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Identification of the Lomofungin Biosynthesis Gene Cluster and Associated Flavin-Dependent Monooxygenase Gene in Streptomyces lomondensis S015.

Zhang C, Sheng C, Wang W, Hu H, Peng H, Zhang X - PLoS ONE (2015)

Bottom Line: Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin.This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position.This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.

ABSTRACT
Streptomyces lomondensis S015 synthesizes the broad-spectrum phenazine antibiotic lomofungin. Whole genome sequencing of this strain revealed a genomic locus consisting of 23 open reading frames that includes the core phenazine biosynthesis gene cluster lphzGFEDCB. lomo10, encoding a putative flavin-dependent monooxygenase, was also identified in this locus. Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin. This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces.

No MeSH data available.


Related in: MedlinePlus

Spectrum of compound A following LC-HRMS analysis.The caculated mass of compound 1 was determined to be 298.25 (m/z 299.07).
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pone.0136228.g005: Spectrum of compound A following LC-HRMS analysis.The caculated mass of compound 1 was determined to be 298.25 (m/z 299.07).

Mentions: To confirm the structure of the new compound, compound A was purified from 10 l of fermentation broth. In total, 80 mg of compound A was obtained following purification. The exact mass of compound A obtained from LC-HRMS was 299.07 for [C15H10N2O5]+ (calculated mass, 298.25; Fig 5). This calculated mass was 16 Da smaller than the known mass of lomofungin, suggesting that compound A contained one less hydroxyl than lomofungin (C15H10N2O6, MW 314 Da) [47]. Structural analysis was performed by 1H NMR, 13C NMR, distortionless enhancement by polarization transfer-90, 1H-1H correlation spectroscopy, heteronuclear multiple bond correlation, and heteronuclear multiple quantum coherence analyses, and the proton and carbon chemical shifts of compound A are shown in Table 4 and in S2 Fig, respectively. The 1H-NMR spectra contained four proton signals (δH, 7.39–8.41 ppm) that are typical of double bonds (ring hydrogen), along with corresponding 13C-NMR spectra of carbon atoms (δC, 110.1–134.5 ppm), suggesting that compound A has a phenazine ring in the core of its structure. Compared with the reported NMR data for lomofungin [47], the main differences were the absence of a hydroxyl hydrogen at δH 11.22, and the presence of hydrogen ring at δH 8.27. Therefore, compound A was predicted to be 1-carbomethoxy-6-formy-4,9-dihydroxy phenazine, a new chemical compound missing the C-7 hydroxyl of lomofungin (Fig 6).


Identification of the Lomofungin Biosynthesis Gene Cluster and Associated Flavin-Dependent Monooxygenase Gene in Streptomyces lomondensis S015.

Zhang C, Sheng C, Wang W, Hu H, Peng H, Zhang X - PLoS ONE (2015)

Spectrum of compound A following LC-HRMS analysis.The caculated mass of compound 1 was determined to be 298.25 (m/z 299.07).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549113&req=5

pone.0136228.g005: Spectrum of compound A following LC-HRMS analysis.The caculated mass of compound 1 was determined to be 298.25 (m/z 299.07).
Mentions: To confirm the structure of the new compound, compound A was purified from 10 l of fermentation broth. In total, 80 mg of compound A was obtained following purification. The exact mass of compound A obtained from LC-HRMS was 299.07 for [C15H10N2O5]+ (calculated mass, 298.25; Fig 5). This calculated mass was 16 Da smaller than the known mass of lomofungin, suggesting that compound A contained one less hydroxyl than lomofungin (C15H10N2O6, MW 314 Da) [47]. Structural analysis was performed by 1H NMR, 13C NMR, distortionless enhancement by polarization transfer-90, 1H-1H correlation spectroscopy, heteronuclear multiple bond correlation, and heteronuclear multiple quantum coherence analyses, and the proton and carbon chemical shifts of compound A are shown in Table 4 and in S2 Fig, respectively. The 1H-NMR spectra contained four proton signals (δH, 7.39–8.41 ppm) that are typical of double bonds (ring hydrogen), along with corresponding 13C-NMR spectra of carbon atoms (δC, 110.1–134.5 ppm), suggesting that compound A has a phenazine ring in the core of its structure. Compared with the reported NMR data for lomofungin [47], the main differences were the absence of a hydroxyl hydrogen at δH 11.22, and the presence of hydrogen ring at δH 8.27. Therefore, compound A was predicted to be 1-carbomethoxy-6-formy-4,9-dihydroxy phenazine, a new chemical compound missing the C-7 hydroxyl of lomofungin (Fig 6).

Bottom Line: Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin.This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position.This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.

ABSTRACT
Streptomyces lomondensis S015 synthesizes the broad-spectrum phenazine antibiotic lomofungin. Whole genome sequencing of this strain revealed a genomic locus consisting of 23 open reading frames that includes the core phenazine biosynthesis gene cluster lphzGFEDCB. lomo10, encoding a putative flavin-dependent monooxygenase, was also identified in this locus. Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin. This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces.

No MeSH data available.


Related in: MedlinePlus