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Genomics-driven discovery of the pneumocandin biosynthetic gene cluster in the fungus Glarea lozoyensis.

Chen L, Yue Q, Zhang X, Xiang M, Wang C, Li S, Che Y, Ortiz-López FJ, Bills GF, Liu X, An Z - BMC Genomics (2013)

Bottom Line: Thus, the pneumocandin biosynthetic gene cluster is significantly more autonomous and organized than that of the recently characterized echinocandin B gene cluster.Characterization of the gene cluster provides a blueprint for engineering new pneumocandin derivatives with improved pharmacological properties.Whole genome estimation of the secondary metabolite-encoding genes from G. lozoyensis provides yet another example of the huge potential for drug discovery from natural products from the fungal kingdom.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China.

ABSTRACT

Background: The antifungal therapy caspofungin is a semi-synthetic derivative of pneumocandin B0, a lipohexapeptide produced by the fungus Glarea lozoyensis, and was the first member of the echinocandin class approved for human therapy. The nonribosomal peptide synthetase (NRPS)-polyketide synthases (PKS) gene cluster responsible for pneumocandin biosynthesis from G. lozoyensis has not been elucidated to date. In this study, we report the elucidation of the pneumocandin biosynthetic gene cluster by whole genome sequencing of the G. lozoyensis wild-type strain ATCC 20868.

Results: The pneumocandin biosynthetic gene cluster contains a NRPS (GLNRPS4) and a PKS (GLPKS4) arranged in tandem, two cytochrome P450 monooxygenases, seven other modifying enzymes, and genes for L-homotyrosine biosynthesis, a component of the peptide core. Thus, the pneumocandin biosynthetic gene cluster is significantly more autonomous and organized than that of the recently characterized echinocandin B gene cluster. Disruption mutants of GLNRPS4 and GLPKS4 no longer produced the pneumocandins (A0 and B0), and the Δglnrps4 and Δglpks4 mutants lost antifungal activity against the human pathogenic fungus Candida albicans. In addition to pneumocandins, the G. lozoyensis genome encodes a rich repertoire of natural product-encoding genes including 24 PKSs, six NRPSs, five PKS-NRPS hybrids, two dimethylallyl tryptophan synthases, and 14 terpene synthases.

Conclusions: Characterization of the gene cluster provides a blueprint for engineering new pneumocandin derivatives with improved pharmacological properties. Whole genome estimation of the secondary metabolite-encoding genes from G. lozoyensis provides yet another example of the huge potential for drug discovery from natural products from the fungal kingdom.

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CAZymes (carbohydrate-active enzymes) analysis in the G. lozoyensis genome and other fungi. (a) Total number of CAZymes in different fungi (Glarea lozoyensis, Neurospora crassa, Aspergillus nidulans, Trichoderma reesei, Fusarium graminearum, Aspergillus oryzae, Verticillium albo-atrum, Sclerotinia sclerotiorum, Glomerella graminicola, Ascocoryne sarcoides, Epichloë festucae, Tuber melanosporum, Laccaria bicolor, Piriformospora indica, and Saccharomyces cerevisiae). (b) Number of different family of CAZymes in different fungi. PL: polysaccharide lyase, GH: glycoside hydrolase, CBM: carbohydrate-binding module, CE: carbohydrate esterases, and GT: glycosyltransferase. See Additional file 2: Table S2 for a detailed tabular summary.
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Figure 5: CAZymes (carbohydrate-active enzymes) analysis in the G. lozoyensis genome and other fungi. (a) Total number of CAZymes in different fungi (Glarea lozoyensis, Neurospora crassa, Aspergillus nidulans, Trichoderma reesei, Fusarium graminearum, Aspergillus oryzae, Verticillium albo-atrum, Sclerotinia sclerotiorum, Glomerella graminicola, Ascocoryne sarcoides, Epichloë festucae, Tuber melanosporum, Laccaria bicolor, Piriformospora indica, and Saccharomyces cerevisiae). (b) Number of different family of CAZymes in different fungi. PL: polysaccharide lyase, GH: glycoside hydrolase, CBM: carbohydrate-binding module, CE: carbohydrate esterases, and GT: glycosyltransferase. See Additional file 2: Table S2 for a detailed tabular summary.

Mentions: Strains of Glarea lozoyensis have been isolated from water, plant litter or soil samples [25,26]. However, the fungus has never been observed in nature, therefore its ecological role and trophic relationships remain unknown. It has been speculated that the fungus may be a plant or plant litter saprobe for the following reasons [25,26]. The fungus belongs to the same phylogenetic lineage as Cyathicula or Crocicreas, an inconspicuous group of fungi that are weak parasites, endophytes of living plants or saprobes of senescent plants and plant litter. In the laboratory, the fungus readily colonized and sporulated on sterilized hardwood [25]. Its asexual sporulation (Figure 1b) resembled that of a heterogeneous group of asexually reproducing fungi known as aero-aquatic fungi that often colonize plant debris in periodically inundated habitats [29,30]. Several recent studies have demonstrated a strong relationship between the suite of carbohydrate active enzymes (CAZymes, http://www.cazy.org) in fungal genomes and their saprobic, parasitic or necrotrophic life strategies [31-33]. Such investigations have focused on those CAZymes involved in polysaccharide degradation and have contributed to a thorough understanding of the ecological role of a fungus. To infer whether G. lozoyensis might be a biotroph, saprotroph or necrotroph, we analyzed its complement of CAZy gene families and genes. The putative CAZymes in G. lozoyensis were identified using the CAZy annotation pipeline (http://mothra.ornl.gov/cgi-bin/cat.cgi) [34,35] and were compared to a selection of ascomycete and basidiomycete fungi (Figure 5a and 5b). At least 345 CAZymes in the five principal category families were identified in the genome (Figure 5a). This value is similar to the number of CAZymes found in known plant cell wall degrading ascomycetes, including the wood-inhabiting endophyte A. sarcoides, but significantly higher than the yeast Saccharomyces cerevisiae, and the plant biotrophic symbionts Laccaria bicolor, Epichloë festucae, and Tuber melanosporum (Figure 5a). A total of 180 glycoside hydrolases (GH) in 70 families were found in the G. lozoyensis genome, which is slightly less than average compared to other filamentous plant associated ascomycetes [36]. Likewise, the number of 67 glycosyl transferases (GT) in 35 families was also comparable to other plant inhabiting ascomycetes (Figure 5b). Average numbers of polysaccharide lyases (PL, 5), carbohydrate esterases (CE, 22) were found. However, a relatively abundant number of carbohydrate binding modules (CMB, 71) were identified. Therefore, its complement of genes associated with carbohydrate degradation and metabolism were consistent with those of other plant-associated ascomycetes.


Genomics-driven discovery of the pneumocandin biosynthetic gene cluster in the fungus Glarea lozoyensis.

Chen L, Yue Q, Zhang X, Xiang M, Wang C, Li S, Che Y, Ortiz-López FJ, Bills GF, Liu X, An Z - BMC Genomics (2013)

CAZymes (carbohydrate-active enzymes) analysis in the G. lozoyensis genome and other fungi. (a) Total number of CAZymes in different fungi (Glarea lozoyensis, Neurospora crassa, Aspergillus nidulans, Trichoderma reesei, Fusarium graminearum, Aspergillus oryzae, Verticillium albo-atrum, Sclerotinia sclerotiorum, Glomerella graminicola, Ascocoryne sarcoides, Epichloë festucae, Tuber melanosporum, Laccaria bicolor, Piriformospora indica, and Saccharomyces cerevisiae). (b) Number of different family of CAZymes in different fungi. PL: polysaccharide lyase, GH: glycoside hydrolase, CBM: carbohydrate-binding module, CE: carbohydrate esterases, and GT: glycosyltransferase. See Additional file 2: Table S2 for a detailed tabular summary.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672099&req=5

Figure 5: CAZymes (carbohydrate-active enzymes) analysis in the G. lozoyensis genome and other fungi. (a) Total number of CAZymes in different fungi (Glarea lozoyensis, Neurospora crassa, Aspergillus nidulans, Trichoderma reesei, Fusarium graminearum, Aspergillus oryzae, Verticillium albo-atrum, Sclerotinia sclerotiorum, Glomerella graminicola, Ascocoryne sarcoides, Epichloë festucae, Tuber melanosporum, Laccaria bicolor, Piriformospora indica, and Saccharomyces cerevisiae). (b) Number of different family of CAZymes in different fungi. PL: polysaccharide lyase, GH: glycoside hydrolase, CBM: carbohydrate-binding module, CE: carbohydrate esterases, and GT: glycosyltransferase. See Additional file 2: Table S2 for a detailed tabular summary.
Mentions: Strains of Glarea lozoyensis have been isolated from water, plant litter or soil samples [25,26]. However, the fungus has never been observed in nature, therefore its ecological role and trophic relationships remain unknown. It has been speculated that the fungus may be a plant or plant litter saprobe for the following reasons [25,26]. The fungus belongs to the same phylogenetic lineage as Cyathicula or Crocicreas, an inconspicuous group of fungi that are weak parasites, endophytes of living plants or saprobes of senescent plants and plant litter. In the laboratory, the fungus readily colonized and sporulated on sterilized hardwood [25]. Its asexual sporulation (Figure 1b) resembled that of a heterogeneous group of asexually reproducing fungi known as aero-aquatic fungi that often colonize plant debris in periodically inundated habitats [29,30]. Several recent studies have demonstrated a strong relationship between the suite of carbohydrate active enzymes (CAZymes, http://www.cazy.org) in fungal genomes and their saprobic, parasitic or necrotrophic life strategies [31-33]. Such investigations have focused on those CAZymes involved in polysaccharide degradation and have contributed to a thorough understanding of the ecological role of a fungus. To infer whether G. lozoyensis might be a biotroph, saprotroph or necrotroph, we analyzed its complement of CAZy gene families and genes. The putative CAZymes in G. lozoyensis were identified using the CAZy annotation pipeline (http://mothra.ornl.gov/cgi-bin/cat.cgi) [34,35] and were compared to a selection of ascomycete and basidiomycete fungi (Figure 5a and 5b). At least 345 CAZymes in the five principal category families were identified in the genome (Figure 5a). This value is similar to the number of CAZymes found in known plant cell wall degrading ascomycetes, including the wood-inhabiting endophyte A. sarcoides, but significantly higher than the yeast Saccharomyces cerevisiae, and the plant biotrophic symbionts Laccaria bicolor, Epichloë festucae, and Tuber melanosporum (Figure 5a). A total of 180 glycoside hydrolases (GH) in 70 families were found in the G. lozoyensis genome, which is slightly less than average compared to other filamentous plant associated ascomycetes [36]. Likewise, the number of 67 glycosyl transferases (GT) in 35 families was also comparable to other plant inhabiting ascomycetes (Figure 5b). Average numbers of polysaccharide lyases (PL, 5), carbohydrate esterases (CE, 22) were found. However, a relatively abundant number of carbohydrate binding modules (CMB, 71) were identified. Therefore, its complement of genes associated with carbohydrate degradation and metabolism were consistent with those of other plant-associated ascomycetes.

Bottom Line: Thus, the pneumocandin biosynthetic gene cluster is significantly more autonomous and organized than that of the recently characterized echinocandin B gene cluster.Characterization of the gene cluster provides a blueprint for engineering new pneumocandin derivatives with improved pharmacological properties.Whole genome estimation of the secondary metabolite-encoding genes from G. lozoyensis provides yet another example of the huge potential for drug discovery from natural products from the fungal kingdom.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China.

ABSTRACT

Background: The antifungal therapy caspofungin is a semi-synthetic derivative of pneumocandin B0, a lipohexapeptide produced by the fungus Glarea lozoyensis, and was the first member of the echinocandin class approved for human therapy. The nonribosomal peptide synthetase (NRPS)-polyketide synthases (PKS) gene cluster responsible for pneumocandin biosynthesis from G. lozoyensis has not been elucidated to date. In this study, we report the elucidation of the pneumocandin biosynthetic gene cluster by whole genome sequencing of the G. lozoyensis wild-type strain ATCC 20868.

Results: The pneumocandin biosynthetic gene cluster contains a NRPS (GLNRPS4) and a PKS (GLPKS4) arranged in tandem, two cytochrome P450 monooxygenases, seven other modifying enzymes, and genes for L-homotyrosine biosynthesis, a component of the peptide core. Thus, the pneumocandin biosynthetic gene cluster is significantly more autonomous and organized than that of the recently characterized echinocandin B gene cluster. Disruption mutants of GLNRPS4 and GLPKS4 no longer produced the pneumocandins (A0 and B0), and the Δglnrps4 and Δglpks4 mutants lost antifungal activity against the human pathogenic fungus Candida albicans. In addition to pneumocandins, the G. lozoyensis genome encodes a rich repertoire of natural product-encoding genes including 24 PKSs, six NRPSs, five PKS-NRPS hybrids, two dimethylallyl tryptophan synthases, and 14 terpene synthases.

Conclusions: Characterization of the gene cluster provides a blueprint for engineering new pneumocandin derivatives with improved pharmacological properties. Whole genome estimation of the secondary metabolite-encoding genes from G. lozoyensis provides yet another example of the huge potential for drug discovery from natural products from the fungal kingdom.

Show MeSH
Related in: MedlinePlus