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      A Gene Cluster for Biosynthesis of Mannosylerythritol Lipids Consisted of 4- O-β-D-Mannopyranosyl-(2 R,3 S)-Erythritol as the Sugar Moiety in a Basidiomycetous Yeast Pseudozyma tsukubaensis

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          Abstract

          Mannosylerythritol lipids (MELs) belong to the glycolipid biosurfactants and are produced by various fungi. The basidiomycetous yeast Pseudozyma tsukubaensis produces diastereomer type of MEL-B, which contains 4- O-β-D-mannopyranosyl-(2 R,3 S)-erythritol ( R-form) as the sugar moiety. In this respect it differs from conventional type of MELs, which contain 4- O-β-D-mannopyranosyl-(2 S,3 R)-erythritol ( S-form) as the sugar moiety. While the biosynthetic gene cluster for conventional type of MELs has been previously identified in Ustilago maydis and Pseudozyma antarctica, the genetic basis for MEL biosynthesis in P. tsukubaensis is unknown. Here, we identified a gene cluster involved in MEL biosynthesis in P. tsukubaensis. Among these genes, PtEMT1, which encodes erythritol/mannose transferase, had greater than 69% identity with homologs from strains in the genera Ustilago, Melanopsichium, Sporisorium and Pseudozyma. However, phylogenetic analysis placed PtEMT1p in a separate clade from the other proteins. To investigate the function of PtEMT1, we introduced the gene into a P. antarctica mutant strain, Δ PaEMT1, which lacks MEL biosynthesis ability owing to the deletion of PaEMT1. Using NMR spectroscopy, we identified the biosynthetic product as MEL-A with altered sugar conformation. These results indicate that PtEMT1p catalyzes the sugar conformation of MELs. This is the first report of a gene cluster for the biosynthesis of diastereomer type of MEL.

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          Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.

          Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.
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            Gene Loss Rather Than Gene Gain Is Associated with a Host Jump from Monocots to Dicots in the Smut Fungus Melanopsichium pennsylvanicum

            Smut fungi are well-suited to investigate the ecology and evolution of plant pathogens, as they are strictly biotrophic, yet cultivable on media. Here we report the genome sequence of Melanopsichium pennsylvanicum, closely related to Ustilago maydis and other Poaceae-infecting smuts, but parasitic to a dicot plant. To explore the evolutionary patterns resulting from host adaptation after this huge host jump, the genome of Me. pennsylvanicum was sequenced and compared with the genomes of U. maydis, Sporisorium reilianum, and U. hordei. Although all four genomes had a similar completeness in CEGMA (Core Eukaryotic Genes Mapping Approach) analysis, gene absence was highest in Me. pennsylvanicum, and most pronounced in putative secreted proteins, which are often considered as effector candidates. In contrast, the amount of private genes was similar among the species, highlighting that gene loss rather than gene gain is the hallmark of adaptation after the host jump to the dicot host. Our analyses revealed a trend of putative effectors to be next to another putative effector, but the majority of these are not in clusters and thus the focus on pathogenicity clusters might not be appropriate for all smut genomes. Positive selection studies revealed that Me. pennsylvanicum has the highest number and proportion of genes under positive selection. In general, putative effectors showed a higher proportion of positively selected genes than noneffector candidates. The 248 putative secreted effectors found in all four smut genomes might constitute a core set needed for pathogenicity, whereas those 92 that are found in all grass-parasitic smuts but have no ortholog in Me. pennsylvanicum might constitute a set of effectors important for successful colonization of grass hosts.
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              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.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                21 June 2016
                2016
                : 11
                : 6
                : e0157858
                Affiliations
                [1 ]Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
                [2 ]Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
                [3 ]Toyobo Co., Ltd., Tsuruga Institute of Biotechnology, Tsuruga, Fukui, Japan
                Tulane University Health Sciences Center, UNITED STATES
                Author notes

                Competing Interests: SY and TK are employees of Toyobo Co., Ltd. HK, TF, SY, TK and TM are inventors of submitted patent which related to this study (PCT/JP2016/061760). This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. The authors have declared that no other competing interests exist.

                Conceived and designed the experiments: AS TM. Performed the experiments: AS HK TF SY TK TM. Analyzed the data: AS HK TF SY TK TM. Wrote the paper: AS TM.

                Article
                PONE-D-16-09819
                10.1371/journal.pone.0157858
                4915680
                27327162
                089a62d8-212d-40dc-99b2-577e31a3db94
                © 2016 Saika et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 March 2016
                : 6 June 2016
                Page count
                Figures: 9, Tables: 3, Pages: 16
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100002241, Japan Science and Technology Agency;
                Award ID: AS2621413N
                Award Recipient :
                This work has been supported by Japan Science and technology Agency (JST) under Adaptable and Seamless Technology Transfer Program through Target-driven R&D (AS2621413N). JST had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Toyobo Co., Ltd. provided support in the form of salaries for authors SY and TK, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
                Categories
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