Primers used in this study.

<p>Primers used in this study.</p

Proposed lomofungin biosynthetic pathway in <i>Streptomyces lomondensis</i> S015.

<p>Proposed lomofungin biosynthetic pathway in <i>Streptomyces lomondensis</i> S015.</p

Chemical shift summarized from ¹H (DMSO-d₆) and ¹³C (MeOD-d₆) analyses recorded by 600 MHz NMR spectrometry.

<p>Chemical shift summarized from ¹H (DMSO-d₆) and ¹³C (MeOD-d₆) analyses recorded by 600 MHz NMR spectrometry.</p

Chemical structure of compound A.

<p>Chemical structure of compound A.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Spectrum of compound A following LC-HRMS analysis.

<p>The caculated mass of compound 1 was determined to be 298.25 (m/z 299.07).</p

Photographs of the different strains grown on MS medium.

<p>(A) Wild-type strain. (B) <i>lomo10</i> inactivated mutant DCC601. (C) <i>lomo10</i> complementation strain DCC602.</p

Putative lomofungin biosynthesis gene cluster in <i>Streptomyces lomondensis</i> S015.

<p>Putative lomofungin biosynthesis gene cluster in <i>Streptomyces lomondensis</i> S015.</p

Identification of the Lomofungin Biosynthesis Gene Cluster and Associated Flavin-Dependent Monooxygenase Gene in <i>Streptomyces lomondensis</i> S015

<div><p><i>Streptomyces lomondensis</i> 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 <i>lphzGFEDCB</i>. <i>lomo10</i>, encoding a putative flavin-dependent monooxygenase, was also identified in this locus. Inactivation of <i>lomo10</i> 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 <i>lomo10</i> is responsible for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phenazine hydroxylation gene in <i>Streptomyces</i>, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in <i>Streptomyces</i>.</p></div

Inactivation and self-complementation of <i>lomo10</i> in <i>Streptomyces lomondensis</i> S015.

<p>(A) Schematic of the in-frame partial deletion of 1032 bp in <i>lomo10</i> to generate the Δ<i>lomo10</i> 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 <i>lomo10</i>, 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 <i>lomo10</i> left arm-For and <i>lomo10</i> right arm-Rev. (B) PCR analysis of WT strain and DCC601. (C) PCR analysis of DCC601 and <i>lomo10</i> 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.</p