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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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Domain prediction and phylogenetic analysis of polyketide synthases (PKSs) and polyketide synthases-nonribosomal peptide synthetase hybrids (PKS-NRPS hybrids) in G. lozoyensis and other characterized fungal PKSs. PKS and PKS-NRPS domains from G. lozoyensis were annotated by SMURF, anti-SMASH and SWISS-MODEL tools. SAT, starter unit acyltransferase domain; KS, ketosynthase domain; AT, acyltransferase domain; PT, product template domain; DH, dehydratase domain; ER, enoylreductase domain; KR, β-ketoacylreductase domain; MT, methyltransferase domain; ACP, acyl carrier protein; TE, thioesterase domain; A, adenylation domain; T, thiolation domain; C, condensation domain; R, reductive domain. Genealogy of PKSs and PKS-NRPSs was inferred by neighbor-joining analysis of the aligned amino acid sequences of the KS domains. Classification of PKSs and PKS-NRPSs sharing a common domain organization are highlighted by gray shading. Branch length indicates number of inferred amino acid changes. Red dots indicate branch nodes with >60% support. PKSs from G. lozoyensis are marked in red. See in Additional file 2: Table S3 for details of gene designations and their corresponding metabolites and references.
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Figure 6: Domain prediction and phylogenetic analysis of polyketide synthases (PKSs) and polyketide synthases-nonribosomal peptide synthetase hybrids (PKS-NRPS hybrids) in G. lozoyensis and other characterized fungal PKSs. PKS and PKS-NRPS domains from G. lozoyensis were annotated by SMURF, anti-SMASH and SWISS-MODEL tools. SAT, starter unit acyltransferase domain; KS, ketosynthase domain; AT, acyltransferase domain; PT, product template domain; DH, dehydratase domain; ER, enoylreductase domain; KR, β-ketoacylreductase domain; MT, methyltransferase domain; ACP, acyl carrier protein; TE, thioesterase domain; A, adenylation domain; T, thiolation domain; C, condensation domain; R, reductive domain. Genealogy of PKSs and PKS-NRPSs was inferred by neighbor-joining analysis of the aligned amino acid sequences of the KS domains. Classification of PKSs and PKS-NRPSs sharing a common domain organization are highlighted by gray shading. Branch length indicates number of inferred amino acid changes. Red dots indicate branch nodes with >60% support. PKSs from G. lozoyensis are marked in red. See in Additional file 2: Table S3 for details of gene designations and their corresponding metabolites and references.

Mentions: An unexpected feature of the G. lozoyensis genome was its remarkable diversity of polyketide biosynthetic pathways and having at least 29 recognizable core PKS genes (Figure 6). Domain structure analysis revealed eight non-reducing PKSs, one partially-reducing PKS, four PKS-NRPS hybrids encoding partially reducing polyketides [37] and 16 PKSs encoding for highly reducing polyketides, including GLPKS4 and one PKS-NRPS hybrid (GLPKS3-NRPS) (Figure 6). A phylogenetic tree based on amino acid sequences of the ketosynthase domains (KS) was constructed for the 24 PKSs and five PKS-NRPS hybrids in G. lozoyensis and 71 functionally characterized fungal PKSs encoding the products with known chemical structures (Figure 6, Additional file 2: Table S3). All four fungal-type PKS-NRPS hybrids (GLPKS26-NRPS, GLPKS27-NRPS, GLPKS28-NRPS, and GLPKS29-NRPS) were grouped with similar PKS-NRPS hybrids, such as those involved in the biosynthesis of the tetramic acids and HIV-1 integrase inhibitor equisetin (EqiS). Interestingly the four PKS-NRPS hybrids were also clustered with the HMG-CoA reductase inhibitor lovastatin (LDKS = LovB) which is proposed to be a truncated PKS-NRPS hybrid [38,39]. GLPKS8 and GLPKS9 were predicted to be non-reducing PKSs related to the PKSs responsible for biosynthesis of the metabolites mycophenolic acid and citrinin. GLPKS13 and three other G. lozoyensis PKSs (GLPKS10, GLPKS18, and GLPKS24) were grouped with the PKSs of lovastatin side chain (LNKS = LovF) [40] and the tetraketide acyl side chain of zaragozic acid A [41]. GLPKS19 and GLPKS11 shared significant homology with the T-toxin encoding gene CHPKS1 of Cochliobolus heterostrophus[42,43]. Six more G. lozoyensis PKSs (GLPKS4, GLPKS25, GLPKS12, GLPKS7, GLPKS14, and GLPKS21) clustered with the hepato- and nephro-toxic fumonisin B1 produced by Gibberella fujikuroi[44] and the solanapyrone Sol1 PKS of Alternaria solani[45]. The previously characterized GLPKS2, encoding for the biosynthesis of 6-methylsalicylic acid [20], grouped tightly with two other fungal 6-methylsalicylic acid PKSs, ATATX from A. terreus and MSAS from Penicillium patulum[46,47]. GLPKS1 has been previously identified as the G. lozoyensis melanin biosynthesis gene [21], and it clustered with other fungal di- and tetra-hydroxynaphthalene melanin biosynthesis genes, e.g. Hypoxylon pulicicidum (formerly Nodulisporium sp.) (NSPKS1) [48] and Colletotrichum lagenarium (CLPKS1) [49]. The ketosynthase sequence of G. lozoyensis GLPKS20 exhibited sequence similarities to genes involved in the biosynthesis of viridicatumtoxin [50]. Adjacent to the large groups of melanin and conidial pigment genes were the mycotoxin sterigmatocystin PKS (ANST) from A. nidulans[51] and the GLPKS5 from G. lozoyensis. Distantly related to the pigment PKSs was the A. nidulans orsellinic acid PKS protein OrsA [52,53], and GLPKS23 shared the same domain structure with OrsA. We speculated that orsellinic acid or related compounds may be produced by G. lozoyensis, and analysis of fermentations of G. lozoyensis confirmed that it produced isolecanoric acid (an orsellinic acid dimer) and pseudogyrophoric acid (a new orsellinic acid trimer) in certain culture media (Additional file 1: Figure S2). Therefore, we propose that GLPKS23 is responsible for orsellinic acid biosynthesis in G. lozoyensis. Cluster analysis revealed that a highly reducing PKS (GLPKS17) was proximal to a non-reducing PKS (GLPKS16) in the same cluster (Additional file 1: Figure S1). This tandem PKS structure was similar to that of the PKSs responsible for the biosynthesis of resorcylic acid lactones, e.g. radicicol and hypothemycin, and in fact, GLPKS16 appeared to be an ortholog of Hpm3 and RDC1 (Figure 6) [54,55].


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)

Domain prediction and phylogenetic analysis of polyketide synthases (PKSs) and polyketide synthases-nonribosomal peptide synthetase hybrids (PKS-NRPS hybrids) in G. lozoyensis and other characterized fungal PKSs. PKS and PKS-NRPS domains from G. lozoyensis were annotated by SMURF, anti-SMASH and SWISS-MODEL tools. SAT, starter unit acyltransferase domain; KS, ketosynthase domain; AT, acyltransferase domain; PT, product template domain; DH, dehydratase domain; ER, enoylreductase domain; KR, β-ketoacylreductase domain; MT, methyltransferase domain; ACP, acyl carrier protein; TE, thioesterase domain; A, adenylation domain; T, thiolation domain; C, condensation domain; R, reductive domain. Genealogy of PKSs and PKS-NRPSs was inferred by neighbor-joining analysis of the aligned amino acid sequences of the KS domains. Classification of PKSs and PKS-NRPSs sharing a common domain organization are highlighted by gray shading. Branch length indicates number of inferred amino acid changes. Red dots indicate branch nodes with >60% support. PKSs from G. lozoyensis are marked in red. See in Additional file 2: Table S3 for details of gene designations and their corresponding metabolites and references.
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Related In: Results  -  Collection

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Figure 6: Domain prediction and phylogenetic analysis of polyketide synthases (PKSs) and polyketide synthases-nonribosomal peptide synthetase hybrids (PKS-NRPS hybrids) in G. lozoyensis and other characterized fungal PKSs. PKS and PKS-NRPS domains from G. lozoyensis were annotated by SMURF, anti-SMASH and SWISS-MODEL tools. SAT, starter unit acyltransferase domain; KS, ketosynthase domain; AT, acyltransferase domain; PT, product template domain; DH, dehydratase domain; ER, enoylreductase domain; KR, β-ketoacylreductase domain; MT, methyltransferase domain; ACP, acyl carrier protein; TE, thioesterase domain; A, adenylation domain; T, thiolation domain; C, condensation domain; R, reductive domain. Genealogy of PKSs and PKS-NRPSs was inferred by neighbor-joining analysis of the aligned amino acid sequences of the KS domains. Classification of PKSs and PKS-NRPSs sharing a common domain organization are highlighted by gray shading. Branch length indicates number of inferred amino acid changes. Red dots indicate branch nodes with >60% support. PKSs from G. lozoyensis are marked in red. See in Additional file 2: Table S3 for details of gene designations and their corresponding metabolites and references.
Mentions: An unexpected feature of the G. lozoyensis genome was its remarkable diversity of polyketide biosynthetic pathways and having at least 29 recognizable core PKS genes (Figure 6). Domain structure analysis revealed eight non-reducing PKSs, one partially-reducing PKS, four PKS-NRPS hybrids encoding partially reducing polyketides [37] and 16 PKSs encoding for highly reducing polyketides, including GLPKS4 and one PKS-NRPS hybrid (GLPKS3-NRPS) (Figure 6). A phylogenetic tree based on amino acid sequences of the ketosynthase domains (KS) was constructed for the 24 PKSs and five PKS-NRPS hybrids in G. lozoyensis and 71 functionally characterized fungal PKSs encoding the products with known chemical structures (Figure 6, Additional file 2: Table S3). All four fungal-type PKS-NRPS hybrids (GLPKS26-NRPS, GLPKS27-NRPS, GLPKS28-NRPS, and GLPKS29-NRPS) were grouped with similar PKS-NRPS hybrids, such as those involved in the biosynthesis of the tetramic acids and HIV-1 integrase inhibitor equisetin (EqiS). Interestingly the four PKS-NRPS hybrids were also clustered with the HMG-CoA reductase inhibitor lovastatin (LDKS = LovB) which is proposed to be a truncated PKS-NRPS hybrid [38,39]. GLPKS8 and GLPKS9 were predicted to be non-reducing PKSs related to the PKSs responsible for biosynthesis of the metabolites mycophenolic acid and citrinin. GLPKS13 and three other G. lozoyensis PKSs (GLPKS10, GLPKS18, and GLPKS24) were grouped with the PKSs of lovastatin side chain (LNKS = LovF) [40] and the tetraketide acyl side chain of zaragozic acid A [41]. GLPKS19 and GLPKS11 shared significant homology with the T-toxin encoding gene CHPKS1 of Cochliobolus heterostrophus[42,43]. Six more G. lozoyensis PKSs (GLPKS4, GLPKS25, GLPKS12, GLPKS7, GLPKS14, and GLPKS21) clustered with the hepato- and nephro-toxic fumonisin B1 produced by Gibberella fujikuroi[44] and the solanapyrone Sol1 PKS of Alternaria solani[45]. The previously characterized GLPKS2, encoding for the biosynthesis of 6-methylsalicylic acid [20], grouped tightly with two other fungal 6-methylsalicylic acid PKSs, ATATX from A. terreus and MSAS from Penicillium patulum[46,47]. GLPKS1 has been previously identified as the G. lozoyensis melanin biosynthesis gene [21], and it clustered with other fungal di- and tetra-hydroxynaphthalene melanin biosynthesis genes, e.g. Hypoxylon pulicicidum (formerly Nodulisporium sp.) (NSPKS1) [48] and Colletotrichum lagenarium (CLPKS1) [49]. The ketosynthase sequence of G. lozoyensis GLPKS20 exhibited sequence similarities to genes involved in the biosynthesis of viridicatumtoxin [50]. Adjacent to the large groups of melanin and conidial pigment genes were the mycotoxin sterigmatocystin PKS (ANST) from A. nidulans[51] and the GLPKS5 from G. lozoyensis. Distantly related to the pigment PKSs was the A. nidulans orsellinic acid PKS protein OrsA [52,53], and GLPKS23 shared the same domain structure with OrsA. We speculated that orsellinic acid or related compounds may be produced by G. lozoyensis, and analysis of fermentations of G. lozoyensis confirmed that it produced isolecanoric acid (an orsellinic acid dimer) and pseudogyrophoric acid (a new orsellinic acid trimer) in certain culture media (Additional file 1: Figure S2). Therefore, we propose that GLPKS23 is responsible for orsellinic acid biosynthesis in G. lozoyensis. Cluster analysis revealed that a highly reducing PKS (GLPKS17) was proximal to a non-reducing PKS (GLPKS16) in the same cluster (Additional file 1: Figure S1). This tandem PKS structure was similar to that of the PKSs responsible for the biosynthesis of resorcylic acid lactones, e.g. radicicol and hypothemycin, and in fact, GLPKS16 appeared to be an ortholog of Hpm3 and RDC1 (Figure 6) [54,55].

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