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

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.

Figure 1

Morphological differentiation of mycelia in Amycolatopsis orientalis HCCB10007 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.

Figure 2

Genome atlas of the A. orientalis and gene clusters for secondary metabolism. The large circle represents the chromosome: the outer scale is numbered in megabases and indicates the core (red), quasi-core (orange), and non-core (sky blue) regions. The circles are numbered from the outside in. The genes in circles 1 and 2 (forward and reverse strands, respectively) are color-coded according to COG functional categories. Circle 3 shows selected essential genes (cell division, replication, transcription, translation, and amino-acid metabolism; the paralogs of essential genes in the non-core regions are not included). Circle 4 shows the secondary metabolic clusters, which are further enlarged outside the circle for detailed illustration. The vcm cluster is further illustrated in Figure 6. Circle 5 depicts the RNAs (blue, tRNA; red, rRNA). Circle 6 shows the mobile genetic elements (transposase, phage). Circle 7 depicts the GC content. Circle 8 shows the GC bias (pink, values > 0; green, values < 0). The small circle on the right side represents the plasmid DNA sequence. The outer scale is numbered in kilobases. All of the genes, regardless of the forward or reverse strands, are illustrated in the same circle. Circles 2 and 3 are the same as circles 7 and 8 of the large chromosome, respectively.

Figure 3

Genome configurations of A. orientalis and A. 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.

Figure 4

Biosynthetic pathways of different types of nitrogenous phospholipids in actinomycetes. (A) The cell membrane of Amycolatopsis belongs to the type PII because PE is the dominant phospholipid detected. Two essential proteins (AORI_7345 and AORI_7346, labeled in red color) involved in the biosynthesis of PE were encoded by the A. orientalis genome, whereas the genes encoding enzymes involved in other types of nitrogenous phospholipids were not found (NF). Actinomycetes of type PI contain no nitrogenous phospholipids in their cell membrane, while type PII, type PIII, type PIV, and type PV actinomycetes contain the following characteristic phospholipids: PE, PC, GluNU, and PG, respectively. Panel (B) illustrates the analysis of isoprenyl diphosphate synthases from type strains of actinomycetes. The names and amino-acid sequences of the strains with different colors represent actinomycetes harboring different-length MKs: red, MK7 (C35); olive-green, MK8 (C40); blue, MK9 (C45). The amino-acid sequences of the chain-length determination (CLD) region are emphasized in green on the right of the panel. The protein sequences were obtained from NCBI at http://www.ncbi.nlm.nih.gov/protein/.

Figure 5

Metabolic pathway of vancomycin biosynthesis. Three steps are involved in the biosynthesis of vancomycin, and the related functional genes in and outside of the vcm cluster were mapped. I) The biosynthesis of its amino-acid precursors (right of the panel). Non-ribosomal peptide synthetase VcmD (AORI_1493) catalyzes free tyrosines to form tyrosyl-S-enzyme, which is hydroxylated by OxyD (AORI_1494) and then release as βHt by the action of Vhp (AORI_1492). Genes of pdh/hpgT/hmaS/hmO (AORI_1476, AORI_1491, AORI_1495-1496) are responsible for Hpg synthesis from prephenate, and dpgA/B/C/D (AORI_1502-AORI_1505) are responsible for Dpg synthesis using malonyl-CoA as the starting unit. II) The modified amino acids are assembled to form linear heptapeptide by NRPSs (VcmABC, AORI_1478-1480) with seven modules (M1-M7, upper left of the panel). A, adenylation domain; C, condensation domain; E, epimerization domain; T, thiolation domain; TE, thioesterase domain. III) The post-modifications of the linear heptapeptide (down the left side of the panel) include cyclization (oxyA/B/C, AORI_1482-AORI_1484), halogenation (vhal, AORI_1485), methylation (vmt, AORI_1490), and glycosylation (gtfDE, AORI_1486-AORI_1487). Finally, vancomycin is generated.

Figure 6

Functional characterization and verification of the modification genes in the vcm cluster. 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.