Identification and heterologous expression of the globomycin biosynthetic gene cluster

Oves-Costales, Daniel; Gren, Tetiana; Sterndorff, Eva Baggesgaard; Martín, Jesús; Ortiz‐López, Francisco Javier; Jørgensen, Tue Sparholt; Jiang, Xinglin; Román‐Hurtado, Fernando; Reyes, Fernando; Genilloud, Olga; Weber, Tilmann

doi:10.1016/j.synbio.2023.02.001

Cited by 7 publications

(9 citation statements)

References 36 publications

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“…Production of Azodyrecins in Heterologous Hosts. To confirm the identity of the azd BGC, it was cloned and heterologously expressed in S. albidof lavus J1074 41 and S. coelicolor M1146, 40 using a modified version 49 of the CATCHcloning 45 procedure. This method involves the targeted extraction of predicted azd BGC using CRISPR-Cas9 and single guide RNAs (sgRNAs) in an in vitro system.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2

Maleckis,

Wibowo,

Gren

et al. 2024

ACS Chem. Biol.

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Azoxy compounds are a distinctive group of bioactive secondary metabolites characterized by a unique RN� N + (O − )R moiety. The azoxy moiety is present in various classes of metabolites that exhibit various biological activities. The enzymatic mechanisms underlying azoxy bond formation remain enigmatic. Azodyrecins are cytotoxic azoxy metabolites produced by Streptomyces mirabilis P8-A2. Here, we cloned and confirmed the putative azd biosynthetic gene cluster through CATCH cloning followed by expression and production of azodyrecins in two heterologous hosts, S. albidoflavus J1074 and S. coelicolor M1146, respectively. We explored the function of 14 enzymes in azodyrecin biosynthesis through gene knockout using CRISPR-Cas9 base editing in the native producer, S. mirabilis P8-A2. The key intermediates were analyzed in the mutants through MS/MS fragmentation studies, revealing azoxy bond formation via the conversion of hydrazine to an azo compound followed by further oxygenation. Enzymes involved in modifications of the precursor could be postulated based on their predicted function and the intermediates identified in the knockout strains. Moreover, the distribution of the azoxy biosynthetic gene clusters across Streptomyces spp. genomes is explored, highlighting the presence of these clusters in over 20% of the Streptomyces spp. genomes and revealing that azoxymycin and valanimycin are scarce, while azodyrecin and KA57A-like clusters are widely distributed across the phylogenetic tree.

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Section: ■ Results and Discussionmentioning

confidence: 99%

“…The putative azodyrecin BGC was cloned according to modified CATCH-cloning procedure. To capture the azodyrecin BGC, Cas9 protospacer sequences were designed using Geneious Prime to find sgRNA bunding sites and score them based on their specificity and activity .…”

Section: Methodsmentioning

confidence: 99%

Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2

Maleckis,

Wibowo,

Gren

et al. 2024

ACS Chem. Biol.

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“…The putative azodyrecin BGC was cloned according to modified 47 CATCH-cloning 72 procedure. To capture the azodyrecin BGC, Cas9 protospacer sequences were designed using Geneious Prime to find sgRNA bunding sites and score them based on their specificity 73 and activity 74 .…”

Section: Methodsmentioning

confidence: 99%

“…To confirm the identity of the azd BGC, it was cloned and heterologously expressed in S. albidoflavus J1074 39 and S. coelicolor M1146 38 , using a modified version 47 of the CATCHcloning 45 procedure. This method involves the targeted extraction of the predicted azd BGC using CRISPR-Cas9 and single guide RNAs (sgRNAs) in an in vitro system.…”

Section: Produc�on Of Azodyrecin In Heterologous Hostsmentioning

confidence: 99%

Biosynthesis of the azoxy compound azodyrecin fromStreptomyces mirabilisP8-A2

Maleckis,

Wibowo,

Gren

et al. 2023

Preprint

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Azoxy compounds are a distinctive group of bioactive secondary metabolites, characterized by a unique RN=N+(O-)R moiety. The azoxy moiety is present in various classes of metabolites that exhibit various biological activities. The enzymatic mechanisms underlying azoxy bond formation remain enigmatic. Azodyrecins are cytotoxic azoxy metabolites produced by Streptomyces mirabilis P8-A2. Here we cloned and confirmed the putative azd biosynthetic gene cluster through CATCH cloning followed by expression and production of azodyrecins in two heterologous hosts, S. albidoflavus J1074 and S. coelicolor M1146, respectively. We explored the function of 14 enzymes in azodyrecin biosynthesis through gene knock-out using CRISPR-Cas9 base editing in the native producer, S. mirabilis P8-A2. The key intermediates were analyzed in the mutants through MS/MS fragmentation studies, revealing azoxy bond formation via the conversion of hydrazine to azo compound; followed by further oxygenation. Additionally, N-oxygenase and dehydrogenase activities were confirmed among 8 core biosynthetic genes and five helper genes. Moreover, the distribution of the azoxy biosynthetic gene clusters across Streptomyces spp. genomes is explored, highlighting the presence of these clusters in over 20% of the Streptomyces spp. genomes and revealing that azoxymycin and valanimycin are scarce, while azodyrecin and KA57A like clusters are widely distributed across the phylogenetic tree.

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“…CA-278952 as a producer of globomycin, allowing us to pin down its hitherto unknown biosynthetic gene cluster (BGC). 25 Close examination of the remaining BGCs from the genome of this strain led to the discovery of an unknown cluster (dil BGC) putatively encoding the biosynthesis of a lipopeptide, which shared some similarity to the cluster responsible for viennamycin biosynthesis 26 (Figure S29 of the Supporting Information). Viennamycins are cysteic/ aspartic-acid-containing lipodepsipeptides with a 7 amino acid macrocycle, which lack the calcium-binding motif and have no antibacterial activity.…”

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confidence: 99%

Dilarmycins A–C, Calcium-Dependent Lipopeptide Antibiotics with a Non-canonical Ca²⁺-Binding Motif

Fernández-Pastor,

Ortiz-López,

Oves-Costales

et al. 2024

Org. Lett.

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Genome analysis of strain Streptomyces sp. CA-278952 revealed a biosynthetic gene cluster encoding a putative lipopeptide with a sequence containing an Asp-Gly-Glu-Ala motif. We envisioned that this motif could mimic the canonical Asp-X-Asp-Gly sequence found in previously reported calcium-dependent lipopeptide antibiotics. Chemical investigation of the producing strain led to the discovery of three novel lipodepsipeptides, dilarmycins A−C. The calcium-dependent antibacterial activity of the new compounds was confirmed against the Gram-positive pathogens methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus.

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Identification and heterologous expression of the globomycin biosynthetic gene cluster

Cited by 7 publications

References 36 publications

Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2

Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2

Biosynthesis of the azoxy compound azodyrecin fromStreptomyces mirabilisP8-A2

Dilarmycins A–C, Calcium-Dependent Lipopeptide Antibiotics with a Non-canonical Ca²⁺-Binding Motif

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Identification and heterologous expression of the globomycin biosynthetic gene cluster

Cited by 7 publications

References 36 publications

Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2

Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2

Biosynthesis of the azoxy compound azodyrecin fromStreptomyces mirabilisP8-A2

Dilarmycins A–C, Calcium-Dependent Lipopeptide Antibiotics with a Non-canonical Ca2+-Binding Motif

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Product

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Dilarmycins A–C, Calcium-Dependent Lipopeptide Antibiotics with a Non-canonical Ca²⁺-Binding Motif