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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

Inactivation and self-complementation of lomo10 in Streptomyces lomondensis S015.(A) Schematic of the in-frame partial deletion of 1032 bp in lomo10 to generate the Δlomo10 mutant DCC601. Primers 1, 2, 3, and 4 were used to amplify the left and right homology arms. GTG and TGA were the start and termination codons for lomo10, respectively. The expected PCR product from the wild-type (WT) strain was 5,106 bp, and that from DCC601 was 4,074 bp using the primers lomo10 left arm-For and lomo10 right arm-Rev. (B) PCR analysis of WT strain and DCC601. (C) PCR analysis of DCC601 and lomo10 complementation strain DCC602. The amplicon generated from the DCC602 genomic DNA gave the expected 1,726 bp fragment, but no band was amplified from the DCC601 strain.
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pone.0136228.g002: Inactivation and self-complementation of lomo10 in Streptomyces lomondensis S015.(A) Schematic of the in-frame partial deletion of 1032 bp in lomo10 to generate the Δlomo10 mutant DCC601. Primers 1, 2, 3, and 4 were used to amplify the left and right homology arms. GTG and TGA were the start and termination codons for lomo10, respectively. The expected PCR product from the wild-type (WT) strain was 5,106 bp, and that from DCC601 was 4,074 bp using the primers lomo10 left arm-For and lomo10 right arm-Rev. (B) PCR analysis of WT strain and DCC601. (C) PCR analysis of DCC601 and lomo10 complementation strain DCC602. The amplicon generated from the DCC602 genomic DNA gave the expected 1,726 bp fragment, but no band was amplified from the DCC601 strain.

Mentions: As there are three hydroxyl groups included in the structure of lomofungin, the function of the putative hydroxylation gene lomo10 was investigated by generating an in-frame partial deletion mutant, DCC601, via double-crossover homologous recombination (Fig 2A). Strain DCC601 was obtained by deletion of the 1032 bp in the coding region of lomo10. No promoter was found in this region by using online software (http://www.fruitfly.org/seq_tools/promoter.html) analysis. Thus, the partial deletion of lomo10 might not affect the expression of other genes, such as lomo11. The genotype of the DCC601 lomo10 deletion mutant, as well as that of the lomo10 self-complementation strain DCC602, was confirmed by PCR analysis as shown in Fig 2B and 2C, respectively and DNA sequencing. The phenotypes of wild-type strain S015, DCC601, and DCC602 were examined following culture on solid MS medium (Fig 3). Wild-type strain S015 produced the olive-yellow lomofungin (Fig 3A), whereas the lomo10 deletion mutant (DCC601) produced light purple colored colonies (Fig 3B). Complementation with lomo10 restored lomofungin production (Fig 3C). There was no obvious difference in the phenotype of mycelium between WT, mutant strain DCC601 and complementary strain DCC602.


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)

Inactivation and self-complementation of lomo10 in Streptomyces lomondensis S015.(A) Schematic of the in-frame partial deletion of 1032 bp in lomo10 to generate the Δlomo10 mutant DCC601. Primers 1, 2, 3, and 4 were used to amplify the left and right homology arms. GTG and TGA were the start and termination codons for lomo10, respectively. The expected PCR product from the wild-type (WT) strain was 5,106 bp, and that from DCC601 was 4,074 bp using the primers lomo10 left arm-For and lomo10 right arm-Rev. (B) PCR analysis of WT strain and DCC601. (C) PCR analysis of DCC601 and lomo10 complementation strain DCC602. The amplicon generated from the DCC602 genomic DNA gave the expected 1,726 bp fragment, but no band was amplified from the DCC601 strain.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0136228.g002: Inactivation and self-complementation of lomo10 in Streptomyces lomondensis S015.(A) Schematic of the in-frame partial deletion of 1032 bp in lomo10 to generate the Δlomo10 mutant DCC601. Primers 1, 2, 3, and 4 were used to amplify the left and right homology arms. GTG and TGA were the start and termination codons for lomo10, respectively. The expected PCR product from the wild-type (WT) strain was 5,106 bp, and that from DCC601 was 4,074 bp using the primers lomo10 left arm-For and lomo10 right arm-Rev. (B) PCR analysis of WT strain and DCC601. (C) PCR analysis of DCC601 and lomo10 complementation strain DCC602. The amplicon generated from the DCC602 genomic DNA gave the expected 1,726 bp fragment, but no band was amplified from the DCC601 strain.
Mentions: As there are three hydroxyl groups included in the structure of lomofungin, the function of the putative hydroxylation gene lomo10 was investigated by generating an in-frame partial deletion mutant, DCC601, via double-crossover homologous recombination (Fig 2A). Strain DCC601 was obtained by deletion of the 1032 bp in the coding region of lomo10. No promoter was found in this region by using online software (http://www.fruitfly.org/seq_tools/promoter.html) analysis. Thus, the partial deletion of lomo10 might not affect the expression of other genes, such as lomo11. The genotype of the DCC601 lomo10 deletion mutant, as well as that of the lomo10 self-complementation strain DCC602, was confirmed by PCR analysis as shown in Fig 2B and 2C, respectively and DNA sequencing. The phenotypes of wild-type strain S015, DCC601, and DCC602 were examined following culture on solid MS medium (Fig 3). Wild-type strain S015 produced the olive-yellow lomofungin (Fig 3A), whereas the lomo10 deletion mutant (DCC601) produced light purple colored colonies (Fig 3B). Complementation with lomo10 restored lomofungin production (Fig 3C). There was no obvious difference in the phenotype of mycelium between WT, mutant strain DCC601 and complementary strain DCC602.

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