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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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Phylogenetic analysis of G. lozoyensis using ITS sequences (brackets on the left) and genome protein sequences (brackets on the right). Left tree: The topology was estimated using neighbor-joining method based on the ITS sequence data from Peláez et al., 2011 [26] and the selected fungi on the right-side of the graphic. Right tree: A maximum likelihood phylogenomic tree showing evolutionary relationship of G. lozoyensis with selected ascomycete fungal species. The tree was constructed from the concatenated amino acid sequences of 878 common orthologous genes (Additional file 2: Table S2). The phylogenetic position of G. lozoyensis wild-type strain ATCC 20868 is marked in red. Branch nodes with greater than 60% support from 1000 bootstrapped pseudoreplicates are indicted with red dots in both trees. Both trees were rooted with S. cerevisiae.
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Figure 3: Phylogenetic analysis of G. lozoyensis using ITS sequences (brackets on the left) and genome protein sequences (brackets on the right). Left tree: The topology was estimated using neighbor-joining method based on the ITS sequence data from Peláez et al., 2011 [26] and the selected fungi on the right-side of the graphic. Right tree: A maximum likelihood phylogenomic tree showing evolutionary relationship of G. lozoyensis with selected ascomycete fungal species. The tree was constructed from the concatenated amino acid sequences of 878 common orthologous genes (Additional file 2: Table S2). The phylogenetic position of G. lozoyensis wild-type strain ATCC 20868 is marked in red. Branch nodes with greater than 60% support from 1000 bootstrapped pseudoreplicates are indicted with red dots in both trees. Both trees were rooted with S. cerevisiae.

Mentions: Sequencing of the G. lozoyensis WT strain ATCC 20868 with an 80× genome coverage revealed a high resolution 39.6-megabase (Mb) genome with 0.5% repeat content. Reads were assembled into 22 scaffolds (>2 kb; N50, 2.45 Mb) incorporating more than 99% of the total genomic base pairs (Figure 2a). The average gene density was 330 genes per Mb (Table 1). The 13,103 putative coding genes were assigned to different functional categories (Figure 2b). Consistent with previous studies by our group [25,26], a combined phylogenomic and phylogenetic analysis confirmed that G. lozoyensis belonged the same major phylogenetic lineage as the plant pathogenic fungi, Sclerotinia sclerotiorum and Botrytis cinerea[27], and the wood endophytic fungus, Ascocoryne sarcoides, the Helotiales [28] (Figure 3). A total of 4931 predicted proteins were assigned by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The top four categories in the KEGG functional classification were “Carbon Metabolism, Energy Metabolism, Amino Acid Metabolism, and Infectious Diseases” (Figure 4).


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)

Phylogenetic analysis of G. lozoyensis using ITS sequences (brackets on the left) and genome protein sequences (brackets on the right). Left tree: The topology was estimated using neighbor-joining method based on the ITS sequence data from Peláez et al., 2011 [26] and the selected fungi on the right-side of the graphic. Right tree: A maximum likelihood phylogenomic tree showing evolutionary relationship of G. lozoyensis with selected ascomycete fungal species. The tree was constructed from the concatenated amino acid sequences of 878 common orthologous genes (Additional file 2: Table S2). The phylogenetic position of G. lozoyensis wild-type strain ATCC 20868 is marked in red. Branch nodes with greater than 60% support from 1000 bootstrapped pseudoreplicates are indicted with red dots in both trees. Both trees were rooted with S. cerevisiae.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Phylogenetic analysis of G. lozoyensis using ITS sequences (brackets on the left) and genome protein sequences (brackets on the right). Left tree: The topology was estimated using neighbor-joining method based on the ITS sequence data from Peláez et al., 2011 [26] and the selected fungi on the right-side of the graphic. Right tree: A maximum likelihood phylogenomic tree showing evolutionary relationship of G. lozoyensis with selected ascomycete fungal species. The tree was constructed from the concatenated amino acid sequences of 878 common orthologous genes (Additional file 2: Table S2). The phylogenetic position of G. lozoyensis wild-type strain ATCC 20868 is marked in red. Branch nodes with greater than 60% support from 1000 bootstrapped pseudoreplicates are indicted with red dots in both trees. Both trees were rooted with S. cerevisiae.
Mentions: Sequencing of the G. lozoyensis WT strain ATCC 20868 with an 80× genome coverage revealed a high resolution 39.6-megabase (Mb) genome with 0.5% repeat content. Reads were assembled into 22 scaffolds (>2 kb; N50, 2.45 Mb) incorporating more than 99% of the total genomic base pairs (Figure 2a). The average gene density was 330 genes per Mb (Table 1). The 13,103 putative coding genes were assigned to different functional categories (Figure 2b). Consistent with previous studies by our group [25,26], a combined phylogenomic and phylogenetic analysis confirmed that G. lozoyensis belonged the same major phylogenetic lineage as the plant pathogenic fungi, Sclerotinia sclerotiorum and Botrytis cinerea[27], and the wood endophytic fungus, Ascocoryne sarcoides, the Helotiales [28] (Figure 3). A total of 4931 predicted proteins were assigned by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The top four categories in the KEGG functional classification were “Carbon Metabolism, Energy Metabolism, Amino Acid Metabolism, and Infectious Diseases” (Figure 4).

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