Identification and biosynthesis of a novel xanthomonadin-dialkylresorcinol-hybrid from Azoarcus sp. BH72

A novel xanthomonadin-dialkylresorcinol hybrid named arcuflavin was identified in Azoarcus sp. BH72 by a combination of feeding experiments, HPLC-MS and MALDI-MS and gene clusters encoding the biosynthesis of this non-isoprenoid aryl-polyene containing pigment are reported. A chorismate-utilizing enzyme from the XanB2-type producing 3- and 4-hydroxybenzoic acid and an AMP-ligase encoded by these gene clusters were characterized, that might perform the first two steps of the polyene biosynthesis. Furthermore, a detailed analysis of the already known or novel biosynthesis gene clusters involved in the biosynthesis of polyene containing pigments like arcuflavin, flexirubin and xanthomonadin revealed the presence of similar gene clusters in a wide range of bacterial taxa, suggesting that polyene and polyene-dialkylresorcinol pigments are more widespread than previously realized

Predicted gene clusters for arcuflavin biosynthesis in <i>Azoarcus</i> sp. BH72. Domain guided annotation is based on conserved domains detected by BLAST-P.

<p>Predicted gene clusters for arcuflavin biosynthesis in <i>Azoarcus</i> sp. BH72. Domain guided annotation is based on conserved domains detected by BLAST-P.</p

Known and proposed gene clusters involved in biosynthesis of DARs, xanthomonadin and xanthomonadin-like compounds in <i>Azoarcus</i> sp. BH72 (A), <i>Xanthomonas campestris pv campestris</i> ATCC 33913 (B), <i>Dechloromonas aromatica</i> RCB (C) <i>Variovorax paradoxus</i> S110 (D) and <i>Sideroxydans lithotrophicus</i> ES-1 (E).

<p>Annotation colors follow the <i>Azoarcus</i> gene clusters and genes are connected by grey lines if their identity was ≥40% in a BLAST-P analysis. All genes are scaled to the depicted size and are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090922#pone.0090922.s001" target="_blank">Table S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090922#pone.0090922.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090922#pone.0090922.s003" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090922#pone.0090922.s004" target="_blank">S4</a>.</p

Natural occurring DARs (1–4) [2]–[6], polyene containing pigments like flexirubin (5) from <i>Chitinophaga filiformes</i>[8] or xanthomonadin I (6) [9], DAR (7) heterologously produced with <i>Azoarcus</i> DAR proteins [1] and the proposed structure of the DAR-xanthomonadin hybrid pigment named arcuflavin (8) from <i>Azoarcus</i> sp. BH72 identified in this study.

<p>Natural occurring DARs (1–4) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090922#pone.0090922-Kanda1" target="_blank">[2]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090922#pone.0090922-Imai1" target="_blank">[6]</a>, polyene containing pigments like flexirubin (5) from <i>Chitinophaga filiformes</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090922#pone.0090922-Achenbach2" target="_blank">[8]</a> or xanthomonadin I (6) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090922#pone.0090922-Andrewes1" target="_blank">[9]</a>, DAR (7) heterologously produced with <i>Azoarcus</i> DAR proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090922#pone.0090922-Fuchs1" target="_blank">[1]</a> and the proposed structure of the DAR-xanthomonadin hybrid pigment named arcuflavin (8) from <i>Azoarcus</i> sp. BH72 identified in this study.</p

GC-MS analysis of <i>E. coli</i> cultures overexpressing <i>arcB</i>.

<p>Total ion chromatograms of extracted supernatants of <i>E. coli</i> BL21 (DE3) Star harboring the vector without insert (I), the <i>arcB</i> overexpression plasmid (II) and of 3-hydroxybenzoic acid (3-HBA) (III) and 4-hydroxybenzoic acid (4-HBA) standards (IV).</p

Mass spectra of 8 from <i>Azoarcus</i> sp. BH72.

<p><b>A</b>: MALDI-orbitrap mass spectra of wildtype <b>8</b> (I) and from labeling experiments with d₃-methionine (II) and 4-Fluoro-3-hydroxybenzoic acid (4F-3HBA) (III). <b>B</b>: MALDI-iontrap-MS² of <b>8</b>. MALDI-iontrap-MS³ of <i>m/z</i> 293.3 (<b>C</b>) and <i>m/z</i> 317.2 (<b>D</b>) with <i>m/z</i> 608.4 [M]^+• as precursor.</p

Identification and Biosynthesis of a Novel Xanthomonadin-Dialkylresorcinol-Hybrid from <i>Azoarcus</i> sp. BH72

<div><p>A novel xanthomonadin-dialkylresorcinol hybrid named arcuflavin was identified in <i>Azoarcus</i> sp. BH72 by a combination of feeding experiments, HPLC-MS and MALDI-MS and gene clusters encoding the biosynthesis of this non-isoprenoid aryl-polyene containing pigment are reported. A chorismate-utilizing enzyme from the XanB2-type producing 3- and 4-hydroxybenzoic acid and an AMP-ligase encoded by these gene clusters were characterized, that might perform the first two steps of the polyene biosynthesis. Furthermore, a detailed analysis of the already known or novel biosynthesis gene clusters involved in the biosynthesis of polyene containing pigments like arcuflavin, flexirubin and xanthomonadin revealed the presence of similar gene clusters in a wide range of bacterial taxa, suggesting that polyene and polyene-dialkylresorcinol pigments are more widespread than previously realized.</p></div

HPLC analysis of <i>Azoarcus</i> sp. BH72 wild type and mutant strains.

<p>HPLC-UV analysis of wildtype (I), BH0260 (II) and BH3911 extracts with traces showing absorption at 420 nm (solid line) or 310 nm (dotted line). All chromatograms were drawn to the same scale.</p