Open Access

Draft genome sequence of Streptomyces sp. MWW064 for elucidating the rakicidin biosynthetic pathway

  • Hisayuki Komaki1Email author,
  • Arisa Ishikawa2,
  • Natsuko Ichikawa3,
  • Akira Hosoyama3,
  • Moriyuki Hamada1,
  • Enjuro Harunari2,
  • Takuya Nihira4, 5,
  • Watanalai Panbangred5, 6 and
  • Yasuhiro Igarashi2
Standards in Genomic Sciences201611:83

https://doi.org/10.1186/s40793-016-0205-3

Received: 24 February 2016

Accepted: 11 October 2016

Published: 21 October 2016

Abstract

Streptomyces sp. MWW064 (=NBRC 110611) produces an antitumor cyclic depsipeptide rakicidin D. 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 7.9 Mb genome of Streptomyces sp. MWW064 encoded 7,135 putative ORFs, of which 6,044 were assigned with COG categories. The genome harbored at least three type I polyketide synthase (PKS) gene clusters, seven nonribosomal peptide synthetase (NRPS) gene clusters, and four hybrid PKS/NRPS gene clusters, from which a hybrid PKS/NRPS gene cluster responsible for rakicidin synthesis was successfully identified. We propose the biosynthetic pathway based on bioinformatic analysis, and experimentally proved that the pentadienoyl unit in rakicidins is derived from serine and malonate.

Keywords

BiosynthesisNonribosomal peptide synthetasePolyketide synthaseRakicidin Streptomyces

Introduction

Rakicidin D is an inhibitor of tumor cell invasion isolated from the culture broth of an actinomycete strain MWW064 of the genus Streptomyces [1]. To date, five congeners rakicidins A, B, and E from Micromonospora and rakicidins C and D from Streptomyces have been reported [14]. Rakicidins share the 15-membered cyclic depsipeptide structure comprising three amino acids and a fatty acid modified with hydroxy and methyl substitutions. The most intriguing part of rakicidins is a rare unusual amino acid, 4-amino-2,4-pentadienoate (APDA), which is present only in a limited range of secondary metabolites of actinomycetes such as BE-43547 [5] and microtermolide [6, 7]. Despite the scarcity of APDA unit in nature, nothing is known about its biosynthesis. Recently, putative biosynthetic genes for rakicidin D were reported [8], but the data is incomplete, no detailed information is shown in the paper, and DNA sequences have not been registered in public databases. Hence, the biosynthesis of rakicidins has been actually unclear yet. In this study, we performed whole genome shotgun sequencing of the strain MWW064 to elucidate the biosynthetic mechanism of rakicidin D. We herein present the draft genome sequence of Streptomyces sp. MWW064, together with the taxonomical identification of the strain, description of its genome properties and annotation of the gene cluster for rakicidin synthesis. We propose the rakicidin-biosynthetic mechanism predicted by bioinformatics analysis and confirmed by precursor-incorporation experiments.

Organism information

Classification and features

In the course of screening for antitumor compounds from actinomycetes, Streptomyces sp. MWW064 was isolated from a marine sediment sample collected in Samut Sakhon province of Thailand and found to produce rakicidin D [1]. The general feature of this strain is shown in Table 1. This strain grew well on ISP 2 and ISP 4 agars. On ISP 5 and ISP 7 agars, the growth was poor. The color of aerial mycelia was white and that of the reverse side was pale red on ISP 2 agar. Diffusible pigments were dark orange on ISP 2 agar medium. Strain MWW064 formed extensively branched- substrate and aerial mycelia. The aerial mycelium formed flexuous spore chains at maturity. The spores were cylindrical, having a smooth surface. A scanning electron micrograph of this strain is shown in Fig. 1. Growth occurred at 15–37 °C (optimum 28 °C) and pH 5–9 (optimum pH 7). Strain MWW064 exhibited growth with 0–3 % (w/v) NaCl (optimum 0 % NaCl). Strain MWW064 utilized glucose and inositol for growth. The gene sequence encoding 16S rRNA was obtained from GenBank/EMBL/DDBJ databases (accession no. GU295447). A phylogenetic tree was reconstructed on the basis of the 16S rRNA gene sequence together with taxonomically close Streptomyces type strains using ClustalX2 [9] and NJPlot [10]. The phylogenetic analysis confirmed that the strain MWW064 belongs to the genus Streptomyces (Fig. 2).
Table 1

Classification and general features of Streptomyces sp. MWW064 [13]

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 undetermined

-

  

strain: MWW064

TAS [1]

 

Gram stain

Gram-positive

NAS

 

Cell shape

Branched mycelia

IDA

 

Motility

Not reported

 
 

Sporulation

Sporulating

IDA

 

Temperature range

15 °C to 37 °C

IDA

 

Optimum temperature

28 °C

IDA

 

pH range; Optimum

5 to 9; 7

IDA

 

Carbon source

D-glucose, inositol

IDA

MIGS-6

Habitat

Marine sediment

TAS [1]

MIGS-6.3

Salinity

0 % to 3 % NaCl

IDA

MIGS-22

Oxygen requirement

Aerobic

IDA

MIGS-15

Biotic relationship

Free-living

IDA

MIGS-14

Pathogenicity

Not reported

 

MIGS-4

Geographic location

Samut Sakhon province, Thailand

TAS [1]

MIGS-5

Sample collection

February 2, 2008

NAS

MIGS-4.1

Latitude

13° 32’ 55” N

NAS

MIGS-4.2

Longitude

100° 16’ 39” E

NAS

MIGS-4.4

Altitude

8.6 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. 1

Scanning electron micrograph of Streptomyces sp. MWW064 grown on 1/2 ISP 2 agar for 7 days at 28 °C. Bar, 2 μm

Fig. 2

Phylogenetic tree of Streptomyces sp. MWW064 and phylogenetically close type strains, showing over 98.5 % similarity, based on 16S rRNA gene sequences. The accession numbers for 16S rRNA genes are shown in parentheses. The tree uses sequences aligned by ClustalX2 [9], and constructed by the neighbor-joining method [35]. All positions containing gaps were eliminated. The building of the tree also involves a bootstrapping process repeated 1,000 times to generate a majority consensus tree, and only bootstrap values above 50 % are shown at branching points. Streptomyces albus NBRC 13014T was used as an outgroup

Chemotaxonomic data

The isomer of diaminopimelic acid in the whole-cell hydrolysate was analyzed according to the method described by Hasegawa et al. [11]. Isoprenoid quinones and cellular fatty acids were analyzed as described previously [12]. The whole-cell hydrolysate of strain MWW064 contained ll-diaminopimelic acid as its diagnostic peptidoglycan diamino acid. The predominant menaquinones were identified as MK-9(H2), MK-9(H4) and MK-9(H6); MK-10(H2), MK-10(H4) and MK-10(H6) were also detected as minor components. The major cellular fatty acids were found to be anteiso-C15:0, iso-C15:0, C16:0, anteiso-C17:0, iso-C17:0 and iso-C16:0.

Genome sequencing information

Genome project history

In collaboration between Toyama Prefectural University and NBRC, the organism was selected for genome sequencing to elucidate the rakicidin biosynthetic pathway. We successfully accomplished the genome project of Streptomyces sp. MWW064 as reported in this paper. The draft genome sequences have been deposited in the INSDC database under the accession number BBUY01000001-BBUY01000099. The project information and its association with MIGS version 2.0 compliance are summarized in Table 2 [13].
Table 2

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

Improved-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

8.9 ×, 93.5 ×, respectively

MIGS 30

Assemblers

Newbler v2.8, GenoFinisher

MIGS 32

Gene calling method

Progidal

 

Locus Tag

SSP35

 

GenBank ID

BBUY00000000

 

GenBank Date of Release

February 20, 2016

 

GOLD ID

Not registered

 

BIOPROJECT

PRJDB3538

MIGS 13

Source Material Identifier

NBRC 110611

 

Project relevance

Industrial

Growth conditions and genomic DNA preparation

Streptomyces sp. MWW064 was deposited in the NBRC culture collection with the registration number of NBRC 110611. Its monoisolate was grown on polycarbonate membrane filter (Advantec) on double diluted 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 70 Mb shotgun sequences and 739 Mb paired-end sequences were assembled using Newbler v2.8 and subsequently finished using GenoFinisher [14] to yield 99 scaffolds larger than 500 bp.

Genome annotation

Coding sequences were predicted by Prodigal [15] and tRNA-scanSE [16]. 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 and their domain organizations were determined as reported previously [17]. Substrates of adenylation (A) and acyltransferase (AT) domains were predicted using antiSMASH [18]. BLASTP search against the NCBI nr databases were also used for predicting function of proteins encoded in the rakicidin biosynthetic gene cluster.

Genome properties

The total size of the genome is 7,870,697 bp and the GC content is 71.1 % (Table 3), similar to other genome-sequenced Streptomyces members. Of the total 7,206 genes, 7,135 are protein-coding genes and 71 are RNA genes. The classification of genes into COGs functional categories is shown in Table 4. As for secondary metabolite pathways by modular PKSs and NRPSs, Streptomyces sp. MWW064 has at least four hybrid PKS/NRPS gene clusters, three type I PKS gene clusters, and seven NRPS gene clusters. According to the assembly line mechanism [19], we predicted the chemical backbones that each cluster will synthesize (Table 5), suggesting the potential of Streptomyces sp. MWW064 to produce diverse polyketide- and nonribosomal peptide-compounds as the secondary metabolites.
Table 3

Genome statistics

Attribute

Value

% of Total

Genome size (bp)

7,904,619

100.0

DNA coding (bp)

6,855,885

86.7

DNA G + C (bp)

5,597,799

70.8

DNA scaffolds

99

-

Total genes

7,206

-

Protein coding genes

7,135

99.0

RNA genes

71

0.99

Pseudogenes

-

-

Genes in internal clusters

2,610

36.2

Genes with function prediction

4,515

62.7

Genes assigned to COGs

6,044

83.9

Genes with Pfam domains

4,870

67.6

Genes with signal peptides

559

7.8

Genes with transmembrane helices

1,550

21.5

CRISPR repeats

1

-

Table 4

Number of genes associated with general COG functional categories

Code

Value

% age

Description

J

279

4.6

Translation, ribosomal structure and biogenesis

A

4

0.1

RNA processing and modification

K

696

11.5

Transcription

L

452

7.5

Replication, recombination and repair

B

6

0.1

Chromatin structure and dynamics

D

55

0.9

Cell cycle control, Cell division, chromosome partitioning

V

132

2.2

Defense mechanisms

T

432

7.1

Signal transduction mechanisms

M

294

4.9

Cell wall/membrane biogenesis

N

33

0.5

Cell motility

U

95

1.6

Intracellular trafficking and secretion

O

223

3.7

Posttranslational modification, protein turnover, chaperones

C

386

6.4

Energy production and conversion

G

474

7.8

Carbohydrate transport and metabolism

E

651

10.8

Amino acid transport and metabolism

F

134

2.2

Nucleotide transport and metabolism

H

253

4.2

Coenzyme transport and metabolism

I

323

5.3

Lipid transport and metabolism

P

404

6.7

Inorganic ion transport and metabolism

Q

385

6.4

Secondary metabolites biosynthesis, transport and catabolism

R

1,032

17.1

General function prediction only

S

440

7.3

Function unknown

-

1,091

18.1

Not in COGs

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

Table 5

Modular PKS and NRPS gene clusters in Streptomyces sp. MWW064

Gene cluster

Encoded in

No. of modular PKS and NRPS genes

No. of modules

Backbone of predicted product

pks/nrps-1 (rak)

scaffold 9

6

7

R-C3-C3-Ser-C2-Gly-X

pks/nrps-2

scaffold 5

6

14

C2-C2-C2-C2-C2-Gly-C2-C2-C2-C2-C2-C2-C2-C2

pks/nrps-3

scaffold 2

4

3

C?-C?-X

pks/nrps-4

scaffold 11

1

2

X-C2

pks-1

scaffold 18

5

5

C?-C3-C2-C2-C?

pks-2

scaffold 23

1

1

C?

other pks a

scaffolds 11, 39, 45

>3

>10

C2-C2-C3-C2, C2-C2, C2-C2-C2, C2

nrps-1

scaffold 11

4

4

X-X-Val-X

nrps-2

scaffold 18

3

3

R-Val-X

nrps-3

scaffold 9

2

3

R-Cys-mCys

nrps-4

scaffold 13

3

4

Val-Gly-Ser-Pro

nrps-5

scaffold 2

1

1

Ser

nrps-6

scaffold 12

1

1

Thr

other nrps a

scaffolds 3, 5

>2

>6

X-X-X-X-X, Cys

anot completely sequenced. R, starter unit; C3, C3 unit derived from methylmalonyl-CoA; C2, C2 unit derived from malonyl-CoA; X, unpredictable amino acid; C?, unpredictable carbon unit derived from acyl-CoA; mCys, methylated cysteine

Insights from the genome sequence

Rakicidin biosynthetic pathway in Streptomyces sp. MWW064

The chemical structure of rakicidin D suggested that it is synthesized by a hybrid PKS/NRPS pathway. Among the four hybrid PKS/NRPS gene clusters present in Streptomyces sp. MWW064 (Table 5), pks/nrps-1 is most likely responsible for rakicidin synthesis because the carbon backbone of the predicted product (R-C3-C3-Ser-C2-Gly-X) is in good accordance with that of rakicidin D. Genes in pks/nrps-1 (Table 6) encode enzymes necessary for rakicidin biosynthesis (Fig. 3). This cluster contains three PKS genes (SSP35_09_01910, SSP35_09_01900, SSP35_09_01880) and three NRPS genes (SSP35_09_01890, SSP35_09_01870, SSP35_09_01860), corresponding to rakAB, rakC, rakEF, rakD, rakG, and rakH [8], respectively. Based on the collinearity rule of modular PKS/NRPS pathways, it is deduced that RakAB loads a starter molecule (‘R’ in Fig. 3), and subsequently RakAB and RakC add a diketide chain to the starter by condensation of two methylmalonyl-CoA molecules, since the substrates of their AT domains are likely methylmalonyl-CoA (‘ATm’ in Fig. 3). An NRPS RakD and the remaining PKS RakEF are most likely involved in the APDA supply: the A domain of RakD has signature amino acid residues for serine, and RakEF contains a set of domains (AT, KR, DH) for malonate incorporation, ketoreduction, and dehydration to provide a double bond between C9 and C10. In addition, the DH domain in RakEF is also proposed to be responsible for the dehydration of the primary hydroxy group of the incorporated serine molecule on the basis of the following reasons although experimental evidences are required. First, no dehydratase gene is present near the rakicidin cluster. In the biosynthesis of dehydroalanine in bacterial peptides such as lantibiotics, a dehydratase catalyzes the exo-methylene formation from serine [20, 21]. Second, the order of KR and DH domains in RakEF is unusual: among the three hundred type I PKS genes for eighty actinomycete polyketides, the order of two domains is exclusively DH-KR [22]. The only exception can be seen in the PKS genes for enediynes in which the chain elongation is iteratively catalyzed as similar to type II PKS [23]. The unusual order of KR-DH may render an undescribed function to the DH domain of RakEF. After formation of APDA moiety, RakG is likely responsible for the condensation of glycine and the following N-methylation, and RakH for asparagine condensation. Hydroxylation of asparagine would be catalyzed by asparagine hydroxylase encoded by rakO in the downstream of the cluster, to yield rakicidin D. On the basis of the above-mentioned bioinfomatic evidences, we here propose the biosynthetic pathway of rakicidin D as shown Fig. 3.
Table 6

ORFs in the rakicidin-biosynthetic gene cluster of Streptomyces sp. MWW064

ORF (locus tag)

Size (aa)

Deduced function

Protein homolog [origin]

Identity/similarity (%)

Accession number

SSP35_09_01970

243

unknown

hypothetical protein [Streptomyces natalensis]

63/73

WP_030067339

SSP35_09_01960

331

transcriptional regulator

hypothetical protein DT87_01625 [Streptomyces sp. NTK 937]

59/69

KDQ65969

SSP35_09_01950

358

3-oxoacyl-ACP synthase

3-oxoacyl-ACP synthase [Streptomyces sp. NRRL S-920]

77/87

WP_030791445

SSP35_09_01940

79

ACP

phosphopantetheine-binding protein [Streptomyces bingchenggensis BCW-1]

68/79

ADI05068

SSP35_09_01930

406

ketosynthase

3-oxoacyl-ACP synthase [Streptomyces sp. NRRL S-15]

81/89

WP_031089521

SSP35_09_01920

146

unknown

methylmalonyl-CoA epimerase [Salinispora pacifica]

80/87

WP_018222873

SSP35_09_01910 (RakAB)

2,902

PKS

hypothetical protein [Streptomyces vitaminophilus]

63/73

WP_018385948

SSP35_09_01900 (RakC)

1,624

PKS

non-ribosomal peptide synthetase [Micromonospora sp. M42]

60/70

EWM63000

SSP35_09_01890 (RakD)

1,126

NRPS

hypothetical protein [Streptomyces vitaminophilus]

68/78

WP_018385946

SSP35_09_01880 (RakEF)

1,950

PKS

hypothetical protein [Streptomyces vitaminophilus]

64/73

WP_018385945

SSP35_09_01870 (RakG)

1,556

NRPS

hypothetical protein, partial [Micromonospora purpureochromogenes]

64/74

WP_030498976

SSP35_09_01860 (RakH)

1,565

NRPS

amino acid adenylation domain protein [Nostoc punctiforme PCC 73102]

38/55

ACC80782

SSP35_09_01850

563

ABC transporter

hypothetical protein [Micromonospora purpureochromogenes]

62/73

WP_030498978

SSP35_09_01840 (RakL)

251

type-II thioesterase

hypothetical protein [Streptomyces vitaminophilus]

64/73

WP_018385940

SSP35_09_01830 (RakN)

1,013

NRPS

non-ribosomal peptide synthetase [Micromonospora sp. M42]

55/63

EWM63010

SSP35_09_01820 (RakO)

331

asparagine oxygenase

clavaminate synthase [Streptomyces sp. LaPpAH-202]

64/75

WP_026235187

SSP35_09_01810

809

unknown

penicillin amidase [Amycolatopsis nigrescens]

63/74

WP_026359955

SSP35_09_01800

205

transcriptional regulator

putative LuxR family transcriptional regulator [Streptomyces glaucescens]

71/81

AIR96926

SSP35_09_01910, SSP35_09_01900, SSP35_09_01890, SSP35_09_01880, SSP35_09_01870, SSP35_09_01860, SSP35_09_01840, SSP35_09_01830, and SSP35_09_01820 are corresponding to RakA plus RakB (RakAB), RakC, RakD, RakE plus RakF (RakEF), RakG, RakH, RakL, RakN, and RakO, previously reported in the reference [8], respectively, and SSP35_09_01940 may possibly be corresponding to RakI

Fig. 3

Genetic map of rakicidin biosynthetic gene cluster of Streptomyces sp. MWW064 and the biosynthetic mechanism of rakicidin D

Identification of biosynthetic precursors of the APDA moiety

To verify the predicted biosynthetic origin of the APDA unit, feeding experiments using 13C-labeled precursors were carried out. Inoculation, cultivation, extraction, and purification were performed in the same manner as previously reported [1]. Addition of sodium [2-13C]acetate or [1-13C]-L-serine (20 mg/100 ml medium/flask, 10 flasks for [2-13C]acetate, 3 flasks for [1-13C]-L-serine) was initiated at 48 h after inoculation and periodically carried out every 24 h for four times. After further incubation for 24 h, the whole culture broths were extracted with 1-butanol and several steps of purification yielded 2.5 mg and 1.7 mg of 13C-labeled rakicidin D, respectively. The 13C NMR spectrum of these labeled rakicidin D is shown in Table 7. Feeding of sodium [2-13C]acetate gave enrichments at C9 of the APDA unit and three carbons C18, C20, and C22 in the aliphatic chain of the fatty acid moiety. [1-13C]-L-serine feeding enriched C10 of the APDA unit and the carbonyl carbon of Gly (C5). These results unambiguously indicated that the APDA unit is derived from an acetate and a serine (Fig. 4). Labeling of C5 by serine-feeding can be explained by the interconversion between glycine and serine by transformylase in primary metabolism for amino acid supply.
Table 7

Incorporation of 13C-labeled precursors into rakicidin D

Position

δC

Relative enrichmentsa

[2-13C]acetate

[1-13C]-L-serine

1

169.2

0.89

1.58

2

54.9

1.14

1.19

3

72.5

1.57

1.07

4

172.7

1.31

1.95

5

167.6

0.68

6.61

6

52.5

0.77

1.15

7

36.5

1.00

1.00

8

166.0

0.77

1.43

9

118.8

3.46

1.54

10

138.4

1.03

13.97

11

137.9

0.95

0.67

12

117.1

1.02

1.33

13

172.5

1.61

1.26

14

41.7

1.86

1.36

15

78.1

1.25

1.21

16

33.9

1.91

1.39

17

32.8

1.30

1.55

18

27.0

2.64

1.26

19

28.9

1.15

1.58

20

31.3

3.37

1.43

21

22.1

1.05

1.28

22

14.0

3.37

1.61

23

15.4

1.51

1.07

24

13.3

1.75

1.32

a 13C signal intensity of each peak in the labeled 1 divided by that of the corresponding signal in the unlabeled 1, respectively, normalized to give an enrichment ratio of 1 for the unenriched peak of C7. The numbers in bold type indicate 13C-enriched atoms from 13C-labeled precursors

Fig. 4

Incorporation of 13C-labeled precursors into rakicidin D

Conclusions

The 7.9 Mb draft genome of Streptomyces sp. MWW064, a producer of rakicidin D isolated from marine segment, has been deposited at GenBank/ENA/DDBJ under the accession number BBUY00000000. We successfully identified the PKS/NRPS hybrid gene cluster for rakicidin synthesis and proposed the plausible biosynthetic pathway. Labeled precursor incorporation experiments showed the APDA moiety is synthesized from serine and malonate. These finding will open up possibilities of genetic engineering to synthesize more potential rakicidin-based antitumor compounds and discovering new bioactive compounds possessing APDA units.

Abbreviations

A: 

Adenylation

ABC: 

ATP-binding cassette

ACP: 

Acyl carrier protein

APDA: 

4-amino-2,4-pentadienoate

AT: 

Acyltransferase

C: 

Condensation

CoA: 

Coenzyme A

CoL: 

CoA ligase

DDBJ: 

DNA Data Bank of Japan

DH: 

Dehydratase

ER: 

Enoylreductase

ISP: 

International Streptomyces project

KR: 

Ketoreductase

KS: 

Ketosynthase

NBRC: 

Biological Resource Center, National Institute of Technology and Evaluation

NMR: 

Nuclear magnetic resonance

NRPS: 

Nonribosomal peptide synthetase

PKS: 

Polyketide synthase

T: 

Thiolation

Declarations

Acknowledgements

This research was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology of Japan to Y.I. We are grateful to Dr. S. Kitani at Osaka University for thoughtful discussion on the biosynthesis of rakicidins. We also thank Ms. Yuko Kitahashi, Ms. Satomi Saitou and Ms. Chiyo Shibata for assistance to this study.

Authors’ contributions

HK elucidated rakicidin-biosynthetic pathway and drafted the manuscript. AI carried out feeding experiments using labeled precursors. NI annotated the genome sequences. AH sequenced the genome. MH performed chemotaxonomic experiments. EH examined the features of the strain. TN coordinated collaboration between Japan and Thailand. WP isolated the strain. YI designed this study and edited the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Biological Resource Center, National Institute of Technology and Evaluation (NBRC)
(2)
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University
(3)
NBRC
(4)
International Center for Biotechnology, Osaka University
(5)
Mahidol University and Osaka University Collaborative Research Center on Bioscience and Biotechnology
(6)
Department of Biotechnology, Faculty of Science, Mahidol University

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© The Author(s). 2016