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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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Related in: MedlinePlus

Genome configurations ofA. orientalisandA. mediterranei. (A) All of the dots in the panels were calculated in a 90-kb sliding window. For the broken X plot (lower right of the panel), the dots represent a reciprocal best match between the genomes of A. orientalis and A. mediterranei, based on the BLASTP comparison. The X-axis (Y-axis) is the nucleotide scale of the A. orientalis (A. mediterranei) chromosome. R1 (4.02-4.28 Mb, AORI_3663-AORI_3909) and R2 (5.55-5.75 Mbp, AORI_4997-AORI_5173) were designated as the two quasi-core regions in the A. orientalis genome. Reciprocally, two regions (AMED_4864-AMED_5049 and AMED_5970-AMED_7071) were defined as the quasi-core in the A. mediterranei genome. The core and quasi-core regions are highlighted in lavender (A. orientalis) or in pink (A. mediterranei). P1 to P4 were designated as the regions containing biosynthesis clusters of rifamycin (rif in A. mediterranei), vancomycin (vcm in A. orientalis), NRPS (nrps10 in A. mediterranei) and polyketide (pks9 in A. orientalis), respectively. In the upper right and lower left panels, the pink triangles represent the coding density of all of the genes; the turquoise squares represent the coding density of orthologs between the genomes of A. orientalis and A. mediterranei; and the yellow circles represent the coding density of the essential genes. The area within the black square frame is the P2 region containing the vcm cluster, with a lower coding density of orthologs and essential genes. (B) Alignment of the P2 region with its flanking genes related to the vancomycin biosynthesis in selected actinomycete genomes. The green arrows represent the omitted genes in the corresponding genomes. (C) Alignment of the P1 region with its flanking genes related to the rifamycin biosynthesis in selected actinomycete genomes. All of the genome data are available at NCBI.
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Fig3: Genome configurations ofA. orientalisandA. mediterranei. (A) All of the dots in the panels were calculated in a 90-kb sliding window. For the broken X plot (lower right of the panel), the dots represent a reciprocal best match between the genomes of A. orientalis and A. mediterranei, based on the BLASTP comparison. The X-axis (Y-axis) is the nucleotide scale of the A. orientalis (A. mediterranei) chromosome. R1 (4.02-4.28 Mb, AORI_3663-AORI_3909) and R2 (5.55-5.75 Mbp, AORI_4997-AORI_5173) were designated as the two quasi-core regions in the A. orientalis genome. Reciprocally, two regions (AMED_4864-AMED_5049 and AMED_5970-AMED_7071) were defined as the quasi-core in the A. mediterranei genome. The core and quasi-core regions are highlighted in lavender (A. orientalis) or in pink (A. mediterranei). P1 to P4 were designated as the regions containing biosynthesis clusters of rifamycin (rif in A. mediterranei), vancomycin (vcm in A. orientalis), NRPS (nrps10 in A. mediterranei) and polyketide (pks9 in A. orientalis), respectively. In the upper right and lower left panels, the pink triangles represent the coding density of all of the genes; the turquoise squares represent the coding density of orthologs between the genomes of A. orientalis and A. mediterranei; and the yellow circles represent the coding density of the essential genes. The area within the black square frame is the P2 region containing the vcm cluster, with a lower coding density of orthologs and essential genes. (B) Alignment of the P2 region with its flanking genes related to the vancomycin biosynthesis in selected actinomycete genomes. The green arrows represent the omitted genes in the corresponding genomes. (C) Alignment of the P1 region with its flanking genes related to the rifamycin biosynthesis in selected actinomycete genomes. All of the genome data are available at NCBI.

Mentions: First, a core region of A. orientalis genome (nucleotide coordinates of 0-3.1 Mbp and 6.3-8.9 Mbp, corresponding to AORI_0001-AORI_2890 and AORI_5565-AORI_8121) was recognized by its good co-linearity of the order of its orthologs, with 14.5% of coding density for essential genes compared with 10.2% in non-core regions (P < 0.01). Meanwhile, the coding density of orthologous genes in the core region (68.2%) was also higher than that in the non-core regions (37.3%) (P < 0.01) (Figure 3A, Additional file 1: Table S2).Figure 3


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)

Genome configurations ofA. orientalisandA. mediterranei. (A) All of the dots in the panels were calculated in a 90-kb sliding window. For the broken X plot (lower right of the panel), the dots represent a reciprocal best match between the genomes of A. orientalis and A. mediterranei, based on the BLASTP comparison. The X-axis (Y-axis) is the nucleotide scale of the A. orientalis (A. mediterranei) chromosome. R1 (4.02-4.28 Mb, AORI_3663-AORI_3909) and R2 (5.55-5.75 Mbp, AORI_4997-AORI_5173) were designated as the two quasi-core regions in the A. orientalis genome. Reciprocally, two regions (AMED_4864-AMED_5049 and AMED_5970-AMED_7071) were defined as the quasi-core in the A. mediterranei genome. The core and quasi-core regions are highlighted in lavender (A. orientalis) or in pink (A. mediterranei). P1 to P4 were designated as the regions containing biosynthesis clusters of rifamycin (rif in A. mediterranei), vancomycin (vcm in A. orientalis), NRPS (nrps10 in A. mediterranei) and polyketide (pks9 in A. orientalis), respectively. In the upper right and lower left panels, the pink triangles represent the coding density of all of the genes; the turquoise squares represent the coding density of orthologs between the genomes of A. orientalis and A. mediterranei; and the yellow circles represent the coding density of the essential genes. The area within the black square frame is the P2 region containing the vcm cluster, with a lower coding density of orthologs and essential genes. (B) Alignment of the P2 region with its flanking genes related to the vancomycin biosynthesis in selected actinomycete genomes. The green arrows represent the omitted genes in the corresponding genomes. (C) Alignment of the P1 region with its flanking genes related to the rifamycin biosynthesis in selected actinomycete genomes. All of the genome data are available at NCBI.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Genome configurations ofA. orientalisandA. mediterranei. (A) All of the dots in the panels were calculated in a 90-kb sliding window. For the broken X plot (lower right of the panel), the dots represent a reciprocal best match between the genomes of A. orientalis and A. mediterranei, based on the BLASTP comparison. The X-axis (Y-axis) is the nucleotide scale of the A. orientalis (A. mediterranei) chromosome. R1 (4.02-4.28 Mb, AORI_3663-AORI_3909) and R2 (5.55-5.75 Mbp, AORI_4997-AORI_5173) were designated as the two quasi-core regions in the A. orientalis genome. Reciprocally, two regions (AMED_4864-AMED_5049 and AMED_5970-AMED_7071) were defined as the quasi-core in the A. mediterranei genome. The core and quasi-core regions are highlighted in lavender (A. orientalis) or in pink (A. mediterranei). P1 to P4 were designated as the regions containing biosynthesis clusters of rifamycin (rif in A. mediterranei), vancomycin (vcm in A. orientalis), NRPS (nrps10 in A. mediterranei) and polyketide (pks9 in A. orientalis), respectively. In the upper right and lower left panels, the pink triangles represent the coding density of all of the genes; the turquoise squares represent the coding density of orthologs between the genomes of A. orientalis and A. mediterranei; and the yellow circles represent the coding density of the essential genes. The area within the black square frame is the P2 region containing the vcm cluster, with a lower coding density of orthologs and essential genes. (B) Alignment of the P2 region with its flanking genes related to the vancomycin biosynthesis in selected actinomycete genomes. The green arrows represent the omitted genes in the corresponding genomes. (C) Alignment of the P1 region with its flanking genes related to the rifamycin biosynthesis in selected actinomycete genomes. All of the genome data are available at NCBI.
Mentions: First, a core region of A. orientalis genome (nucleotide coordinates of 0-3.1 Mbp and 6.3-8.9 Mbp, corresponding to AORI_0001-AORI_2890 and AORI_5565-AORI_8121) was recognized by its good co-linearity of the order of its orthologs, with 14.5% of coding density for essential genes compared with 10.2% in non-core regions (P < 0.01). Meanwhile, the coding density of orthologous genes in the core region (68.2%) was also higher than that in the non-core regions (37.3%) (P < 0.01) (Figure 3A, Additional file 1: Table S2).Figure 3

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