Cellobiose Lipids Research Articles

Discovery of novel cellobiose lipid gene clusters from Basidiomycetes: How chemical variation is reflected in gene cluster architecture.

Cellobiose lipids are surface-active compounds or biological detergents produced by distinct Basidiomycetes yeasts, of which the most and best-described ones belong to the Ustilaginomycetes class. The molecules display slight variation in congener type, which is linked to the hydroxylation position of the long fatty acid, acetylation profile of the cellobiose unit, and presence or absence of the short fatty acid. In general, this variation is strain specific. Although cellobiose lipid biosynthesis has been described for about 11 yeast species, hitherto only two types of biosynthetic gene clusters are identified, and this for only three species. This work adds six more biosynthetic gene clusters and describes for the first time a novel type of cellobiose lipid biosynthetic cluster with a simplified architecture related to specific cellobiose lipids synthesized by Trichosporonaceae family members.

Life cycle assessment as a driver for process optimisation of cellobiose lipids fermentation and purification

PurposeCellobiose lipids (CL) are biosurfactants produced by various Ustilaginaceae species in aerobic fermentations. They show high potential for application as alternatives to conventional oleochemical- or petrochemical surfactants. To ensure their environmentally friendly performance, we aimed to assess CL production from a life cycle perspective at an early developmental stage to identify process steps that have the highest impact on the environment. With this information, optimisation approaches can be derived.Materials and methodsFollowing a cradle-to-gate approach, we modelled the CL fermentation and purification process based on experimental data from the lab scale and process simulation data at a 10 m3 scale. For LCA, the impact categories (IC) abiotic depletion potential (ADP), eutrophication potential, photochemical ozone creation potential, global warming potential, acidification potential, and the primary energy demand were calculated for all process steps. Based on the obtained results, process bottlenecks were identified, and alternative process scenarios varying the related process parameters were simulated. These were used to assess the environmental impact reduction potential (EIRP) of an optimised process and draw recommendations for experimental process optimisation.Results and discussionThe obtained results showed that the fermentation caused ~ 73% of ADP and more than 85% of all other ICs. The major contributor was the electricity consumption for continuous fermenter aeration. Thus, reducing the fermentation duration from the initial 14 to 5 days would result in a decrease in all investigated ICs of up to ~ 27–52%. An increase in CL concentration results in a decrease in all ICs of a similar magnitude due to the higher yield per batch at comparable energy and material consumption. Although the share of purification process steps to all ICs is overall relatively small, implementing foam fractionation for in situ product recovery showed an additional EIRP of 18–27% in all purification IC shares.ConclusionsThe conducted LCA showed that overall, more EIRP can be achieved by optimising fermentation process parameters compared to purification process steps. This is mainly due to the long fermentation duration and large energy consumption for fermenter aeration. This highlights the importance of using LCA as a driver for process optimisation to identify process steps with high EIRP. While some of the results are specific to CL, other obtained results can be transferred to other fermentations.

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.

Fermentation and recovery of cellobiose lipids using foam fractionation

Cellobiose lipids (CL) are glycolipids secreted by many Ustilaginaceae species in aerobic fermentations characterised by excessive foaming. While increasing CL concentrations remains an aim for its industrial production, excessive foaming during fermentation presents a challenge even at laboratory scale. Foam fractionation (FF) provides a solution to the foaming problem and facilitates the proceeding purification of CL. Here, we present a first CL fermentation process applying FF. With our set-up, we manage to exploit the excessive foaming for continuous product separation. The set-up includes a foam collecting vessel (FCV) with inserts for CL accumulation and foamate recirculation to minimise biomass and nutrient loss. Integrating a foam column (FC) into the fermenter headspace enabled foam enrichment, resulting in the recovery of > 90% of the produced CL from the separated fractions consisting of foam depositions in the fermenter headspace and the FCV. We also increased the fermenter filling volume and thus achieved a higher fermentation capacity. The separated CL fraction was purified via ethanol extraction to obtain CL with purities > 90%. We further examined the effects of different culture media constituents, including biomass and CL, on foam generation and decay and assessed the effect of FC geometries on product enrichment and recovery. In this work, a FF set-up is presented that enables a stable CL fermentation without additional foam mitigation methods. At the same time, the application of FF separated a fraction that was highly enriched in CL during fermentation, resulting in highly pure CL after a simple ethanol extraction.

Techno-Economic Analysis as a Driver for Optimisation of Cellobiose Lipid Fermentation and Purification.

Cellobiose lipids (CL) are glycolipids synthesized by Ustilaginaceae species with potential application as detergents or in cosmetics. This study identified process optimisation potential for CL fermentation based on process modelling and techno-economic analysis. Using a stoichiometric equation based on laboratory data, we calculated the maximum possible CL yield YP/S of 0.45 gCL·gglucose −1 at the biomass yield of 0.10 gBiomass·gglucose −1 with an Ustilago maydis strain. Due to substrate inhibition that may occur at high glucose concentrations, a fed-batch process to increase biomass and CL concentrations was considered in our model. Simulation of different process scenarios showed that the choice of aeration units with high oxygen transfer rates and adaptation of power input to oxygen uptake can significantly decrease electricity consumption. We further assessed scenarios with different fermentation media and CL purification methods, suggesting additional process optimisation potential. Here the omission of vitamins from the fermentation medium proved to be a possible mean to enhance process economy, without compromising CL productivity.

Fungal biosurfactants, from nature to biotechnological product: bioprospection, production and potential applications.

Biosurfactants are in demand by the global market as natural commodities that can be added to commercial products or use in environmental applications. These biomolecules reduce the surface/interfacial tension between fluid phases and exhibit superior stability to chemical surfactants under different physico-chemical conditions. Biotechnological production of biosurfactants is still emerging. Fungi are promising producers of these molecules with unique chemical structures, such as sophorolipids, mannosylerythritol lipids, cellobiose lipids, xylolipids, polyol lipids and hydrophobins. In this review, we aimed to contextualize concepts related to fungal biosurfactant production and its application in industry and the environment. Concepts related to the thermodynamic and physico-chemical properties of biosurfactants are presented, which allows detailed analysis of their structural and application. Promising niches for isolating biosurfactant-producing fungi are presented, as well as screening methodologies are discussed. Finally, strategies related to process parameters and variables, simultaneous production, process optimization through statistical and genetic tools, downstream processing and some aspects of commercial products formulations are presented.

Factors Affecting the Synthesis of Cellobiose Lipids by Sporisorium scitamineum.

Cellobiose lipids (CL) are extracellular glycolipids that are produced by many microorganisms from the family Ustilaginaceae. The sugarcane smut fungus Sporisorium scitamineum has been long known as a producer of the glycolipids mannosylerythritol lipids (MEL) and was recently described to additionally secrete CL as a byproduct. In fact, we identified 11 homologous genes in S. scitamineum by in silico analysis sharing a high similarity to the CL biosynthesis gene cluster of Ustilago maydis. We here report the first systematic cultivation of S. scitamineum targeting the synthesis of CL with high product titers and its transfer to the bioreactor. In an initial screening we examined different fermentation media compositions, consisting of a mineral salts solution with vitamins and/or trace elements, three carbon sources (glucose, fructose, sucrose), three pH values (2.5, 4.0, 6.7) and three levels of C/N values (42.2, 83.8, 167.2 molC⋅molN–1) with urea as nitrogen source. A pH of 2.5 proved to result in the highest product titers. An increase of urea concentration from 0.6 to 1.2 g⋅L–1 had a positive effect on biomass formation, however the glycolipid formation was favored at a C/N ratio of 83.8 molC⋅molN–1, using 0.6 g⋅L–1 urea. Amongst the examined carbon sources, sucrose resulted in an increase in the secretion of cellobiose lipids, compared to glucose. Comparing different media compositions, vitamins were identified as not necessary for CL synthesis. We obtained a concentration of cellobiose lipids of 8.3 ± 1.0 g⋅L–1 in shaking flasks. This increased to 17.6 g⋅L–1 in the 1 L bioreactor with additional feeding of carbon source, with a final purity of 85–93%. As a side product, erythritol and mannosylerythritol lipids (MEL) were also synthesized. Via HPTLC coupled MALDI-TOF MS we were able to analyze the secreted CL structures. S. scitamineum produces a mixture of acylated low molecular weight D-glucolipids, linked to a 2,15,16-trihydroxy-hexadecanoic acid via their ω-hydroxyl group (CL-B). The produced cellobiose lipids precipitate as needle like crystals at an acidic pH value of 2.5.

A New Screening Approach for Glycolipid-type Biosurfactant Producers Using MALDI-TOF/MS.

We applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) to screen for glycolipid-type biosurfactant (BS) producers. A crude extract of Pseudozyma antarctica, a well-known mannosylerythritol lipid (MEL) producer, was initially subjected to MALDI-TOF/MS. The spectrum of the extract showed the accumulation of diacylated MELs in culture. We then screened 80 environmental samples for BS-producing yeasts, and extracts from broth cultures of the selected five strains were examined using MALDI-TOF/MS. The results showed that all five strains produced MELs, whereas four strains also produced cellobiose lipids (CLs). By D1/D2 region sequence analysis, the MEL-producing strain was assigned to P. antarctica while the four MEL- and CL-producing strains were assigned to P. hubeiensis. These results demonstrate that MALDI-TOF/MS is a rapid and reliable tool to detect BS molecules in crude extracts of broth cultures to screen for glycolipid-type BS producers.

Application of Glycolipid Biosurfactants as Surface Modifiers in Bioplastics

Surface properties of cast films of poly(lactic acid) (PLA) containing 1 wt% of several glycolipid-type biosurfactants (BSs) were investigated. The wettability of PLA films containing a homologue of mannosylerythritol lipids (MEL-B), lactone-form sophorolipid (LSL), or cellobiose lipid (CL) was drastically higher than that of untreated PLA and several synthetic surfactants-containing PLA. Surface wettability was also dependent on the hydrophilicity of the substrate used during solvent casting of the PLA films. The wetting behavior of the opposing sides of MEL-B-containing films prepared on glass substrates differed significantly; the contact angle on the side of the film that had been in contact with the glass surface was significantly lower than that obtained on the side of the film that had been in contact with air. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis results showed that the MEL in MEL-B-containing thin PLA cast films was localized to a thin surface layer. These results suggest self-assembly of MEL-B and micro-phase separation between the PLA matrix and MEL-B domains. This resulted in the localization and orientation of MEL-B at the surface of the cast PLA film, which determined its specific wetting behavior.

Yeast glycolipid biosurfactants

Various yeasts, both conventional and exotic ones, are known to produce compounds useful to mankind. Ethanol is the most known of these compounds, but more complex molecules such as amphiphilic biosurfactants can also be derived from eukaryotic microorganisms at an industrially and commercially relevant scale. Among them, glycolipids are the most promising, due to their attractive properties and high product titers. Many of these compounds can be considered as secondary metabolites with a specific function for the host. Hence, a dedicated biosynthetic process enables regulation and combines pathways delivering the lipidic moiety and the hydrophilic carbohydrate part of the glycolipid. In this Review, we will discuss the biosynthetic and regulatory aspects of the yeast-derived sophorolipids, mannosylerythritol lipids, and cellobiose lipids, with special emphasis on the relation between glycolipid synthesis and the general lipid metabolism.

Fungal Glycolipids as Biosurfactants

Background: Fungi are key organisms in the biotechnological production of a plethora of products relevant for mankind. Recently several new products from fungi have entered the markets, including biosurfactants. Biosurfactants are microbial produced surface active compounds with emulsifying properties which proved to be an interesting alternative to petrochemically or palm oil derived surfactants. Keywords: Biosurfactants, Ustilago spp., Pseudozyma spp., mannosylerythritol lipids, cellobiose lipids, APG.

Resistance to cellobiose lipids and specific features of lipid composition in yeast

The significance of the fatty acid composition and ergosterol content in cells for resistance to cellobiose lipids has been investigated in the cells of mutant Saccharomyces cerevisiae strains that are unable to produce ergosterol or sphingomyelin and in the cells of microorganisms that produce cellobiose lipids. S. cerevisiae mutants were shown to be less sensitive to cellobiose lipids from Cryptococcus humicola than the wild-type strain, and the strains that produced cellobiose lipids were virtually insensitive to this compound as well. The sensitivity of Pseudozyma fusiformata yeast to its own cellobiose lipids was reduced under conditions that favored the production of these compounds. No correlation between the content of ergosterol and sensitivity to cellobiose lipids was observed in S. cerevisiae or in the strains that produced cellobiose lipids. The ratio between the levels of saturated and unsaturated fatty acids in the cells of the mutant strains was correlated to the sensitivity of the cells to cellobiose lipids.

Glycolipid biosurfactants: main properties and potential applications in agriculture and food industry.

Glycolipids, consisting of a carbohydrate moiety linked to fatty acids, are microbial surface active compounds produced by various microorganisms. They are characterized by high structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface, respectively. Rhamnolipids, trehalolipids, mannosylerythritol lipids and cellobiose lipids are among the most popular glycolipids. They have received much practical attention as biopesticides for controlling plant diseases and protecting stored products. As a result of their antifungal activity towards phytopathogenic fungi and larvicidal and mosquitocidal potencies, glycolipid biosurfactants permit the preservation of plants and plant crops from pest invasion. Also, as a result of their emulsifying and antibacterial activities, glycolipids have great potential as food additives and food preservatives. Furthermore, the valorization of food byproducts via the production of glycolipid biosurfactant has received much attention because it permits the bioconversion of byproducts on valuable compounds and decreases the cost of production. Generally, the use of glycolipids in many fields requires their retention from fermentation media. Accordingly, different strategies have been developed to extract and purify glycolipids. © 2016 Society of Chemical Industry.

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.

Biosurfactant gene clusters in eukaryotes: regulation and biotechnological potential

Biosurfactants (BSs) are a class of secondary metabolites representing a wide variety of structures that can be produced from renewable feedstock by a wide variety of micro-organisms. They have (potential) applications in the medical world, personal care sector, mining processes, food industry, cosmetics, crop protection, pharmaceuticals, bio-remediation, household detergents, paper and pulp industry, textiles, paint industries, etc. Especially glycolipid BSs like sophorolipids (SLs), rhamnolipids (RLs), mannosylerythritol lipids (MELs) and cellobioselipids (CBLs) have been described to provide significant opportunities to (partially) replace chemical surfactants. The major two factors currently limiting the penetration of BSs into the market are firstly the limited structural variety and secondly the rather high production price linked with the productivity. One of the keys to resolve the above mentioned bottlenecks can be found in the genetic engineering of natural producers. This could not only result in more efficient (economical) recombinant producers, but also in a diversification of the spectrum of available BSs as such resolving both limiting factors at once. Unraveling the genetics behind the biosynthesis of these interesting biological compounds is indispensable for the tinkering, fine tuning and rearrangement of these biological pathways with the aim of obtaining higher yields and a more extensive structural variety. Therefore, this review focuses on recent developments in the investigation of the biosynthesis, genetics and regulation of some important members of the family of the eukaryotic glycolipid BSs (MELs, CBLs and SLs). Moreover, recent biotechnological achievements and the industrial potential of engineered strains are discussed.

Genome Sequence of the Basidiomycetous Fungus Pseudozyma aphidis DSM70725, an Efficient Producer of Biosurfactant Mannosylerythritol Lipids

Pseudozyma aphidis is an efficient producer of mannosylerythritol lipids exceeding concentrations of >100 g/liter from renewable feed stocks. Additionally, a biosurfactant cellobiose lipid is also secreted during nitrogen limitation. Here, we describe the sequencing of P. aphidis to unravel the genomic basis of biosurfactant metabolism in P. aphidis.

The antibiotic and membrane-damaging activities of cellobiose lipids and sophorose lipids.

Antibiotic activity was compared for Cryptococcus humicola cellobiose lipids, the mixture of 2,3,4-О-triacetyl-β-D-glucopyranosyl-(1→4)-(6-О-acetyl-β-D-glucopyranosyl-(1→16)-2,16-dihydroxyhexodecanoic acid and 2,3,4-О-triacetyl-β-D-glucopyranosyl-(1→4)-(6-О-acetyl-β-D-glucopyranosyl-(1→16)-2,17,18-trihydroxyoctotodecanoic acid, and the commercial sophorose lipid mixture of a mono-acetylated acidic sophorose lipid and a di-acetylated acidic sophorose lipid, both containing the C18:1 fatty acid residue. The MIC values of cellobiose lipids were 0.005 and 0.04 mg/mL for Filobasidiella neoformans and Candida tropicalis, respectively. The MIC values of sophorose lipids were 1 and 15 mg/mL for F. neoformans and C. tropicalis, respectively. MIC values for some bacteria were in the range of 10-30 mg/mL for both glycolipid preparations. Both sophorose lipids and cellobiose lipids displayed a membrane-damaging activity against F. neoformans. The treatment with these glycolipids reduces the content of ATP in the cells of test cultures and results in their staining with ethidium bromide.

Aqueous gel formation from sodium salts of cellobiose lipids.

Cellobiose lipids (CLs) are asymmetric bolaform biosurfactants, which are produced by Cryptococcus humicola JCM 10251 and have fungicidal activity. In this study, the sodium salts of CLs (CLNa) were prepared to improve aqueous solubility of the CLs, and their surface and gelation properties in aqueous solutions were examined by surface tension, rheology, and freeze-fracture transmission electron microscopy (FF-TEM) measurements. The surface tension measurements revealed that the CLNa have high surface activity: CMC1 and γCMC1 are 0.1 mg/mL and 34.7 mN/m, respectively. It was also found that the CLNa form giant micelles above their CMC, whose average size is 116.6 ± 31.9 nm. Unlike conventional surfactants, the surface tension reduced further with an increase in concentration and the aqueous solution became viscous at the minimum gelation concentration (MGC: 5.0 mg/mL). In rheological studies, the obtained gels proved to be rather soft and their sol-gel temperature was found to be approximately 50℃. FF-TEM observation of the gels showed 3D supramolecular structures with an entangled fibrous network. Since the present CLNa aqueous gels have a degree of fungicidal activity, they could be useful for novel multifunctional soft materials applicable to the food and cosmetic industries.

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.

The antifungal effect of cellobiose lipid on the cells of Saccharomyces cerevisiae depends on carbon source

The cellobiose lipid of Cryptococcus humicola, 16-(tetra-O-acetyl-β-cellobiosyloxy)-2-hydroxyhexadecanoic acid, is a natural fungicide. Sensitivity of the cells of Saccharomyces cerevisiae to the fungicide depends on a carbon source. Cellobiose lipid concentrations inducing the leakage of potassium ions and ATP were similar for the cells grown in the medium with glucose and ethanol. However, the cells grown on glucose and ethanol died at 0.05 mg ml-1 and 0.2 mg ml-1 cellobiose lipid, respectively. Inorganic polyphosphate (PolyP) synthesis was 65% of the control with 0.05 mg ml-1 cellobiose lipid during cultivation on ethanol. PolyP synthesis was not observed during the cultivation on glucose at the same cellobiose lipid concentration. The content of longer-chain polyP was higher during cultivation on ethanol. We speculate the long-chained polyP participate in the viability restoring of ethanol-grown cells after treatment with the cellobiose lipid.