Genome and transcriptome analysis of the basidiomycetous yeast Pseudozyma antarctica producing extracellular glycolipids, mannosylerythritol lipids.
ABSTRACT: Pseudozyma antarctica is a non-pathogenic phyllosphere yeast known as an excellent producer of mannosylerythritol lipids (MELs), multi-functional extracellular glycolipids, from vegetable oils. To clarify the genetic characteristics of P. antarctica, we analyzed the 18 Mb genome of P. antarctica T-34. On the basis of KOG analysis, the number of genes (219 genes) categorized into lipid transport and metabolism classification in P. antarctica was one and a half times larger than that of yeast Saccharomyces cerevisiae (140 genes). The gene encoding an ATP/citrate lyase (ACL) related to acetyl-CoA synthesis conserved in oleaginous strains was found in P. antarctica genome: the single ACL gene possesses the four domains identical to that of the human gene, whereas the other oleaginous ascomycetous species have the two genes covering the four domains. P. antarctica genome exhibited a remarkable degree of synteny to U. maydis genome, however, the comparison of the gene expression profiles under the culture on the two carbon sources, glucose and soybean oil, by the DNA microarray method revealed that transcriptomes between the two species were significantly different. In P. antarctica, expression of the gene sets relating fatty acid metabolism were markedly up-regulated under the oily conditions compared with glucose. Additionally, MEL biosynthesis cluster of P. antarctica was highly expressed regardless of the carbon source as compared to U. maydis. These results strongly indicate that P. antarctica has an oleaginous nature which is relevant to its non-pathogenic and MEL-overproducing characteristics. The analysis and dataset contribute to stimulate the development of improved strains with customized properties for high yield production of functional bio-based materials.
Project description:Pseudozyma antarctica is a nonpathogenic phyllosphere yeast known as an excellent producer of industrial lipases and mannosylerythritol lipids (MELs), which are multi-functional glycolipids. The fungus produces a much higher amount of MELs from vegetable oil than from glucose, whereas its close relative, Ustilago maydis UM521, produces a lower amount of MELs from vegetable oil. In the present study, we used previous gene expression profiles measured by DNA microarray analyses after culturing on two carbon sources, glucose and soybean oil, to further characterize MEL biosynthesis in P. antarctica T-34. A total of 264 genes were found with induction ratios and expression intensities under oily conditions with similar tendencies to those of MEL cluster genes. Of these, 93 were categorized as metabolic genes using the Eukaryotic Orthologous Groups classification. Within this metabolic category, amino acids, carbohydrates, inorganic ions, and secondary metabolite metabolism, as well as energy production and conversion, but not lipid metabolism, were enriched. Furthermore, genes involved in central metabolic pathways, such as glycolysis and the tricarboxylic acid cycle, were highly induced in P. antarctica T-34 under oily conditions, whereas they were suppressed in U. maydis UM521. These results suggest that the central metabolism of P. antarctica T-34 under oily conditions contributes to its excellent oil utilization and extracellular glycolipid production.
Project description: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.
Project description: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 <i>Ustilago maydis</i>. 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 <i>U. maydis</i> 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.
Project description:The basidiomycetous yeast-like fungus Pseudozyma hubeiensis strain SY62 is capable of producing an abundant amount of the glycolipid biosurfactant mannosylerythritol lipids (MELs), which are a major component of monoacetylated MEL (MEL-C). To reveal the synthetic pathway of the MELs of strain SY62, we present the 18.44-Mb draft genome sequence.
Project description:The basidiomycetous yeast Pseudozyma antarctica (currently designated Moesziomyces antarcticus) produces extracellular enzymes and glycolipids, including mannosylerythritol lipids (MELs), which are biosurfactants. Strain GB-4(0) of this species was previously isolated from rice husks and produces biodegradable plastic-degrading enzyme (Pseudozyma antarctica esterase; PaE). In this study, we generated a MEL biosynthesis-deficient strain (?PaEMT1) by deleting the gene PaEMT1, which is essential to MEL biosynthesis in strain GB-4(0). The resulting ?PaEMT1 strain showed deficient PaE activity, and the corresponding signal was hardly detected in its culture supernatant through western blotting analysis using rabbit anti-PaE serum. On the other hand, the relative expression of the gene PaCLE1, encoding PaE, was identical between GB-4(0) and ?PaEMT1 based on quantitative real-time PCR. When strain ?PaEMT1 was grown in culture media supplemented with various surfactants, i.e., Tween20, BRIJ35 and TritonX-100, and MELs, PaE activity and secretion recovered. We also attempted to detect intracellular PaE using cell-free extract, but observed no signal in the soluble or insoluble fractions of ?PaEMT1. This result suggested that the PaCLE1 gene was not translated to PaE, or that expressed PaE was degraded immediately in ?PaEMT1. Based on these results, MEL biosynthesis is an important contributor to PaE production.
Project description: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.
Project description:The basidiomycetous yeast Pseudozyma antarctica T-34 is an excellent producer of mannosylerythritol lipids (MELs), members of the multifunctional extracellular glycolipids, from various feedstocks. Here, the genome sequence of P. antarctica T-34 was determined and annotated. Analysis of the sequence might provide insights into the properties of this yeast that make it superior for use in the production of functional glycolipids, leading to the further development of P. antarctica for industrial applications.
Project description: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 [Formula: see text], 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.
Project description:Pseudozyma aphidis is one of the most productive microbial producers of mannosylerythritol lipids on vegetable oils with a unique product spectrum that contains all four main variants MEL-A, -B, -C and -D. Secretion of MEL is thereby accompanied by a morphologenetic switch from yeast to hyphal growth. To investigate the genetic characteristics of MEL secretion and dimorphic transition, we analyzed the transcriptome of P. aphidis during production of MEL after the morphologenetic switch. The analysis revealed that the strong activation of 4 of the 5 genes within the MEL-cluster is clearly dependent on the presence of a hydrophobic carbon source. Only the acetyltransferase mat1 is not induced. This may explain the heterogeneous mixture of MEL with different degree of acetylation. In parallel to the MEL-Cluster, we saw a significant induction of a large group of genes which are coding for cell wall modifying enzymes. These genes were mainly grouped in two large gene clusters typical for the concerted cellular switch. In addition, a group of transcription factors was activated which may be key regulator candidates for MEL-synthesis and cell development. The induction of nitrogen metabolism and assimilation processes for phosphate, iron and other nutrients draw a picture of further cellular requirements at this time point. As part of this work, we present the manually annotated and experimentally verified genome and transcriptome of P. aphidis DSM 70725 with a total number of 6942 genes. 82 % of the genes are othologs to Pseudozyma antarctica and 73 % orthologs to Ustilago maydis. Experimental annotation of transcriptional landscape combined with examination of gene expression on two different carbon sources.
Project description:Mannosylerythritol lipids-A (MEL-A) is a novel biosurfactant with excellent surface activity and potential biomedical applications. In this study, we explored the antibacterial activity and the underlying mechanisms of MEL-A against the important food-borne pathogen Listeria monocytogenes. The bacterial growth and survival assays revealed a remarkable antibacterial activity of MEL-A. Since MEL-A is a biosurfactant, we examined the cell membrane integrity and morphological changes of MEL-A-treated bacteria by biochemical assays and flow cytometry analysis and electron microscopes. The results showed obvious damaging effects of MEL-A on the cell membrane and morphology. To further explore the antibacterial mechanism of MEL-A, a transcriptome analysis was performed, which identified 528 differentially expressed genes (DEGs). Gene ontology (GO) analysis revealed that the gene categories of membrane, localization and transport were enriched among the DEGs, and the analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways demonstrated significant changes in the maltodextrin ABC transporter system and stress response system. Furthermore, the growth of L. monocytogenes could also be significantly inhibited by MEL-A in milk, a model of a real food system, suggesting that MEL-A could be potentially applied as an natural antimicrobial agent to control food-borne pathogens in the food industry.