Dataset Information

Heterologous Expression and Biochemical Characterization of a New Chloroperoxidase Isolated from the Deep-Sea Hydrothermal Vent Black Yeast Hortaea werneckii UBOCC-A-208029.

ABSTRACT: The initiation of this study relies on a targeted genome-mining approach to highlight the presence of a putative vanadium-dependent haloperoxidase-encoding gene in the deep-sea hydrothermal vent fungus Hortaea werneckii UBOCC-A-208029. To date, only three fungal vanadium-dependent haloperoxidases have been described, one from the terrestrial species Curvularia inaequalis, one from the fungal plant pathogen Botrytis cinerea, and one from a marine derived isolate identified as Alternaria didymospora. In this study, we describe a new vanadium chloroperoxidase from the black yeast H. werneckii, successfully cloned and overexpressed in a bacterial host, which possesses higher affinity for bromide (K_m = 26 µM) than chloride (K_m = 237 mM). The enzyme was biochemically characterized, and we have evaluated its potential for biocatalysis by determining its stability and tolerance in organic solvents. We also describe its potential three-dimensional structure by building a model using the AlphaFold 2 artificial intelligence tool. This model shows some conservation of the 3D structure of the active site compared to the vanadium chloroperoxidase from C. inaequalis but it also highlights some differences in the active site entrance and the volume of the active site pocket, underlining its originality.

SUBMITTER: Cochereau B

PROVIDER: S-EPMC10427571 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Heterologous Expression and Biochemical Characterization of a New Chloroperoxidase Isolated from the Deep-Sea Hydrothermal Vent Black Yeast Hortaea werneckii UBOCC-A-208029.

Cochereau Bastien B Le Strat Yoran Y Ji Qiaolin Q Pawtowski Audrey A Delage Ludovic L Weill Amélie A Mazéas Lisa L Hervé Cécile C Burgaud Gaëtan G Gunde-Cimerman Nina N Pouchus Yves François YF Demont-Caulet Nathalie N Roullier Catherine C Meslet-Cladiere Laurence L

Marine biotechnology (New York, N.Y.) 20230624 4

The initiation of this study relies on a targeted genome-mining approach to highlight the presence of a putative vanadium-dependent haloperoxidase-encoding gene in the deep-sea hydrothermal vent fungus Hortaea werneckii UBOCC-A-208029. To date, only three fungal vanadium-dependent haloperoxidases have been described, one from the terrestrial species Curvularia inaequalis, one from the fungal plant pathogen Botrytis cinerea, and one from a marine derived isolate identified as Alternaria didymospo ...[more]

PMID: 37354383

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Project description:The polymorphic black yeast Hortaea werneckii (Capnodiales, Ascomycota) is extremely halotolerant (growth from 0 to 30% [w/v] NaCl) and has been extensively studied as a model for halotolerance in Eukaryotes for over two decades. Its most frequent sources are hypersaline environments and adjacent sea-water habitats in temperate, subtropical and tropical climates. Although typically saprobic, H. werneckii can also act as a commensal coloniser on human skin, causing tinea nigra on hands and soles. Here, we report that addition of NaCl to culture media expands the growth range of H. werneckii to 37 °C, which explains its colonisation of human skin, with its increased salinity. The morphological and physiological plasticity/ versatility of H. werneckii indicate that a species complex might be involved. This was investigated in this polyphasic taxonomic analysis based on the global diversity of H. werneckii strains collected from hypersaline environments, and from humans and animals. Analysis of D1/D2domains of 28S and internal transcribed spacer rDNA revealed 10 and 17 genotypes, respectively, that were not always compliant. The genotypes have global distributions. Human and environmental strains with the same genotypes are intermingled. Due to the limited number of phylogenetically informative characters in the ribosomal DNA dataset, the partial genes encoding for β-tubulin (BTB) and mini-chromosome maintenance protein (MCM7) were also sequenced. The use of these genes was hampered by ambiguous sequences obtained by Sanger sequencing, as a consequence of the diploid and highly heterozygous genome of many H. werneckii strains. Analysis of the BTB and MCM7 genes showed that in some cases two copies of the gene from the same genome are positioned in distant phylogenetic clusters of the intraspecific gene tree. Analysis of whole-genome sequences of selected H. werneckii strains generally confirmed the phylogenetic distances estimated on the basis of ribosomal genes, but also showed substantial reticulation within the phylogenetic history of the strains. This is in line with the hypothesis that the diploid genomes of H. werneckii were formed by hybridizations, which have sometimes occurred between relatively divergent strains.

Project description:BackgroundHortaea werneckii is one of the most salt-tolerant species among microorganisms. It has been isolated from hypersaline waters of salterns as one of the predominant species of a group of halophilic and halotolerant melanized yeast-like fungi, arbitrarily named as "black yeasts". It has previously been shown that H. werneckii has distinct mechanisms of adaptation to high salinity environments that are not seen in salt-sensitive and only moderately salt-tolerant fungi. In H. werneckii, the HOG pathway is important for sensing the changes in environmental osmolarity, as demonstrated by identification of three main pathway components: the mitogen-activated protein kinase (MAPK) HwHog1, the MAPK kinase HwPbs2, and the putative histidine kinase osmosensor HwHhk7.ResultsIn this study, we show that the expression of HwHOG1 in salt-adapted cells depends on the environmental salinity and that HwHOG1 transcription responds rapidly but reciprocally to the acute hyper-saline or hypo-saline stress. Molecular modelling of HwHog1 reveals an overall structural homology with other MAPKs. HwHog1 complements the function of ScHog1 in the Saccharomyces cerevisiae multistress response. We also show that hyper-osmolar, oxidative and high-temperature stresses activate the HwHog1 kinase, although under high-temperature stress the signal is not transmitted via the MAPK kinase Pbs2. Identification of HOG1-like genes from other halotolerant fungi isolated from solar salterns demonstrates a high degree of similarity and excellent phylogenetic clustering with orthologues of fungal origin.ConclusionThe HOG signalling pathway has an important role in sensing and responding to hyper-osmolar, oxidative and high-temperature stresses in the halophilic fungi H. werneckii. These findings are an important advance in our understanding of the HOG pathway response to stress in H. werneckii, a proposed model organism for studying the salt tolerance of halophilic and halotolerant eukaryotes.

Project description:BackgroundFluctuations in external salinity force eukaryotic cells to respond by changes in the gene expression of proteins acting in protective biochemical processes, thus counteracting the changing osmotic pressure. The high-osmolarity glycerol (HOG) signaling pathway is essential for the efficient up-regulation of the osmoresponsive genes. In this study, the differential gene expression of the extremely halotolerant black yeast Hortaea werneckii was explored. Furthermore, the interaction of mitogen-activated protein kinase HwHog1 and RNA polymerase II with the chromatin in cells adapted to an extremely hypersaline environment was analyzed.ResultsA cDNA subtraction library was constructed for H. werneckii, adapted to moderate salinity or an extremely hypersaline environment of 4.5 M NaCl. An uncommon osmoresponsive set of 95 differentially expressed genes was identified. The majority of these had not previously been connected with the adaptation of salt-sensitive S. cerevisiae to hypersaline conditions. The transcriptional response in hypersaline-adapted and hypersaline-stressed cells showed that only a subset of the identified genes responded to acute salt-stress, whereas all were differentially expressed in adapted cells. Interaction with HwHog1 was shown for 36 of the 95 differentially expressed genes. The majority of the identified osmoresponsive and HwHog1-dependent genes in H. werneckii have not been previously reported as Hog1-dependent genes in the salt-sensitive S. cerevisiae. The study further demonstrated the co-occupancy of HwHog1 and RNA polymerase II on the chromatin of 17 up-regulated and 2 down-regulated genes in 4.5 M NaCl-adapted H. werneckii cells.ConclusionExtremely halotolerant H. werneckii represents a suitable and highly relevant organism to study cellular responses to environmental salinity. In comparison with the salt-sensitive S. cerevisiae, this yeast shows a different set of genes being expressed at high salt concentrations and interacting with HwHog1 MAP kinase, suggesting atypical processes deserving of further study.

Project description:Hortaea werneckii, ascomycetous yeast from the order Capnodiales, shows an exceptional adaptability to osmotically stressful conditions. To investigate this unusual phenotype we obtained a draft genomic sequence of a H. werneckii strain isolated from hypersaline water of solar saltern. Two of its most striking characteristics that may be associated with a halotolerant lifestyle are the large genetic redundancy and the expansion of genes encoding metal cation transporters. Although no sexual state of H. werneckii has yet been described, a mating locus with characteristics of heterothallic fungi was found. The total assembly size of the genome is 51.6 Mb, larger than most phylogenetically related fungi, coding for almost twice the usual number of predicted genes (23333). The genome appears to have experienced a relatively recent whole genome duplication, and contains two highly identical gene copies of almost every protein. This is consistent with some previous studies that reported increases in genomic DNA content triggered by exposure to salt stress. In hypersaline conditions transmembrane ion transport is of utmost importance. The analysis of predicted metal cation transporters showed that most types of transporters experienced several gene duplications at various points during their evolution. Consequently they are present in much higher numbers than expected. The resulting diversity of transporters presents interesting biotechnological opportunities for improvement of halotolerance of salt-sensitive species. The involvement of plasma P-type H? ATPases in adaptation to different concentrations of salt was indicated by their salt dependent transcription. This was not the case with vacuolar H? ATPases, which were transcribed constitutively. The availability of this genomic sequence is expected to promote the research of H. werneckii. Studying its extreme halotolerance will not only contribute to our understanding of life in hypersaline environments, but should also identify targets for improving the salt- and osmotolerance of economically important plants and microorganisms.

Dataset Information

Heterologous Expression and Biochemical Characterization of a New Chloroperoxidase Isolated from the Deep-Sea Hydrothermal Vent Black Yeast Hortaea werneckii UBOCC-A-208029.

Publications

Heterologous Expression and Biochemical Characterization of a New Chloroperoxidase Isolated from the Deep-Sea Hydrothermal Vent Black Yeast Hortaea werneckii UBOCC-A-208029.

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