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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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Chemical and functional analysis of pneumocandins produced by wild-type and mutant strains of G. lozoyensis. (a) HPLC-MS profiles of chemical extracts from the wild-type (WT), glnrps4 deletion mutant, and glpks4 deletion mutant of G. lozoyensis. Full-scan + mode spectra were acquired in over a scan range of m/z 80–1,200. When grown in FGY broth, pneumocandin B0 (peak 1 m/z = 1065) and pneumocandin A0 (peak 2 m/z = 1079) were detected in the WT strain. Deletion of glnrps4 and glpks4 abolished pneumocandin B0 and pneumocandin A0 production in the mutant strains. (b) Antifungal activity of culture extracts from the WT and corresponding inactive extracts from glnrps4 and glpks4 mutants of G. lozoyensis. Purified pneumocandin B0 (5 mg mL-1) and DMSO (100%) were used as positive and negative controls, respectively.
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Figure 9: Chemical and functional analysis of pneumocandins produced by wild-type and mutant strains of G. lozoyensis. (a) HPLC-MS profiles of chemical extracts from the wild-type (WT), glnrps4 deletion mutant, and glpks4 deletion mutant of G. lozoyensis. Full-scan + mode spectra were acquired in over a scan range of m/z 80–1,200. When grown in FGY broth, pneumocandin B0 (peak 1 m/z = 1065) and pneumocandin A0 (peak 2 m/z = 1079) were detected in the WT strain. Deletion of glnrps4 and glpks4 abolished pneumocandin B0 and pneumocandin A0 production in the mutant strains. (b) Antifungal activity of culture extracts from the WT and corresponding inactive extracts from glnrps4 and glpks4 mutants of G. lozoyensis. Purified pneumocandin B0 (5 mg mL-1) and DMSO (100%) were used as positive and negative controls, respectively.

Mentions: To verify whether the gene cluster was responsible for pneumocandin biosynthesis, glnrps4 and glpks4 were knocked out by homologous replacement with an Agrobacterium-mediated transformation protocol developed previously for G. lozoyensis, and the deletions were verified by PCR analysis (Additional file 1: Figure S4b and S4c). Twelve and ten positive transformants were recovered for the GLNRPS4 and GLPKS4 knockouts, respectively. After growing the fungi in FGY medium and comparative analysis of the extracts by HPLC-MS using purified pneumocandin B0 as a standard, the two major pneumocandins (A0 and B0) were produced by the G. lozoyensis WT strain as expected, but the pneumocandins were absent in the glnrps4 and glpks4 knockout mutants (Figure 9a). Consistent with earlier observations [5], the WT strain produced pneumocandin A0 in larger quantities than pneumocandin B0 (Figure 9a, Additional file 1: Figure S4d). Antifungal assays showed that crude extracts from the WT strain caused zones of inhibition against the yeast C. albicans, whereas the crude extracts from mutants Δglnrps4 and Δglpks4 were inactive (Figure 9b). These results demonstrated that both glnrps4 and glpks4 were essential for biosynthesis of the pneumocandin core structure as predicted.


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)

Chemical and functional analysis of pneumocandins produced by wild-type and mutant strains of G. lozoyensis. (a) HPLC-MS profiles of chemical extracts from the wild-type (WT), glnrps4 deletion mutant, and glpks4 deletion mutant of G. lozoyensis. Full-scan + mode spectra were acquired in over a scan range of m/z 80–1,200. When grown in FGY broth, pneumocandin B0 (peak 1 m/z = 1065) and pneumocandin A0 (peak 2 m/z = 1079) were detected in the WT strain. Deletion of glnrps4 and glpks4 abolished pneumocandin B0 and pneumocandin A0 production in the mutant strains. (b) Antifungal activity of culture extracts from the WT and corresponding inactive extracts from glnrps4 and glpks4 mutants of G. lozoyensis. Purified pneumocandin B0 (5 mg mL-1) and DMSO (100%) were used as positive and negative controls, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 9: Chemical and functional analysis of pneumocandins produced by wild-type and mutant strains of G. lozoyensis. (a) HPLC-MS profiles of chemical extracts from the wild-type (WT), glnrps4 deletion mutant, and glpks4 deletion mutant of G. lozoyensis. Full-scan + mode spectra were acquired in over a scan range of m/z 80–1,200. When grown in FGY broth, pneumocandin B0 (peak 1 m/z = 1065) and pneumocandin A0 (peak 2 m/z = 1079) were detected in the WT strain. Deletion of glnrps4 and glpks4 abolished pneumocandin B0 and pneumocandin A0 production in the mutant strains. (b) Antifungal activity of culture extracts from the WT and corresponding inactive extracts from glnrps4 and glpks4 mutants of G. lozoyensis. Purified pneumocandin B0 (5 mg mL-1) and DMSO (100%) were used as positive and negative controls, respectively.
Mentions: To verify whether the gene cluster was responsible for pneumocandin biosynthesis, glnrps4 and glpks4 were knocked out by homologous replacement with an Agrobacterium-mediated transformation protocol developed previously for G. lozoyensis, and the deletions were verified by PCR analysis (Additional file 1: Figure S4b and S4c). Twelve and ten positive transformants were recovered for the GLNRPS4 and GLPKS4 knockouts, respectively. After growing the fungi in FGY medium and comparative analysis of the extracts by HPLC-MS using purified pneumocandin B0 as a standard, the two major pneumocandins (A0 and B0) were produced by the G. lozoyensis WT strain as expected, but the pneumocandins were absent in the glnrps4 and glpks4 knockout mutants (Figure 9a). Consistent with earlier observations [5], the WT strain produced pneumocandin A0 in larger quantities than pneumocandin B0 (Figure 9a, Additional file 1: Figure S4d). Antifungal assays showed that crude extracts from the WT strain caused zones of inhibition against the yeast C. albicans, whereas the crude extracts from mutants Δglnrps4 and Δglpks4 were inactive (Figure 9b). These results demonstrated that both glnrps4 and glpks4 were essential for biosynthesis of the pneumocandin core structure as predicted.

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