask level within 7 days with the optimized nitrogen and carbon source of 2.0 g/L NaNO3 and 70 g/L soybean oil. Furthermore, during a 10-L two-stage fed-batch fermentation, the final MEL titer reached 113.6 ± 3.1 g/L within 8 days, with prominent productivity and yield of 14.2 g·L−1·day−1 and 94.6 g/g(glucose and soybean oil). Structural analysis indicated that the produced MELs were mainly MEL-A and its fatty acid profile was composed of only medium-chain fatty acids (C8–C12), especially C10 acids (77.81%). Further applications of this compound were evaluated as one-step self-assembly nanomicelles. The obtained MEL nanomicelles showed good physicochemical stability and antibacterial activity. In addition, using clarithromycin as a model hydrophobic drug, the MEL nanomicelles exhibited high loading capacity and could be used for the controlled and sustained drug release in low-pH environments. Therefore, M. aphidis XM01 is an excellent candidate for efficient MEL production, and the prepared MEL nanomicelles have broad application prospects in the pharmaceutical and cosmetic fields.

Moesziomyces spp. cultivation using cheese whey: new yeast extract-free media, β-galactosidase biosynthesis and mannosylerythritol lipids production.

Mannosylerythritol lipids (MELs) are biosurfactants with excellent biochemical properties and a wide range of potential applications. However, high production costs, low productivity and unsatisfactory scale-up production have hampered commercial adoption. Herein, we report for the first time the β-galactosidase production by Moesziomyces spp. from different sugars (D-galactose, D-glucose and D-lactose), with D-galactose being the best β-galactosidase inducer, with 11.2 and 63.1 IU/mgbiomass, for Moesziomyces aphidis 5535 T and Moesziomyces antarcticus 5048 T, respectively. The production of this enzyme allows to break down D-lactose and thus to produce MEL directly from D-lactose or cheese whey (a cheese industry by-product). Remarkably, when CW was used as sole media component (carbon and mineral source), in combination with waste frying oil, MEL productivities were very close (1.40 and 1.31 gMEL/L/day) to the ones obtained with optimized medium containing yeast extract (1.92 and 1.50 gMEL/gsusbtrate), both for M. antarcticus and M. aphidis. The low-cost, facile and efficient process which generates large amounts of MELs potentiates its industrialization.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13399-022-02837-y.

Non-traditional oils with water-soluble substrate as cell growth booster for the production of mannosylerythritol lipids by Pseudozyma antarctica (ATCC 32657) with their antimicrobial activity

Abstract Among glycolipids, mannosylerythritol lipids (MEL), are mild and environmentally friendly surfactants used in various industrial applications. MELs are produced by biofermentation using non-traditional oils with various water-soluble carbon sources as cell growth booster. This substrate affects the production yield and cost of MEL. In this research work, the non-traditional oils jatropha oil, karanja oil and neem oil were used as new substrates along with glucose, glycerol and honey as new water-soluble substrates. All these oils are new feedstocks for the production of MEL using Pseudozyma antarctica (ATCC 32657). Jatropha oil, karanja oil and neem oil with honey as substrates resulted in higher MEL yields of (8.07, 7.75, and 1.86) g/L and better cell growth of (8.07, 7.75, and 1.86) g/L, respectively, than non-traditional oils with glucose and glycerol as substrates. Neem oil gave a lower yield of MEL (1.54 g/L) as well as cell growth (6.06 g/L) compared to jatropha oil and karanja oil (7.03 and 6.17) g/L, respectively. Crude MEL from the fermentation broth was detected by thin-layer chromatography (TLC), Fourier transform infrared spectrommetry (FT-IR), high performance liquid chromatography (HPLC) and proton nuclear magnetic resonance spectroscopy (1H NMR). Purified MEL has been used as an antimicrobial agent in cosmetic products associated with gram-positive and gram-negative bacteria and fungi.

Detection and diversity of the mannosylerythritol lipid (MEL) gene cluster and lipase A and B genes of Moesziomyces antarcticus isolated from terrestrial sites chronically contaminated with crude oil in Trinidad

BackgroundMannosylerythritol lipids (MELs) belong to the class of glycolipid biosurfactants and are produced by members of the Ustilago and Moesziomyces genera. Production of MELs is regulated by a biosynthetic gene cluster (MEL BGC). Extracellular lipase activity is also associated with MEL production. Most microbial glycolipid-producers are isolated from oil-contaminated environments. MEL-producing yeast that are capable of metabolizing crude oil are understudied, and there is very limited data on indigenous strains from tropical climates. Analysis of the MEL BGC and lipase genes in Trinidad M. antarcticus strains, using a gene-targeted approach, revealed a correlation between their intrinsic capability to degrade crude oil and their adaptation to survive in a chronically polluted terrestrial environment.ResultsM. antarcticus was isolated from naturally-occurring crude oil seeps and an asphaltic mud volcano in Trinidad; these are habitats that have not been previously reported for this species. Genus identification was confirmed by the large-subunit (LSU) and the small-subunit (SSU) sequence comparisons and species identification was confirmed by ITS sequence comparisons and phylogenetic inference. The essential genes (Emt1, Mac1, Mac2, Mmf1) of the MEL BGC were detected with gene-specific primers. Emt1p, Mac1p and Mmf1p sequence analyses confirmed that the Trinidad strains harboured novel synonymous amino acid (aa) substitutions and structural comparisons revealed different regions of disorder, specifically for the Emt1p sequence. Functionality of each protein sequence was confirmed through motif mining and mutation prediction. Phylogenetic relatedness was inferred for Emt1p, Mac1p and Mmf1p sequences. The Trinidad strains clustered with other M. antarcticus sequences, however, the representative Trinidad M. antarcticus sequences consistently formed a separate, highly supported branch for each protein. Similar phylogenetic placement was indicated for LipA and LipB nucleotide and protein sequences. The Trinidad strains also demonstrated lipolytic activity in culture, with an ability to utilize different carbon sources. Comparative evolution of MEL BGC and LipA gene suggested early and late duplication events, depending on the gene, followed by a number of speciation events within Ustilaginaceae. M. antarcticus and M. aphidis were separated from all other members of Ustilaginaceae and two gene homologues were detected, one for each species.ConclusionsSequence analyses was based on a novel gene-targeted approach to analyze the essential genes of the MEL BGC and LipA and LipB genes of M. antarcticus strains from Trinidad. The findings indicated that these strains accumulated nucleotide mutations to a threshold level that did not affect the function of specific proteins encoded by the MEL BGC and LipA and LipB genes. The biosurfactant and lipase enzymes secreted by these Trinidad M. antarcticus strains facilitated their survival in oil-contaminated terrestrial environments. These findings suggest that the Trinidad strains should be explored as promising candidates for the commercial production of MEL biosurfactants and lipase enzymes.

Production of mannosylerythritol lipids: biosynthesis, multi-omics approaches, and commercial exploitation.

The production of mannosylerythritol lipids (MELs) has been reported on different smut fungi. These biomolecules possess four typical conformations to which key commercially interesting properties have been attributed. In vivo, MEL production could only be explained by the chain-shortening pathway, a new route, unlike the main three fatty acid synthesis pathways described. The production of MELs requires nitrogen starvation and a carbon source, usually a fatty acid, the principals. The first MEL biosynthetic gene cluster was elucidated in U. maydis by the combination of different methodologies, which resulted in a reference genome, on which five genes comprising the MEL cluster were annotated. Subsequently, the evolution of DNA sequencing technologies advanced genome and transcriptome assembly, allowing the annotation of more MEL producers' genomes. These, in combination with different experimental techniques, coupled to bioinformatic methods offer a plethora of genomic resources for further manipulation and commercial exploitation. In this review, we present the main findings, which unraveled MEL production and multi-omics studies, leading to molecular tools for further genomic manipulation and exploitation in smut fungi.

Metabolomic Evaluation of the Central Metabolic Pathways of Mannosylerythritol Lipid Biosynthesis in Moesziomyces antarcticus T-34.

Moesziomyces antarcticus is a basidiomycetous yeast that produces mannosylerythritol lipids (MELs), which have potential applications as bio-based functional materials in various oleochemical industries, the cosmetics, toiletry, agriculture, and pharmaceutical industries. To better understand the MEL producer, we characterized the central metabolic pathways of M. antarcticus strain T-34 grown on glucose or olive oil via metabolomics. The relative fatty acid content was higher in the cells cultured in olive oil compared to glucose, while the acetyl-CoA content was lower in cells cultured in olive oil. The levels of the tricarboxylic acid cycle metabolites citrate/isocitrate, α-ketoglutarate, and succinate were lower in olive oil compared to glucose, while fumarate and malate levels exhibited the opposite pattern. Pyruvate was not detected in olive oil compared to glucose culture. The levels of glycerol, as well as trehalose, myo-inositol, threitol/erythritol, and mannitol/sorbitol, were higher in olive oil compared to glucose cultures. The ATP level was lower in olive oil compared to glucose culture, although the assimilation of fatty acids produced by digestion of olive oil should promote large amounts of ATP production. The possibility that ATP regeneration by respiratory chain complex promote oil utilization and MEL production in M. antarcticus T-34 was found based on the results of this metabolomic analysis.

Improvement of Oil Degradation and MEL Production in a Yeast Strain, Pseudozyma tsukubaensis, by Translation Elongation Factor 1 Promoter-driven Expression of a Lipase.

The basidiomycetous yeast Pseudozyma tsukubaensis produces a mannosylerythritol lipid (MEL) homologue, a diastereomer type of MEL-B, from olive oil. In a previous study, MEL-B production was increased by the overexpression of lipase PaLIPAp in P. tsukubaensis 1E5, through the enhancement of oil consumption. In the present study, RNA sequence analysis was used to identify a promoter able to induce high-level PaLIPA expression. The recombinant strain, expressing PaLIPA via the translation elongation factor 1 alpha/Tu promoter, showed higher lipase activity, rates of oil degradation, and MEL-B production than the strain which generated in our previous study.

Fermentative Production of Mannosylerythritol Lipids using Sweetwater as Waste Substrate by Pseudozyma antarctica (MTCC 2706)

Abstract Mannosylerythritol lipids are glycolipid biosurfactants with promising industrial applications. However, their commercial production is hindered due to its high production cost. The current study investigates the use of sweetwater, a by-product of the fat-splitting industry in combination with soybean oil for the production of mannosylerythritol lipids using Pseudozyma antarctica (MTCC 2706). The optimum sweetwater and soybean oil concentration of 22% and 7% (w/v) yielded 7.52 g L–1and 21.5 g L–1 mannosylerythritol lipids at shake flask and fermenter level respectively. The structure and functional groups of mannosylerythritol lipids were confirmed by fourier transform infrared (FTIR) spectroscopy, liquid chromatography-mass spectrometry (LC/MS) and 1H- and 13C-nuclear magnetic resonance (NMR) analysis. Surfactant properties, such as surface tension, critical micelle concentration, foaming and emulsification of mannosylerythritol lipids were also explored.

Novel mannosylerythritol lipid biosurfactant structures from castor oil revealed by advanced structure analysis.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by fungi of the Ustilaginaceae family in the presence of hydrophobic carbon sources like plant oils. In the present study, we investigated the structural composition of MELs produced from castor oil using seven different microorganisms and compared them to MEL structures resulting from other plant oils. Castor oil is an industrially relevant plant oil that presents as an alternative to currently employed edible plant oils like rapeseed or soybean oil. The main fatty acid in castor oil is the mono-hydroxylated ricinoleic acid, providing the possibility to produce novel MEL structures with interesting features. Analysis of the produced MELs from castor oil by different chromatographic and mass spectrometry techniques revealed that all seven microorganisms were generally able to integrate hydroxylated fatty acids into the MEL molecule, although at varying degrees. These novel MELs containing a hydroxy fatty acid (4-O-[2′-O-alka(e)noyl-3′-O-hydroxyalka(e)noyl-4′/6′-O-acetyl-β-D-mannopyranosyl]-erythritol) were more hydrophilic than conventional MEL and therefore showed a different elution behavior in chromatography. Large shares of novel hydroxy MELs (around 50% of total MELs) were found for the two MEL-B/C producing species Ustilago siamensis and Ustilago shanxiensis, but also for the MEL-A/B/C producer Moesziomyces aphidis (around 25%). In addition, tri-acylated hydroxylated MELs with a third long-chain fatty acid esterified to the free hydroxyl group of the hydroxy fatty acid were identified for some species. Overall, production of MEL from castor oil with the investigated organisms provided a complex mixture of various novel MEL structures that can be exploited for further research.

Engineering Ustilago maydis for production of tailor-made mannosylerythritol lipids

Mannosylerythritol lipids (MELs) are surface active glycolipids secreted by various fungi. MELs can be used as biosurfactants and are a biodegradable resource for the production of detergents or pharmaceuticals. Different fungal species synthesize a unique mixture of MELs differing in acetyl- and acyl-groups attached to the sugar moiety. Here, we report the construction of a toolbox for production of glycolipids with predictable fatty acid side chains in the basidiomycete Ustilago maydis. Genes coding for acyl-transferases involved in MEL production (Mac1 and Mac2) from different fungal species were combined to obtain altered MEL variants with distinct physical properties and altered antimicrobial activity. We also demonstrate that a U. maydis paralog of the acyltransferase Mac2 with a different substrate specificity can be employed for the biosynthesis of modified MEL variants. In summary, our data showcase how the fungal repertoire of Mac enzymes can be used to engineer tailor-made MELs according to specific biotechnological or pharmaceutical requirements.

Acid pH Strategy Adaptation through NRG1 in Ustilago maydis.

The role of the Ustilago maydis putative homolog of the transcriptional repressor ScNRG1, previously described in Saccharomyces cerevisiae, Candida albicans and Cryptococcus neoformans, was analyzed by means of its mutation. In S. cerevisiae this gene regulates a set of stress-responsive genes, and in C. neoformans it is involved in pathogenesis. It was observed that the U. maydis NRG1 gene regulates several aspects of the cell response to acid pH, such as the production of mannosyl-erythritol lipids, inhibition of the expression of the siderophore cluster genes, filamentous growth, virulence and oxidative stress. A comparison of the gene expression pattern of the wild type strain versus the nrg1 mutant strain of the fungus, through RNA Seq analyses, showed that this transcriptional factor alters the expression of 368 genes when growing at acid pH (205 up-regulated, 163 down-regulated). The most relevant genes affected by NRG1 were those previously reported as the key ones for particular cellular stress responses, such as HOG1 for osmotic stress and RIM101 for alkaline pH. Four of the seven genes included WCO1 codifying PAS domain ( These has been shown as the key structural motif involved in protein-protein interactions of the circadian clock, and it is also a common motif found in signaling proteins, where it functions as a signaling sensor) domains sensors of blue light, two of the three previously reported to encode opsins, one vacuolar and non-pH-responsive, and another one whose role in the acid pH response was already known. It appears that all these light-reactive cell components are possibly involved in membrane potential equilibrium and as virulence sensors. Among previously described specific functions of this transcriptional regulator, it was found to be involved in glucose repression, metabolic adaptation to adverse conditions, cellular transport, cell rescue, defense and interaction with an acidic pH environment.

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.

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.