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Complete genome sequence and comparative genomic analyses of the vancomycin-producing Amycolatopsis orientalis.

Xu L, Huang H, Wei W, Zhong Y, Tang B, Yuan H, Zhu L, Huang W, Ge M, Yang S, Zheng H, Jiang W, Chen D, Zhao GP, Zhao W - BMC Genomics (2014)

Bottom Line: Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster.The broad substrate spectra characteristics of these modification enzymes were inferred.This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Laiyi Center for Biopharmaceutical R&D, Shanghai 200240, China. whjiang@sibs.ac.cn.

ABSTRACT

Background: Amycolatopsis orientalis is the type species of the genus and its industrial strain HCCB10007, derived from ATCC 43491, has been used for large-scale production of the vital antibiotic vancomycin. However, to date, neither the complete genomic sequence of this species nor a systemic characterization of the vancomycin biosynthesis cluster (vcm) has been reported. With only the whole genome sequence of Amycolatopsis mediterranei available, additional complete genomes of other species may facilitate intra-generic comparative analysis of the genus.

Results: The complete genome of A. orientalis HCCB10007 comprises an 8,948,591-bp circular chromosome and a 33,499-bp dissociated plasmid. In total, 8,121 protein-coding sequences were predicted, and the species-specific genomic features of A. orientalis were analyzed in comparison with that of A. mediterranei. The common characteristics of Amycolatopsis genomes were revealed via intra- and inter-generic comparative genomic analyses within the domain of actinomycetes, and led directly to the development of sequence-based Amycolatopsis molecular chemotaxonomic characteristics (MCCs). The chromosomal core/quasi-core and non-core configurations of the A. orientalis and the A. mediterranei genome were analyzed reciprocally, with respect to further understanding both the discriminable criteria and the evolutionary implementation. In addition, 26 gene clusters related to secondary metabolism, including the 64-kb vcm cluster, were identified in the genome. Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster. The broad substrate spectra characteristics of these modification enzymes were inferred.

Conclusions: This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.

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Functional characterization and verification of the modification genes in thevcmcluster. The 64-kb vcm cluster is illustrated in detail. AORI_1490 (vmt), AORI_1486 (gtfD), AORI_1487 (gtfE), and AORI_1485 (vhal) were replaced in-frame by selection markers, and AORI_1490 was overexpressed in vitro using demethylvancomycin/aglucovancomycin as the substrate. The vancomycin standards (A) and the corresponding variants obtained by isolation from mutant strains or the in vitro treatments were detected by HPLC-MS: (B) dechlorovancomycin, (C) desvancosamine vancomycin, (D) aglucovancomycin, (E) dimethylaglucovancomycin, (F) demethylvancomycin, and (G) dimethylvancomycin. The structural formulae of the variants are shown in the table of Figure 1. A mass of 20 μg of each compound was used to assay its activity against MRSA, and the picture is representative of three independent experiments.
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Fig6: Functional characterization and verification of the modification genes in thevcmcluster. The 64-kb vcm cluster is illustrated in detail. AORI_1490 (vmt), AORI_1486 (gtfD), AORI_1487 (gtfE), and AORI_1485 (vhal) were replaced in-frame by selection markers, and AORI_1490 was overexpressed in vitro using demethylvancomycin/aglucovancomycin as the substrate. The vancomycin standards (A) and the corresponding variants obtained by isolation from mutant strains or the in vitro treatments were detected by HPLC-MS: (B) dechlorovancomycin, (C) desvancosamine vancomycin, (D) aglucovancomycin, (E) dimethylaglucovancomycin, (F) demethylvancomycin, and (G) dimethylvancomycin. The structural formulae of the variants are shown in the table of Figure 1. A mass of 20 μg of each compound was used to assay its activity against MRSA, and the picture is representative of three independent experiments.

Mentions: Morphological differentiation of mycelia inAmycolatopsis orientalisHCCB10007 and chemical structures of vancomycin variants. Scanning electron micrograph of A. orientalis HCCB10007 cultured for one or three incubation days (upper left of the panel). The red arrow indicates the sporulation of A. orientalis cultured for three days. The core structural formula proposed for vancomycin and its variants (upper right of the panel) shows minor modifications of the heptapeptide core of vancomycin. Table below shows the specific formulae and radical compositions of each vancomycin variant compounds. Alphabetic numbering in the table are corresponding to the legend of Figure 6.


Complete genome sequence and comparative genomic analyses of the vancomycin-producing Amycolatopsis orientalis.

Xu L, Huang H, Wei W, Zhong Y, Tang B, Yuan H, Zhu L, Huang W, Ge M, Yang S, Zheng H, Jiang W, Chen D, Zhao GP, Zhao W - BMC Genomics (2014)

Functional characterization and verification of the modification genes in thevcmcluster. The 64-kb vcm cluster is illustrated in detail. AORI_1490 (vmt), AORI_1486 (gtfD), AORI_1487 (gtfE), and AORI_1485 (vhal) were replaced in-frame by selection markers, and AORI_1490 was overexpressed in vitro using demethylvancomycin/aglucovancomycin as the substrate. The vancomycin standards (A) and the corresponding variants obtained by isolation from mutant strains or the in vitro treatments were detected by HPLC-MS: (B) dechlorovancomycin, (C) desvancosamine vancomycin, (D) aglucovancomycin, (E) dimethylaglucovancomycin, (F) demethylvancomycin, and (G) dimethylvancomycin. The structural formulae of the variants are shown in the table of Figure 1. A mass of 20 μg of each compound was used to assay its activity against MRSA, and the picture is representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4048454&req=5

Fig6: Functional characterization and verification of the modification genes in thevcmcluster. The 64-kb vcm cluster is illustrated in detail. AORI_1490 (vmt), AORI_1486 (gtfD), AORI_1487 (gtfE), and AORI_1485 (vhal) were replaced in-frame by selection markers, and AORI_1490 was overexpressed in vitro using demethylvancomycin/aglucovancomycin as the substrate. The vancomycin standards (A) and the corresponding variants obtained by isolation from mutant strains or the in vitro treatments were detected by HPLC-MS: (B) dechlorovancomycin, (C) desvancosamine vancomycin, (D) aglucovancomycin, (E) dimethylaglucovancomycin, (F) demethylvancomycin, and (G) dimethylvancomycin. The structural formulae of the variants are shown in the table of Figure 1. A mass of 20 μg of each compound was used to assay its activity against MRSA, and the picture is representative of three independent experiments.
Mentions: Morphological differentiation of mycelia inAmycolatopsis orientalisHCCB10007 and chemical structures of vancomycin variants. Scanning electron micrograph of A. orientalis HCCB10007 cultured for one or three incubation days (upper left of the panel). The red arrow indicates the sporulation of A. orientalis cultured for three days. The core structural formula proposed for vancomycin and its variants (upper right of the panel) shows minor modifications of the heptapeptide core of vancomycin. Table below shows the specific formulae and radical compositions of each vancomycin variant compounds. Alphabetic numbering in the table are corresponding to the legend of Figure 6.

Bottom Line: Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster.The broad substrate spectra characteristics of these modification enzymes were inferred.This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Laiyi Center for Biopharmaceutical R&D, Shanghai 200240, China. whjiang@sibs.ac.cn.

ABSTRACT

Background: Amycolatopsis orientalis is the type species of the genus and its industrial strain HCCB10007, derived from ATCC 43491, has been used for large-scale production of the vital antibiotic vancomycin. However, to date, neither the complete genomic sequence of this species nor a systemic characterization of the vancomycin biosynthesis cluster (vcm) has been reported. With only the whole genome sequence of Amycolatopsis mediterranei available, additional complete genomes of other species may facilitate intra-generic comparative analysis of the genus.

Results: The complete genome of A. orientalis HCCB10007 comprises an 8,948,591-bp circular chromosome and a 33,499-bp dissociated plasmid. In total, 8,121 protein-coding sequences were predicted, and the species-specific genomic features of A. orientalis were analyzed in comparison with that of A. mediterranei. The common characteristics of Amycolatopsis genomes were revealed via intra- and inter-generic comparative genomic analyses within the domain of actinomycetes, and led directly to the development of sequence-based Amycolatopsis molecular chemotaxonomic characteristics (MCCs). The chromosomal core/quasi-core and non-core configurations of the A. orientalis and the A. mediterranei genome were analyzed reciprocally, with respect to further understanding both the discriminable criteria and the evolutionary implementation. In addition, 26 gene clusters related to secondary metabolism, including the 64-kb vcm cluster, were identified in the genome. Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster. The broad substrate spectra characteristics of these modification enzymes were inferred.

Conclusions: This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.

Show MeSH
Related in: MedlinePlus