Draft genome sequence of Streptomyces hyaluromycini MB-PO13T, a hyaluromycin producer

Streptomyces hyaluromycini MB-PO13T (=NBRC 110483T = DSM 100105T) is type strain of the species, which produces a hyaluronidase inhibitor, hyaluromycin. Here, we report the draft genome sequence of this strain together with features of the organism and generation, annotation and analysis of the genome sequence. The 11.5 Mb genome of Streptomyces hyaluromycini MB-PO13T encoded 10,098 putative ORFs, of which 5317 were assigned with COG categories. The genome harbored at least six type I PKS clusters, three type II PKS gene clusters, two type III PKS gene clusters, six NRPS gene clusters, and one hybrid PKS/NRPS gene cluster. The type II PKS gene cluster including 2-amino-3-hydroxycyclopent-2-enone synthetic genes was identified to be responsible for hyaluromycin synthesis. We propose the biosynthetic pathway based on bioinformatic analysis.


Introduction
Hyaluromycin is a hyaluronidase inhibitor isolated from the culture broth of an actinomycete strain MB-PO13 T of the genus Streptomyces [1]. The structure consists of a γ-rubromycin core possessing a C 5 N unit as an amide substituent of the carboxyl functionality. Rubromycins have inhibitory activities against human telomerase and the reverse transcriptase of human immunodeficiency virus-1 [2]. The core structure possesses a hexacyclic ring system and a 5,6-bisbenzannelated spiroketal structure. The most intriguing part of hyaluromycin is the C 5 N moiety, which is present only in a limited range of secondary metabolites of actinomycetes [3]. As for the rubromycin family biosynthesis, putative biosynthetic genes for griseorhodin A were reported [4], but there is no report on the rubromycins. Hence, the biosynthesis of rubromycin family remains unclear. In this study, we performed whole genome shotgun sequencing of the strain MB-PO13 T to elucidate the biosynthetic mechanism of hyaluromycin. We herein present the draft genome sequence of Streptomyces hyaluromycini MB-PO13 T , together with the taxonomical identification of the strain, description of its genome properties and annotation of the gene cluster for hyaluromycin synthesis. The biosynthetic pathway of hyaluromycin is also proposed on the basis of the bioinformatic prediction.

Classification and features
During the course of screening for hyaluronidase inhibitors from actinomycetes, Streptomyces hyaluromycini MB-PO13 T was isolated from a tunicate (Molgula manhattensis) collected in Tokyo Bay, Japan and found to produce hyaluromycin [1]. Colony appearance was examined after incubation at 28°C for 14 days on an agar plate of ISP 4. Morphological features were observed under a light microscope (model BX-51; Olympus) and a scanning electron microscope (model JSM-6060; JEOL). The temperature range and optimum temperature for growth were determined by incubating the strain at 5,10,15,20,28,37,42, and 50°C on ISP 2 agar plates for 14 days. The pH range for growth was determined at 28°C in ISP 2 broth, of which pH was adjusted to 3 to 12 by 1 N HCl or 1 M Na 2 CO 3 . Tolerance to NaCl was tested on ISP 2 agar plates containing 2, 3, 5, 7, 9, and 12% (w/v) NaCl at 28°C. Carbohydrate utilization was determined on ISP 9 supplemented with sterilized carbon sources [5]. The strain grow well on ISP 3, ISP 4 and yeast-starch agars but poor on ISP 2, ISP 5, ISP 6, ISP 7, glucose-asparagine, nutrient, sucrose-nitrate and skim milk agars. Soluble red pigments are produced on ISP 2, ISP 3, ISP 4, ISP 7, glucose-asparagine, nutrient and yeast-starch agars. Cells are aerobic and Gramstain-positive. The aerial mycelia are branched and yellowish white in color, which become light grey at sporulation and the substrate mycelia are deep red on ISP 4 agar plate. Smooth surface spores (0.5-0.8 × 1.0-1.5 μm) in spiral chains are formed when cultured on nutritionally poor media. A scanning electron micrograph of the strain is shown in Fig. 1. Growth occurs at 10-37°C (optimum 28°C), at pH 4.0-9.0 (optimum pH 7.0) and in the presence of less than 2% NaCl (w/v). The strain utilizes L-arabinose, D-fructose, D-glucose, inositol, D-mannitol, rhamnose and D-xylose as sole carbon source for energy and growth, but not raffinose and sucrose (all at 1%, w/v). These results are summarized in Table 1. The genes encoding 16S rRNA were amplified by PCR using two universal primers, 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) [6]. GoTaq Green Master Mix (Promega) was used as described by the manufacture for the PCR. The reaction was started with denaturation at 94°C for 5 min followed by a total 27 cycles that consisted of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 1.5 min, and extension at 72°C for 7 min. The PCR product was purified by Wizard SV Gel and PCR Clean-Up System (Promega) and sequenced with a BigDye cycle sequencing ready reaction kit (Appled Biosystems) on an ABI PRISM 310 Genetic analyzer (Applied Biosystems). The sequence was deposited into DDBJ under the accession number AB184533. BLAST search of the sequence by the EzTaxon-e server [7] indicated the highest similarity to that of Streptomyces graminisoli JR-19 T (HQ267975, 99.79%, 1440/1443). A phylogenetic tree was reconstructed on the basis of the  Phylum Actinobacteria TAS [25] Class Actinobacteria TAS [26] Order Actinomycetales TAS [26][27][28][29] Suborder Streptomycineae TAS [26,29] Family Streptomycetaceae TAS [26,[28][29][30][31] Genus Streptomyces TAS [28,[31][32][33] Species Streptomyces hyaluromycini TAS [12] Strain: MB-PO13 TAS [1] Gram stain Gram-positive TAS [12] Cell shape Branched mycelia TAS [12] Motility Not reported Sporulation Sporulating TAS [12] Temperature range 10°C to 37°C TAS [12] Optimum temperature 28°C TAS [12] pH range; Optimum , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [34] 16S rRNA gene sequence together with taxonomically close Streptomyces type strains using CLUSTAL-W program [8] and by the neighbor-joining method [9] using the MEGA 6.0 program [10]. The resultant tree topologies were evaluated by bootstrap analysis [11] based on 1000 replicates. The phylogenetic tree is shown in Fig. 2. On the basis of these findings, strain MB-PO13 T was proposed to be classified as a representative of a novel species of the genus Streptomyces, with the name Streptomyces hyaluromycini sp. nov. [12].

Genome sequencing information
Genome project history In collaboration between Toyama Prefectural University and NBRC, the organism was selected for genome sequencing to elucidate the hyaluromycin biosynthetic pathway. We successfully accomplished the genome project of Streptomyces hyaluromycini MB-PO13 T as reported in this paper. The draft genome sequences have been deposited in the INSDC database under the accession number BCFL01000001-BCFL01000052. The project information and its association with MIGS version 2.0 compliance are summarized in Table 2 [15].

Growth conditions and genomic DNA preparation
Streptomyces hyaluromycini MB-PO13 T was deposited in the NBRC culture collection with the registration number of NBRC 110483 T . Its monoisolate was grown on polycarbonate membrane filter (Advantec) on 1/2 ISP 2 agar medium (0.2% yeast extract, 0.5% malt extract, 0.2% glucose, 2% agar, pH 7.3) at 28°C. High quality genomic DNA for sequencing was isolated from the mycelia with an EZ1 DNA Tissue Kit and a Bio Robot EZ1 (Qiagen) according to the protocol for extraction of nucleic acid from Gram-positive bacteria. The size, purity, and double-strand DNA concentration of the genomic DNA were measured by pulsed-field gel electrophoresis, ratio of absorbance values at 260 nm and 280 nm, and Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies), respectively, to assess the quality of genomic DNA.

Genome sequencing and assembly
Shotgun and paired-end libraries were prepared and subsequently sequenced using 454 pyrosequencing technology and HiSeq1000 (Illumina) paired-end technology, respectively ( Table 2). The 77 Mb shotgun sequences and 881 Mb paired-end sequences were assembled using Newbler v2.8 and subsequently finished using GenoFinisher [16] to yield 52 scaffolds larger than 500 bp.

Genome annotation
Coding sequences were predicted by Prodigal [17] and tRNA-scanSE [18]. The gene functions were annotated using an in-house genome annotation pipeline, and PKS and NRPS-related domains were searched using the SMART and PFAM domain databases. PKS and NRPS gene clusters were determined as reported previously [19]. BLASTP search against the NCBI nr databases were also used for predicting function of  Fig. 3 Gene organizations of rubromycin-, hyarulomycin-and griseorhodin-biosynthetic gene clusters. Homologs are linked by gray dotted lines. The rub, Orf1-and grh are rubromycin-, hyarulomycin-and griseorhodin-biosynthetic gene clusters, respectively. Hyarulomycin-biosynthetic genes are indicated with orf numbers as shown in Table 5  proteins encoded in the hyaluromycin biosynthetic gene cluster.

Genome properties
The total size of the genome of Streptomyces hyaluromycini MB-PO13 T is 11,525,033 bp and the GC content is 71.0% (Table 3), similar to other genome-sequenced Streptomyces members such as Streptomyces violaceoniger Tu4133, Streptomyces bingchenggensis BCW-1 [20] and Streptomyces rapamycinicus NRRL 5491 T . Of the total 10,201 genes, 10,098 are protein-coding genes and 103 are RNA genes. The classification of genes into COGs functional categories is shown in Table 4. As for secondary metabolite pathways by PKSs and NRPSs, Streptomyces hyaluromycini MB-PO13 T has at least six type I PKS gene clusters, three type II PKS gene clusters, two type III PKS gene clusters, six NRPS gene clusters, and one hybrid PKS/NRPS gene cluster.

Insights from the genome sequence
Hyaluromycin biosynthetic pathway in Streptomyces hyaluromycini MB-PO13 T Hyarulomycin is a derivative of γ-rubromycin, possessing a C 5 N unit instead of a methoxy group as a side chain. The rubromycin-biosynthetic (rub) gene cluster is published in the GenBank (accession no. AF293355.2), but the biosynthetic mechanism has not been reported yet. Among the members of rubromycin family, only the griseorhodin-biosynthetic (grh) pathway has been extensively studied: griseorhodin A is synthesized by type II PKSs and modification enzymes [4,21]. In the genome sequence of S. hyaluromycini MB-PO13 T , three type II PKS gene clusters are present. Among them, the type II PKS gene cluster in scaffold000001 resembles those of rubromycin and griseorhodin as shown in Fig. 3 and Table 5. But, unlike rub and grh gene clusters, the cluster also encodes amide synthase (Orf1-763), 5aminolevulinate synthase (Orf1-762) and AMPdependent synthase (Orf1-761) essential for C 5 N unit synthesis [22]. Thus, we considered it to be the biosynthetic gene cluster for hyarulomycin. According to the proposed biosynthetic mechanisms of griseorhodin [4] and C 5 N [22,23], we predicted the biosynthetic pathway of hyarulomycin as shown in Fig. 4. The polyketide chain is synthesized by the iterative condensation of an acyl-CoA starter and 12 malonyl-CoA units. This elongation cycle is catalyzed by KSα, KSβ (chain length factor) and acyl carrier protein. Since almost all the homologs of Grh enzymes are present in the putative hyarulomycin-biosynthetic gene cluster (Table 5, Fig. 3),