Biosynthetic pathway of the final Er product, Er-A.

<p>Three DEBSs, EryAI-AIII, are responsible for the generation of the 16-membered lactone, 6-dEB; Tailoring enzymes catalyze sequential reactions, including two hydroxylations, two glycosylations, and one methylation, to obtain the final product, Er-A.</p

Identification and Characterization of a New Erythromycin Biosynthetic Gene Cluster in <i>Actinopolyspora erythraea</i> YIM90600, a Novel Erythronolide-Producing Halophilic Actinomycete Isolated from Salt Field

<div><p>Erythromycins (Ers) are clinically potent macrolide antibiotics in treating pathogenic bacterial infections. Microorganisms capable of producing Ers, represented by <i>Saccharopolyspora erythraea</i>, are mainly soil-dwelling actinomycetes. So far, <i>Actinopolyspora erythraea</i> YIM90600, a halophilic actinomycete isolated from Baicheng salt field, is the only known Er-producing extremophile. In this study, we have reported the draft genome sequence of <i>Ac. erythraea</i> YIM90600, genome mining of which has revealed a new Er biosynthetic gene cluster encoding several novel Er metabolites. This Er gene cluster shares high identity and similarity with the one of <i>Sa. erythraea</i> NRRL2338, except for two absent genes, <i>eryBI</i> and <i>eryG</i>. By correlating genotype and chemotype, the biosynthetic pathways of 3′-demethyl-erythromycin C, erythronolide H (EH) and erythronolide I have been proposed. The formation of EH is supposed to be sequentially biosynthesized via C-6/C-18 epoxidation and C-14 hydroxylation from 6-deoxyerythronolide B. Although an <i>in vitro</i> enzymatic activity assay has provided limited evidence for the involvement of the cytochrome P450 oxidase EryF^Ac (derived from <i>Ac. erythraea</i> YIM90600) in the catalysis of a two-step oxidation, resulting in an epoxy moiety, the attempt to construct an EH-producing <i>Sa. erythraea</i> mutant via gene complementation was not successful. Characterization of EryK^Ac (derived from <i>Ac. erythraea</i> YIM90600) <i>in vitro</i> has confirmed its unique role as a C-12 hydroxylase, rather than a C-14 hydroxylase of the erythronolide. Genomic characterization of the halophile <i>Ac. erythraea</i> YIM90600 will assist us to explore the great potential of extremophiles, and promote the understanding of EH formation, which will shed new insights into the biosynthesis of Er metabolites.</p></div

The identified Er metabolites in <i>Ac. erythraea</i> YIM90600.

<p>EB and Er-C are normal Er intermediates, while 3′-<i>N</i>-demethyl-Er-C, EH, and EI are novel Er congeners that have rarely been reported.</p

HPLC-ESI-MS analysis of the fermentation culture of <i>Sa. erythraea</i> EX103.

<p>Total ion current chromatogram (<i>i</i>), and reconstructed base peak chromatograms for 6-deoxy-Er-A (<i>ii</i>), Er-A (<i>iii</i>), 6-deoxy-Er-B (<i>iv</i>), Er-B (<i>v</i>), 6-deoxy-Er-C (<i>vi</i>), Er-C (<i>vii</i>), 6-deoxy-Er-D (<i>viii</i>), and Er-D (<i>ix</i>) are recorded. Note that 6-deoxy-Er-A and Er-B, as well as 6-deoxy-Er-C and Er-D share the same molecular weights and similar polarities, their base peaks are thus overlapping.</p

HPLC-ESI-MS analyses of the fermentation cultures of <i>Sa. erythraea</i> EX101 and EX102.

<p>(A) Total ion current chromatogram (<i>i</i>), and reconstructed base peak chromatogram for 6-dEB (<i>ii</i>) of the fermentation products of EX101. ESI-MS data recorded at the retention time of 27.06 min (<i>iii</i>). (B) Total ion current chromatogram (<i>iv</i>), and reconstructed base peak chromatogram for EB (<i>v</i>) of the fermentation products of EX102. ESI-MS data recorded at the retention time of 15.96 min (<i>vi</i>).</p

HPLC-ESI-MS analyses of the <i>in vitro</i> enzymatic reactions catalyzed by EryF^Sa and EryF^Ac, respectively.

<p>(A) Total ion current chromatograms indicating the <i>in vitro</i> conversion of 6-dEB to EB in the absence of active EryF^Ac (<i>i</i>), in the presence of active EryF^Ac (<i>ii</i>), or in the presence of active EryF^Sa (<i>iii</i>). (B) Total ion current chromatogram (<i>iv</i>) and reconstructed base peak chromatogram for 6, 18-epoxy-EB (<i>v</i>) of the EryF^Ac reaction mixture. (C) Total ion current chromatogram (<i>vi</i>) and reconstructed base peak chromatogram for 6, 18-epoxy-EB (<i>vii</i>) of the EryF^Sa reaction mixture.</p

Bacterial strains and plasmids used and constructed in this study.

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

HPLC-ESI-MS analyses of the <i>in vitro</i> enzymatic reactions catalyzed by EryK^Sa and EryK^Ac, respectively.

<p>Total ion current chromatograms indicating standard Er-B (red circle, <i>i</i>) and Er-A (blue lozenge, <i>ii</i>), the <i>in vitro</i> conversion of Er-B to Er-A in the absence of active EryK^Sa (<i>iii</i>), in the presence of active EryK^Sa (<i>iv</i>), in the absence of active EryK^Ac (<i>v</i>), or in the presence of active EryK^Ac (<i>vi</i>).</p