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  • Short genome report
  • Open Access

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

  • 1,
  • 2,
  • 3,
  • 3,
  • 3,
  • 2 and
  • 1Email author
Standards in Genomic Sciences201813:2

https://doi.org/10.1186/s40793-017-0286-7

  • Received: 14 September 2017
  • Accepted: 23 November 2017
  • Published:

Abstract

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.

Keywords

  • Biosynthesis
  • C5N
  • Polyketide synthase
  • Rubromycin
  • Streptomyces

Introduction

Hyaluromycin is a hyaluronidase inhibitor isolated from the culture broth of an actinomycete strain MB-PO13T of the genus Streptomyces [1]. The structure consists of a γ-rubromycin core possessing a C5N 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 C5N 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-PO13T to elucidate the biosynthetic mechanism of hyaluromycin. We herein present the draft genome sequence of Streptomyces hyaluromycini MB-PO13T, 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.

Organism information

Classification and features

During the course of screening for hyaluronidase inhibitors from actinomycetes, Streptomyces hyaluromycini MB-PO13T 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 Na2CO3. 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 Gram-stain-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-19T (HQ267975, 99.79%, 1440/1443). A phylogenetic tree was reconstructed on the basis of the 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-PO13T was proposed to be classified as a representative of a novel species of the genus Streptomyces , with the name Streptomyces hyaluromycini sp. nov. [12].
Fig. 1
Fig. 1

Scanning electron micrograph of Streptomyces hyaluromycini MB-PO13T grown on 1/10 ISP 2 agar for 14 days at 28 °C. Bar, 5 μm

Table 1

Classification and general features of Streptomyces hyaluromycini MB-PO13T

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [24]

  

Phylum Actinobacteria

TAS [25]

  

Class Actinobacteria

TAS [26]

  

Order Actinomycetales

TAS [2629]

  

Suborder Streptomycineae

TAS [26, 29]

  

Family Streptomycetaceae

TAS [26, 2831]

  

Genus Streptomyces

TAS [28, 3133]

  

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

4 to 9; 7

TAS [12]

 

Carbon source

Glucose, inositol, arabinose, fructose, glucose, inositol, mannitol, rhamnose, xylose

TAS [12]

MIGS-6

Habitat

Tunicate (Molgula manhattensis)

TAS [1]

MIGS-6.3

Salinity

0% to 2% NaCl

TAS [12]

MIGS-22

Oxygen requirement

Aerobic

TAS [12]

MIGS-15

Biotic relationship

Free-living

TAS [12]

MIGS-14

Pathogenicity

Not reported

 

MIGS-4

Geographic location

Tokyo Bay, Minato-ku, Tokyo, Japan

TAS [1]

MIGS-5

Sample collection

August 13, 2007

NAS

MIGS-4.1

Latitude

35° 37′ 33″ N

NAS

MIGS-4.2

Longitude

139° 45′ 5″ E

NAS

MIGS-4.4

Altitude

−1.0 m. above sea level

NAS

a Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., 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]

Fig. 2
Fig. 2

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of strain MB-PO13T and its taxonomic neighbors. Kitasatospora setae KM-6054T (AB022868) was used as the outgroup. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. Bar, 0.01 substitutions per nucleotide position

Chemotaxonomic data

The isomer of diaminopimelic acid in the whole-cell hydrolysate was analyzed according to the method described by Hasegawa et al. [13]. Isoprenoid quinones and cellular fatty acids were analyzed as described previously [14]. The whole-cell hydrolysate of strain MB-PO13T contained LL-A2pm, glucose and mannose. The detected menaquinones were identified as MK-9(H8), MK-9(H6), MK-9(H4) and MK-9(H10) (5:37:57:1). The principal polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Six unidentified phospholipids were also detected. The major cellular fatty acids (>10%) were anteiso-C15:0 (24.9%), iso-C16:0 (23.4%), iso-C14:0 (15.0%) and C16:0 (10.7%). These chemotaxonomic features corresponded to those of the genus Streptomyces .

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-PO13T 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].
Table 2

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

High-Quality Draft

MIGS-28

Libraries used

454 shotgun library, Illumina paired-end library

MIGS 29

Sequencing platforms

454 GS FLX+, Illumina HiSeq1000

MIGS 31.2

Fold coverage

77×

MIGS 30

Assemblers

Newbler v2.6, GenoFinisher

MIGS 32

Gene calling method

Prodigal

 

Locus Tag

MB-PO13

 

Genbank ID

BCFL01000001-BCFL01000052

 

GenBank Date of Release

July 1, 2017

 

GOLD ID

Not registered

 

BIOPROJECT

PRJDB4283

MIGS 13

Source Material Identifier

NBRC 110483

 

Project relevance

Industrial

Growth conditions and genomic DNA preparation

Streptomyces hyaluromycini MB-PO13T was deposited in the NBRC culture collection with the registration number of NBRC 110483T. 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 proteins encoded in the hyaluromycin biosynthetic gene cluster.

Genome properties

The total size of the genome of Streptomyces hyaluromycini MB-PO13T 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 5491T. 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-PO13T 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.
Table 3

Genome statistics

Attribute

Value

% of Total

Genome size (bp)

11,525,033

100.0

DNA coding (bp)

10,176,135

88.3

DNA G + C (bp)

8,184,694

71.0

DNA scaffolds

52

Total genes

10,201

100.0

Protein coding genes

10,098

99.0

RNA genes

103

1.0

Pseudo genes

Genes in internal clusters

4827

47.3

Genes with function prediction

7049

69.1

Genes assigned to COGs

5317

52.1

Genes with Pfam domains

7836

77.6

Genes with signal peptides

1003

9.9

Genes with transmembrane helices

2326

23.0

CRISPR repeats

2

0

Table 4

Number of genes associated with general COG functional categories

Code

Value

%age

Description

J

244

2.4

Translation, ribosomal structure and biogenesis

A

0

0

RNA processing and modification

K

948

9.4

Transcription

L

129

1.3

Replication, recombination and repair

B

1

0

Chromatin structure and dynamics

D

45

0.4

Cell cycle control, cell division, chromosome partitioning

V

205

2.0

Defense mechanisms

T

477

4.7

Signal transduction mechanisms

M

279

2.8

Cell wall/membrane biogenesis

N

25

0.2

Cell motility

U

24

0.2

Intracellular trafficking and secretion

O

176

1.7

Posttranslational modification, protein turnover, chaperones

C

397

3.9

Energy production and conversion

G

563

5.6

Carbohydrate transport and metabolism

E

480

4.8

Amino acid transport and metabolism

F

108

1.1

Nucleotide transport and metabolism

H

332

3.3

Coenzyme transport and metabolism

I

497

4.9

Lipid transport and metabolism

P

281

2.8

Inorganic ion transport and metabolism

Q

380

3.8

Secondary metabolites biosynthesis, transport and catabolism

R

708

7.0

General function prediction only

S

82

0.8

Function unknown

4781

47.3

Not in COGs

The total is based on the total number of protein coding genes in the genome

Insights from the genome sequence

Hyaluromycin biosynthetic pathway in Streptomyces hyaluromycini MB-PO13T

Hyarulomycin is a derivative of γ-rubromycin, possessing a C5N 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-PO13T, 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), 5-aminolevulinate synthase (Orf1-762) and AMP-dependent synthase (Orf1-761) essential for C5N 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 C5N [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), the resulting polyketide chain is likely cyclized and modified to the polycyclic intermediate bearing a spiroketal moiety in the similar fashion to griseorhodin biosynthesis. Unlike griseorhodin A, the epoxide functionality is not present in the spiroketal moiety of rubromycin and hyaluromycin. This can be explained by the absence of  homolog of grhO4 encoding ferredoxin responsible for epoxide formation of griseorhodin A in rubromycin- and hyarulomycin-biosynthetic gene clusters. It was unable to predict a gene responsible for the removal of the hydroxyl group at the spiroketal only by this bioinformatic analysis. 5-Aminolevulinate synthase (Orf1-762), 5-aminolevulinate CoA ligase (Orf1-761) and amide synthase (Orf1-763) are involved in the formation of C5N unit and its coupling with the aromatic core.
Fig. 3
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

Table 5

Putative hyaluromycin biosynthetic gene cluster and the neighboring genes

Orf1-

Size (aa)

Proposed function

Closest homolog

Homolog (I/S, %) in

Description, Origin, Accession number

I/Sb (%)

grh cluster

rub cluster

769

230

cyclase

hypothetical protein, Streptomyces fulvoviolaceus, WP_052425082

54/63

RubK (53/63)

768a

656

ABC transporter ATP-binding protein

multidrug ABC transporter ATP-binding protein, Actinopolymorpha alba, WP_020576731

70/83

767a

577

multidrug ABC transporter ATPase

multidrug ABC transporter ATPase, Streptomyces varsoviensis, WP_030881385

69/81

766a

117

MarR family transcriptional regulator

MarR family transcriptional regulator, Actinomadura macra, WP_067468911

45/63

765a

72

unknown

hypothetical protein, Streptomyces aurantiacus, WP_055507532.

56/60

764a

498

transcriptional regulator

hypothetical protein, Streptomyces sp. NRRL WC-3742, WP_051836320

55/63

GrhR2 (34/48)

 

763a

533

amide synthetase

hypothetical protein, partial, Streptomyces sp. NRRL WC-3742, WP_078910860

60/70

762a

405

5-aminolevulinate synthase

AsuD2, Streptomyces nodosus subsp. asukaensis, ADI58646

77/85

761

515

5-aminolevulinate CoA ligase

AMP-dependent synthetase, Streptomyces uncialis, OKH94380

77/83

760

183

unknown

hypothetical protein, Streptomyces prunicolor, WP_019061819

50/60

759

122

unknown

hypothetical protein, Streptomyces fulvoviolaceus, WP_030615859

72/82

GrhI (61/73)

758

477

oxygenase

hypothetical protein, Streptomyces yerevanensis, WP_033324694

72/82

GrhO1 (72/80)

RubI (71/80)

757

257

3-oxoacyl-ACP reductase

SDR family oxidoreductase, Streptomyces fulvoviolaceus, WP_030615854

83/92

GrhO2 (73/81)

RubJ (83/91)

756

325

acetyltransferase

GrhJ, Streptomyces sp. CN48+, AIE76926

68/74

GrhJ (67/73)

755

540

monooxygenase

hypothetical protein, Streptomyces prunicolor, WP_026151147

73/80

GrhO5 (69/75)

RubL (73/80)

754a

161

transcriptional regulator

putative transcriptional repressor GrhR3, Streptomyces sp. CN48+, AIE76928

76/88

GrhR3 (76/88)

RubM (74/83)

753

501

monooxygenase

RubN, Streptomyces collinus, AAM97364

80/86

GrhO6 (73/80)

RubN (80/86)

752

325

oxidoreductase

hypothetical protein, Streptomyces sp. TSRI0261, WP_073806081

86/93

GrhO7 (78/89)

751

343

methyltransferase

hypothetical protein, Streptomyces fulvoviolaceus, WP_030615823

81/86

GrhL (77/83)

750

535

monooxygenase

hypothetical protein, Streptomyces prunicolor, WP_019061807

74/82

GrhO8 (70/79)

RubO (63/72)p

749a

534

oxidoreductase

hypothetical protein, Streptomyces sp. TP-A0875, WP_053912978

74/80

GrhO9 (71/79)

RubP (74/80)

748

161

unknown

hypothetical protein, Streptomyces prunicolor, WP_019061805

81/85

GrhM (80/86)

RubQ (80/85)

747

174

unknown

hypothetical protein, Streptomyces fulvoviolaceus, WP_030615810

67/74

GrhN (56/64)

RubW (64/74)

746

623

asparagine synthase

RubR, Streptomyces collinus, AAM97368

80/86

GrhP (74/81)

RubR (80/86)

745

669

transcriptional regulator

RubS, Streptomyces collinus, AAM97369

63/75

GrhR2 (43/56)

RubS (63/75)

744

123

cyclase

putative cyclaseI, Streptomyces collinus, AAG03065

83/88

GrhQ (75/88)

RubE (83/88)

743

143

cyclase

cupin, Streptomyces sp. TSRI0261, OKJ01252

83/90

GrhS (66/77)

RubD (79/85)

742

424

ketosynthase α subunit

type II polyketide synthase 4, Streptomyces sp., APD71740

89/95

GrhA (85/91)

RubA (89/93)

741

420

ketosynthase β subunit

type II polyketide synthase 5, Streptomyces sp., APD71741

82/88

GrhB (76/83)

RubB (79/85)

740

87

acyl carrier protein

acyl carrier protein, Streptomyces collinus, AAG03069

68/79

GrhC (34/61)

RubC (68/79)

739

398

cyclase/reductase

hypothetical protein, Streptomyces prunicolor, WP_019061796

79/87

GrhT (67/78)

RubF (78/85)

738

249

ketoreductase

SDR family oxidoreductase, Streptomyces prunicolor, WP_019061795

86/94

GrhO10 (79/89)

RubG (86/93)

737

108

monooxygenase

hypthetical protein, Streptomyces collinus, AAG03072

88/93

GrhU (75/84)

RubH (88/93)

736

113

unknown

hypothetical protein, Streptomyces fulvoviolaceus, WP_078655944

73/80

GrhV (67/76)

RubT (70/81)

735

417

cytochrome P450

cytochrome P450, Streptomyces fulvoviolaceus, WP_030615776

80/86

GrhO3 (37/53)

RubU (80/86)

734

301

unknown

DUF1963 domain-containing protein, Streptacidiphilus carbonis, WP_042397320

78/85

733

155

cupin

cupin, Streptomyces prunicolor, WP_019056246

93/97

732

322

esterase

alpha/beta hydrolase, Actinobacteria bacterium OK074, KPI24488

83/88

731

313

transcriptional regulator

transcriptional regulator, Streptomyces hokutonensis, WP_043260174

79/85

730a

491

unknown

dolichyl-phosphate-mannose-protein mannosyltransferase, Micromonospora auratinigra, SBT53146

57/67

729

42

unknown

728a

333

transcriptional regulator

LacI family transcriptional regulator, ‘Streptomyces humi’, WP_046734674

93/96

aencoded in complementary strand, bI/S, identity/similarity. Orf1-763 also shows 48% sequence identity/61% sequence similarity to AsuD1 of Streptomyces nodosus subsp. asukaensis (ADI58645); Orf1-761 shows 73% sequence identity/81% sequence similarity to AsuD3 of S. nodosus subsp. asukaensis (ADI58647)

Fig. 4
Fig. 4

Putative biosynthetic pathways of hyarulomycin, rubromycin and griseorhodin. Each step is catalyzed by enzymes encoded following genes as proposed in griseorhodin biosynthesis [4]. 1grhA/rubA/orf1-742 (KSα), grhB/rubB/orf1-741 (KSβ) and grhC/rubC/orf1-740 (ACP); 2grhE/rubK?/orf1-769?, grhQ/rubE/orf1-744, grhS/rubD/orf1-743 and grhT/rubF/orf1-739, 3grhO8/rubO/orf1-750, grhO9/rubP/orf1-749 (monooxygenases), grhL/−/orf1-751 (MT), grhM/rubQ/orf1-748 (unknown) and grhP/rubR/orf1-746 (asparagine synthase); 4grhO5/rubL/orf1-755 (monooxygenase) and grhO1/rubI/orf1-758 (oxygenase)?; 5grhO6/rubN/orf1-753 (monooxygenase) and grhJ/−/orf1-756 (acetyltransferase)?; 6grhO10/rubG/orf1-738 (KR) or grhT/rubF/orf1-739 (cyclase/reductase); 7grhO3/rubU/orf1-735 (cytochrome P450), grhO4/−/− (ferredoxin) and grhO7/−/orf1-752 (oxidoreductase). Homologs are connected with slashes in order of rubromycin/griseorhodin/hyarulomycin. ACP, acyl carrier protein; CLF, chain length factor; Fd, ferredoxin; KS, ketosynthase; KR, ketoreductase; MT, methyltransferase; −, no homolog in the sequence

Conclusions

The 11.5 Mb draft genome of Streptomyces hyaluromycini MB-PO13T, a producer of hyaluromycin, isolated from tunicate ( Molgula manhattensis ) has been deposited at GenBank/ENA/DDBJ under the accession number BCFL00000000. We successfully identified the gene cluster for hyaluromycin synthesis and proposed the plausible biosynthetic pathway. These findings provide useful information for genetic engineering to synthesize more potential hyaluronidase inhibitors and discovering new bioactive aromatic polyketides possessing the C5N unit.

Abbreviations

A2pm: 

Diaminopimelic acid

ABC: 

ATP-binding cassette

ACP: 

Acyl carrier protein

C5N: 

2-amino-3-hydroxycyclopent-2-enone

CLF: 

Chain length factor

CoA: 

Coenzyme A

DDBJ: 

DNA Data Bank of Japan

Fd: 

Ferredoxin

ISP: 

International Streptomyces project

KR: 

Ketoreductase

KS: 

Ketosynthase

MK: 

Menaquinone

MT: 

Methyltransferase

NBRC: 

Biological Resource Center, National Institute of Technology and Evaluation

NRPS: 

Nonribosomal peptide synthetase

PKS: 

Polyketide synthase

Declarations

Acknowledgements

This research was supported by the Japan Society for the Promotion of Science (JSPS) for Young Scientists (15 K18692) and Institute for Fermentation, Osaka (IFO) for Young Scientists to E.H. We are grateful to Ms. Yuko Kitahashi for finishing the genome sequence and helping the search of secondary metabolite genes. We would like to thank Ms. Satomi Hirakata for technical assistance on whole genome sequencing. We also thank Ms. Mariko Ozu for registering the sequences on DDBJ.

Authors’ contributions

EH performed chemotaxonomic experiments, examined features of the strain, and drafted the manuscript. KH elucidated the hyaluromycin-biosynthetic pathway. NI annotated the genome sequences. AH sequenced the genome. AK analysed secondary metabolite-biosynthetic genes. MH supervised taxonomic study of the strain. YI designed this study and edited the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare no competing interest.

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Authors’ Affiliations

(1)
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Toyama, Japan
(2)
Biological Resource Center, National Institute of Technology and Evaluation (NBRC), Chiba, Japan
(3)
NBRC, Tokyo, Japan

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Copyright

© The Author(s). 2018

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