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. 2020 Feb 13;7:55. doi: 10.1038/s41597-020-0395-9

Thirty complete Streptomyces genome sequences for mining novel secondary metabolite biosynthetic gene clusters

Namil Lee 1,#, Woori Kim 1,#, Soonkyu Hwang 1, Yongjae Lee 1, Suhyung Cho 1, Bernhard Palsson 3,4,5, Byung-Kwan Cho 1,2,5,✉
PMCID: PMC7018776  PMID: 32054853

Abstract

Streptomyces are Gram-positive bacteria of significant industrial importance due to their ability to produce a wide range of antibiotics and bioactive secondary metabolites. Recent advances in genome mining have revealed that Streptomyces genomes possess a large number of unexplored silent secondary metabolite biosynthetic gene clusters (smBGCs). This indicates that Streptomyces genomes continue to be an invaluable source for new drug discovery. Here, we present high-quality genome sequences of 22 Streptomyces species and eight different Streptomyces venezuelae strains assembled by a hybrid strategy exploiting both long-read and short-read genome sequencing methods. The assembled genomes have more than 97.4% gene space completeness and total lengths ranging from 6.7 to 10.1 Mbp. Their annotation identified 7,000 protein coding genes, 20 rRNAs, and 68 tRNAs on average. In silico prediction of smBGCs identified a total of 922 clusters, including many clusters whose products are unknown. We anticipate that the availability of these genomes will accelerate discovery of novel secondary metabolites from Streptomyces and elucidate complex smBGC regulation.

Subject terms: Genomic analysis, Systems biology, Antibiotics

Measurement(s) DNA • genome • sequence_assembly • sequence feature annotation
Technology Type(s) DNA sequencing • sequence assembly process • sequence annotation
Factor Type(s) strain
Sample Characteristic - Organism Streptomyces
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Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11791323

Background & Summary

With the rapid emergence of antibiotic microbial resistance (AMR) to all major classes of antibiotics and the decline in number of potential candidates for new antibiotics, there is a pressing need for the discovery of novel antibacterial compounds1. Streptomyces, soil dwelling gram-positive bacteria, continue to be promising microorganisms for the production of clinically important secondary metabolites, including not only antibiotics, but also antiviral, antifungal, and antiparasitic agents, and antitumorals and immunosuppressant compounds2. Streptomyces are distinguished by their complex life cycle and high G + C content (often over 70%) in their linear genomes3,4. Traditionally, drug discovery from Streptomyces has been based on bioactivity screening followed by mass spectrometry and NMR-based molecular identification5. However, recent advances in genomics-based approaches revealed that most of the secondary metabolite biosynthetic gene clusters (smBGCs) of streptomycetes are inactive under laboratory conditions, suggesting that the ability of streptomycetes to produce secondary metabolites has been under-estimated5,6. Each Streptomyces species has the genetic potential to produce more than 30 secondary metabolites on average, which are diverse and differ between species7,8. Considering Streptomyces is the largest genus of actinobacteria with approximately 900 species characterized so far, streptomycetes are a valuable resource for the discovery of novel secondary metabolites9.

SmBGCs, especially polyketide and non-ribosomal peptide synthetase types, are often composed of extraordinarily long genes (>5 kb) encoding multi-modular enzymes with repetitive domain structures. Therefore, accurate gene annotations based on high quality genome sequences are essential for the precise identification of smBGCs10. Gene annotation with the high quality genome of S. clavuligerus revealed that 30% out of a total of 7,163 protein coding genes were incorrectly annotated in the previous draft genome of S. clavuligerus containing ambiguous and inaccurate nucleotides, indicating the importance of high quality genome sequences11. In addition, high quality genome sequences are essential for multi-omics analysis, which facilitates the understanding of the complex regulation on smBGCs and rational engineering for increasing secondary metabolites production11,12.

Among the 1,614 streptomycetes genomes that have been deposited in the NCBI Assembly database to date (as of 9th December 2019), only 189 and 35 assemblies were designated as complete genome level and chromosome level, respectively. More than 86% of assemblies were draft-quality genome sequences, which contain fragmented multiple contigs or ambiguous sequences4,13–15. One of the main obstacles to obtaining high quality genomic information of streptomycetes is the low fidelity of sequencing techniques when dealing with high G w C genomes and frequently repetitive sequences such as terminal inverted repeats13. In addition, since streptomycetes have linear chromosome, it is difficult to confirm the completeness of the assembled chromosome.

In this study, we present the high-quality genome sequences of 30 streptomycetes, increasing the total number of reported complete Streptomyces genome by about 10%. The target streptomycetes were 22 Streptomyces type strains and eight different Streptomyces venezuelae strains, most of which are currently used as industrial strains for producing various bioactive compounds. We applied hybrid assembly strategy with long-read (PacBio) and short-read (Illumina) sequencing techniques to obtain complete genome sequences. PacBio sequencing provides long reads of several kb in length which allows the readthrough of regions with low complexity, enabling the assembly of repetitive regions, which are difficult to assemble by using Illumina sequencing reads, even with the high coverage data16. However, Illumina sequencing provides reads with a lower error rate compared to the PacBio sequencing, and assembled contigs based on the Illumina sequencing reads are not simply a subset of the contigs from PacBio sequencing reads13,17. Therefore, reconciling PacBio and Illumina sequencing methods enables one to generate more complete genomes by overcoming the shortcomings of each method. During the genome assembly using reads from PacBio (0.46~5.18 Gbp) and Illumina (0.5~3.0 Gbp) sequencing, we constructed 6.7 to 10.1 Mbp of streptomycetes genomes, most of which consist of single chromosomes with 72% G + C contents on average. Inaccurate sequences in the assembled genome were corrected using Illumina sequencing reads. The complete streptomycetes genomes have more than 97.4% gene space completeness and on average 7,000 protein coding genes, 20 rRNAs, and 68 tRNAs were annotated. Finally, based on the complete genome sequences and annotations, we predicted a total of 922 smBGCs. The complete genome sequences and newly determined smBGCs in this study should prove to be a fundamental resource for understanding the genetic basis of streptomycetes and for discovering novel secondary metabolites.

Methods

Genomic DNA (gDNA) extraction

Total 30 streptomycetes were purchased from Korean Collection for Type Cultures (KCTC, Korea). A stock of streptomycetes were inoculated to 50 mL of liquid culture medium with 0.16 g mL−1 of glass beads (3 ± 0.3 mm diameter) in 250 mL baffled flask and grown at 30 °C in a 200 rpm orbital shaker. Each streptomycetes was grown in one of four different culture medium, R5(–) medium (25 mM TES (pH 7.2), 103 g L−1 sucrose, 1% (w/v) glucose, 5 g L−1 yeast extract, 10.12 g L−1 MgCl2∙6H2O, 0.25 g L−1 K2SO4, 0.1 g L−1 casamino acids, 0.08 g L−1 ZnCl2, 0.4 mg L−1 FeCl3, 0.02 mg L−1 CuCl2∙2H2O, 0.02 mg L−1 MnCl2∙4H2O, 0.02 mg L−1 Na2B4O7∙10H2O, and 0.02 mg L−1 (NH4)6Mo7O24∙4H2O), 1 × sporulation medium (3.33 g L−1 glucose, 1 g L−1 yeast extract, 1 g L−1 beef extract, 2 g L−1 tryptose, and 0.006 g L−1 FeSO4∙7H2O), YEME medium (340 g L−1 sucrose, 10 g L−1 glucose, 3 g L−1 yeast extract, 5 g L−1 bacto peptone, and 3 g L−1 oxoid malt extract), and MYM medium (4 g L−1 maltose, 4 g L−1 yeast extract, 10 g L−1 malt extract). For gDNA extraction, 25 mL cultured cells were harvested at the exponential growth phase and washed twice with same volume of 10 mM EDTA, followed by the lysozyme (10 mg mL−1) treatment at 37 °C for 45 min. gDNA was extracted using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instruction. Quality and quantity of extracted gDNA samples were evaluated using 1% agarose gel electrophoresis and Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA), respectively.

Short-read (Illumina) genome sequencing

For construction of short-read genome sequencing library, 2.5 μg of gDNA was sheared to approximately 350 bp by a Covaris instrument (Covaris Inc., Woburn, MA, USA) with the following conditions; Power 175, Duty factor 20%, C. burst 200, Time 23 s, 8 times. The library was constructed using a TruSeq DNA PCR-Free LT kit (Illumina Inc., San Diego, CA, USA) following manufacturer’s instruction. Briefly, the fragmented DNA samples were cleaned and end-repaired, followed by the adaptor ligation and bead-based size selection ranging from 400 to 500 bp. Quantity of final libraries was measured using Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific) and the library size was determined using Agilent 2200 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Among the constructed sequencing libraries, 29 libraries were sequenced with the HiSeq. 2500 (Illumina Inc.) as 100 bp single-end reads and remaining one library for S. tsukubaensis was sequenced with the Miseq v.2 (Illumina Inc.) with 50 bp single-read recipe. Finally, 0.46 to 5.18 Gbp of raw sequence data were obtained and the read qualities were examined by creating sequencing QC reports function of CLC genomic workbench version 6.5.1 (CLC bio, Denmark) (Online-only Table 1 and Fig. 1a).

Online-only Table 1.

Summary of PacBio and Illumina genome sequencing data for 30 streptomycetes.

No. Species Strain Platform Raw reads (No.) Mean raw reads length (bp) Clean reads (No.) Mean clean reads length (bp) SRA accession number
1 Streptomyces clavuligerus ATCC27064 Illumina 9,853,205 100 9,852,872 100 SRR9192366
pacBio 150,292 6,572 60,834 15,473 SRR9290551
2 Streptomyces tsukubaensis NRRL18488 Illumina 9,181,843 50 9,176,299 50 SRR9192343
pacBio 150,292 8,163 93,481 12,715 SRR9290550
3 Streptomyces galilaeus ATCC14969 Illumina 15,603,988 100 15,603,366 100 SRR9192357
pacBio 150,292 5,458 95,691 8,339 SRR9290549
4 Streptomyces nitrosporeus ATCC12769 Illumina 15,858,551 100 15,857,973 100 SRR9192358
pacBio 150,292 5,007 101,572 7,222 SRR9290548
5 Streptomyces subrutilus ATCC27467 Illumina 23,157,475 100 23,157,337 100 SRR9192359
pacBio 150,292 8,757 92,569 13,591 SRR9290547
6 Streptomyces viridosporus T7A ATCC39115 Illumina 24,919,393 100 24,918,458 100 SRR9192360
pacBio 150,292 7,910 92,139 12,571 SRR9290546
7 Streptomyces kanamyceticus ATCC12853 Illumina 14,877,606 100 14,877,044 100 SRR9192361
pacBio 150,292 5,044 59,627 12,291 SRR9290545
8 Streptomyces aureofaciens ATCC11989 Illumina 19,155,832 100 19,155,718 100 SRR9192362
pacBio 150,292 5,890 88,462 9,681 SRR9290544
9 Streptomyces prasinus ATCC13879 Illumina 18,655,418 100 18,654,718 100 SRR9192388
pacBio 150,292 5,560 84,107 9,288 SRR9290553
10 Streptomyces fradiae ATCC10745 Illumina 9,446,513 100 9,445,832 100 SRR9192354
pacBio 150,292 6,843 94,568 10,538 SRR9290561
11 Streptomyces alboniger ATCC12461 Illumina 16,425,356 100 16,425,241 100 SRR9192355
pacBio 300,584 3,638 159,270 6,351 SRR9290560
12 Streptomyces coeruleorubidus ATCC13740 Illumina 17,546,981 100 17,546,866 100 SRR9192339
pacBio 300,584 2,836 121,377 6,164 SRR9290563
13 Streptomyces cinereoruber ATCC19740 Illumina 20,182,754 100 20,181,890 100 SRR9192340
pacBio 150,292 3,319 52,279 9,266 SRR9290562
14 Streptomyces nodosus ATCC14899 Illumina 14,034,927 100 14,034,843 100 SRR9192341
pacBio 150,292 8,387 94,719 12,692 SRR9290557
15 Streptomyces vinaceus ATCC27476 Illumina 6,612,160 100 6,610,219 100 SRR9192335
pacBio 150,292 4,984 75,928 9,432 SRR9290559
16 Streptomyces platensis ATCC23948 Illumina 14,819,357 100 14,819,243 100 SRR9192336
pacBio 150,292 4,869 72,573 9,619 SRR9290558
17 Streptomyces spectabilis ATCC27465 Illumina 18,717,018 100 18,716,280 100 SRR9192337
pacBio 300,584 10,049 202,966 14,047 SRR9290555
18 Streptomyces chartreusis ATCC14922 Illumina 16,988,966 100 16,988,850 100 SRR9192338
pacBio 150,292 8,813 88,869 14,106 SRR9290554
19 Stretpomyces rimosus ATCC10970 Illumina 25,109,758 100 25,108,764 100 SRR9192333
pacBio 300,584 7,066 164,891 12,342 SRR9290564
20 Streptomyces albofaciens ATCC23873 Illumina 19,360,703 100 19,360,564 100 SRR9192364
pacBio 150,292 11,091 110,521 14,419 SRR9290552
21 Streptomyces filamentosus ATCC23958 Illumina 21,181,460 100 21,180,615 100 SRR9192342
pacBio 150,292 4,805 70,677 8,178 SRR9290556
22 Streptomyces venezuelae ATCC 10712 ATCC 10712 Illumina 18,667,122 100 18,664,663 100 SRR9192374
pacBio 150,292 9,101 87,860 15,328 SRR9290565
23 Streptomyces venezuelae ATCC 21113 ATCC 21113 Illumina 21,561,533 100 21,559,491 100 SRR9192334
pacBio 150,292 10,148 104,695 14,180 SRR9290566
24 Streptomyces venezuelae ATCC 10595 ATCC 10595 Illumina 16,798,923 100 16,797,236 100 SRR9192369
pacBio 150,292 7,829 92,681 12,197 SRR9290567
25 Streptomyces venezuelae ATCC 15068 ATCC 15068 Illumina 15,310,620 100 15,308,905 100 SRR9192368
pacBio 150,292 10,754 107,412 14,515 SRR9290568
26 Streptomyces venezuelae ATCC 14583 ATCC 14583 Illumina 19,423,668 100 19,421,332 100 SRR9192371
pacBio 150,292 10,888 106,813 14,647 SRR9290569
27 Streptomyces venezuelae ATCC 14584 ATCC 14584 Illumina 15,447,783 100 15,446,240 100 SRR9192370
pacBio 150,292 8,844 100,523 12,580 SRR9290540
28 Streptomyces venezuelae ATCC 14585 ATCC 14585 Illumina 51,795,644 100 51,791,331 100 SRR9192373
pacBio 150,292 8,275 96,243 12,388 SRR9290541
29 Streptomyces venezuelae ATCC 21782 ATCC 21782 Illumina 19,569,337 100 19,567,069 100 SRR9192372
pacBio 150,292 6,539 70,655 13,089 SRR9290542
30 Streptomyces venezuelae ATCC 21018 ATCC 21018 Illumina 16,469,406 100 16,467,790 100 SRR9192375
pacBio 150,292 10,754 107,412 14,515 SRR9290543
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Fig. 1.

Fig. 1

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Quality of the genome sequencing data. (a) Distribution of Illumina reads quality based on Phred score. (b) Read quality distribution of PacBio reads. Black line indicates total number of bases in the reads which have greater read quality than the corresponding read quality value on x-axis.

Long-read (PacBio) genome sequencing

A total of 5 μg gDNA was used as input for PacBio genome sequencing library preparation. The sequencing library was constructed with the PacBio SMRTbellTM Template Prep Kit (Pacific Biosciences, Menlo Park, CA, USA) following manufacturer’s instructions. Fragments smaller than 20 kbp were removed using the Blue Pippin Size selection system (Sage Science, Beverly, MA, USA) and the constructed libraries were validated using Agilent 2100 Bioanalyzer (Agilent Technologies). Final SMRTbell libraries were sequenced using one or two SMRT cells with P6-C4-chemistry (DNA Sequencing Reagent 4.0) on the PacBio RS II sequencing platform (Pacific Biosciences). Approximately, 0.5 to 3.0 Gbp of raw sequence data were generated (Online-only Table 1).

Genome assembly

Among the raw PacBio sequencing reads, only the reads with a read quality value greater than 0.75 and a length longer than 50 bp were filtered (Fig. 1b). Post filtered reads were assembled by the hierarchical genome assembly process workflow (HGAP, Version 2.3), including consensus polishing with Quiver18. For each assembled contig, error correction was performed based on their estimated genome size and average coverage. Raw reads from the Illumina sequencing were quality trimmed using CLC genomic workbench version 6.5.1 (ambiguous limit 2 and quality limit 0.05) and assembled using de novo assembly function of CLC genomic workbench version 6.5.1 with default parameters. To expand the assembled contigs, all of assembled PacBio and Illumina contigs were aligned using MAUVE 2.4.019 and linked using GAP5 program (Staden package)20.

Genome correction

Quality trimmed Illumina sequencing reads were mapped to the assembled genome using CLC genomic workbench version 6.5.1 (mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.9, and similarity fraction 0.9). Conflicts showing more than 80% frequency for Illumina reads were corrected as Illumina sequence (Table 1). In addition, percentage of mapped Illumina reads on to the assembled genome represents degree of completeness (Table 1 and Fig. 2b). Completeness of gene space was estimated using the BUSCO v3 (Table 2)21.

Table 1.

The statistics of genome assembly and correction.

No. Species Final scaffolds (No.) Scaffold length before correction (bp) Mapped Illumina reads (%) Conflict positions (No.) Added bases (No.) Deleted bases (No.) Scaffold length after correction (bp) G + C contets (%) Assembly accession number
1 Streptomyces clavuligerus 2 6,748,589 and 1,795,496 71.16 and 14.03 7 4 3 6,748,591 and 1,795,495 72.5 GCA_005519465.1
2 Streptomyces tsukubaensis 1 7,963,727 95.13 15 15 0 7,963,742 71.9 GCA_003932715.1
3 Streptomyces galilaeus 1 7,756,176 90.56 51 34 16 7,756,194 71.4 GCA_008704575.1
4 Streptomyces nitrosporeus 1 7,581,543 93.50 51 35 16 7,581,562 72.2 GCA_008704555.1
5 Streptomyces subrutilus 1 7,604,705 96.41 286 269 0 7,604,974 73.4 GCA_008704535.1
6 Streptomyces viridosporus T7A 1 7,280,447 90.44 90 89 0 7,280,536 72.6 GCA_008704515.1
7 Streptomyces kanamyceticus 1 10,133,525 99.09 376 375 3 10,133,897 71.0 GCA_008704495.1
8 Streptomyces aureofaciens 1 7,757,873 84.86 16 9 5 7,757,877 72.6 GCA_008704475.1
9 Streptomyces prasinus 1 7,646,576 89.70 1,025 1,021 5 7,647,592 72.0 GCA_008704445.1
10 Streptomyces fradiae 1 6,725,574 97.63 5 5 0 6,725,579 74.7 GCA_008704425.1
11 Streptomyces alboniger 1 7,962,594 99.12 193 193 1 7,962,786 71.2 GCA_008704395.1
12 Streptomyces coeruleorubidus 1 9,334,399 99.67 1,297 1,299 0 9,335,698 71.1 GCA_008705135.1
13 Streptomyces cinereoruber 1 7,516,474 99.74 178 178 0 7,516,652 72.9 GCA_009299385.1
14 Streptomyces nodosus 1 7,772,564 99.51 26 25 2 7,772,587 70.9 GCA_008704995.1
15 Streptomyces vinaceus 1 7,673,329 92.46 180 180 0 7,673,509 72.3 GCA_008704935.1
16 Streptomyces platensis 1 8,500,673 99.75 354 352 13 8,501,012 71.1 GCA_008704855.1
17 Streptomyces spectabilis 1 9,806,222 95.30 934 938 0 9,807,160 72.4 GCA_008704795.1
18 Streptomyces chartreusis 1 9,911,637 98.42 461 461 0 9,912,098 71.0 GCA_008704715.1
19 Stretpomyces rimosus 1 9,361,132 96.22 22 22 0 9,361,154 72.0 GCA_008704655.1
20 Streptomyces albofaciens 2 4,757,761 and 4,494,336 53.36 and 45.53 504 501 3 4,757,978 and 4,494,617 72.3 GCA_008634025.1
21 Streptomyces filamentosus 2 5,742,252 and 2,129,928 75.22 and 24.28 3,218 3,228 1 5,744,022 and 2,131,385 73.6 GCA_008634015.1
22 Streptomyces venezuelae ATCC 10712 1 8,223,439 99.84 96 81 15 8,223,505 72.5 GCA_008639165.1
23 Streptomyces venezuelae ATCC 21113 1 7,893,622 99.85 173 181 0 7,893,803 72.5 GCA_008639045.1
24 Streptomyces venezuelae ATCC 10595 1 7,871,449 95.50 35 34 3 7,871,480 72.5 GCA_008705255.1
25 Streptomyces venezuelae ATCC 15068 1 8,557,615 99.71 587 587 0 8,558,202 71.9 GCA_008642375.1
26 Streptomyces venezuelae ATCC 14583 1 8,018,461 87.17 29 27 4 8,018,484 71.3 GCA_008642355.1
27 Streptomyces venezuelae ATCC 14584 1 8,941,823 99.00 255 255 0 8,942,078 71.2 GCA_008642315.1
28 Streptomyces venezuelae ATCC 14585 1 8,048,139 82.34 64 41 26 8,048,154 71.3 GCA_008642335.1
29 Streptomyces venezuelae ATCC 21782 1 7,525,235 90.50 87 87 0 7,525,322 71.9 GCA_008642295.1
30 Streptomyces venezuelae ATCC 21018 1 7,746,214 91.61 59 57 4 7,746,267 72.1 GCA_008642275.1
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Fig. 2.

Fig. 2

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Genome assembly of 30 streptomycetes. (a) Strategy for genome assembly and corrections. (b) Profile of Illumina reads mapped on assembled genomes. Data were visualized using SignalMap (Roche NimbleGen, Inc.). Red line indicates the average Illumina read coverage of all genomic positions.

Table 2.

Gene space completeness of completed genomes.

No. Species Complete and single-copy Complete and duplicated Fragmented Missing Total Gene space completeness (%)
1 Streptomyces clavuligerus 343 0 0 9 352 97.4
2 Streptomyces tsukubaensis 350 0 0 2 352 99.4
3 Streptomyces galilaeus 351 0 0 1 352 99.7
4 Streptomyces nitrosporeus 352 0 0 0 352 100.0
5 Streptomyces subrutilus 349 0 0 3 352 99.1
6 Streptomyces viridosporus T7A 351 0 0 1 352 99.7
7 Streptomyces kanamyceticus 352 0 0 0 352 100.0
8 Streptomyces aureofaciens 350 0 0 2 352 99.4
9 Streptomyces prasinus 350 0 0 2 352 99.4
10 Streptomyces fradiae 351 0 0 1 352 99.7
11 Streptomyces alboniger 351 0 0 1 352 99.7
12 Streptomyces coeruleorubidus 351 0 0 1 352 99.7
13 Streptomyces cinereoruber 351 0 0 1 352 99.7
14 Streptomyces nodosus 350 0 1 1 352 99.4
15 Streptomyces vinaceus 349 0 1 2 352 99.1
16 Streptomyces platensis 351 0 0 1 352 99.7
17 Streptomyces spectabilis 350 0 1 1 352 99.4
18 Streptomyces chartreusis 351 0 0 1 352 99.7
19 Stretpomyces rimosus 351 0 0 1 352 99.7
20 Streptomyces albofaciens 346 4 0 2 352 99.4
21 Streptomyces filamentosus 351 0 0 1 352 99.7
22 Streptomyces venezuelae ATCC 10712 352 0 0 0 352 100.0
23 Streptomyces venezuelae ATCC 21113 352 0 0 0 352 100.0
24 Streptomyces venezuelae ATCC 10595 352 0 0 0 352 100.0
25 Streptomyces venezuelae ATCC 15068 351 0 0 1 352 99.7
26 Streptomyces venezuelae ATCC 14583 351 0 0 1 352 99.7
27 Streptomyces venezuelae ATCC 14584 351 0 0 1 352 99.7
28 Streptomyces venezuelae ATCC 14585 351 0 0 1 352 99.7
29 Streptomyces venezuelae ATCC 21782 349 0 0 3 352 99.1
30 Streptomyces venezuelae ATCC 21018 350 0 0 2 352 99.4
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Genome annotation and secondary metabolite biosynthetic gene cluster prediction

The complete genome sequences of streptomycetes were submitted to the NCBI GenBank database and annotated by the latest updated version of NCBI Prokaryotic Genome Annotation Pipeline (PGAP)22. Using the GenBank formatted files of each genomes as input, secondary metabolite biosynthetic gene clusters were predicted by antiSMASH 4.023.

Data Records

Raw reads from short-read (Illumina) and long-read (PacBio) sequencing were deposited in the NCBI Sequence Read Archive (SRA) (Online-only Table 1)24,25. 30 complete genome sequences were deposited in GenBank via the NCBI’s submission portal (Table 3)26–55. Detailed information on the predicted 922 smBGCs in 30 streptomycetes genomes has been deposited in FigShare56.

Table 3.

Summary of genome annotation.

No. Species CDS (No.) 16s rRNA (No.) tRNA (No.) Genome accession number BioProject accession number
1 Streptomyces clavuligerus 6,880 18 66 CP027858 PRJNA414136
2 Streptomyces tsukubaensis 6,376 18 66 CP020700 PRJNA382016
3 Streptomyces galilaeus 6,725 18 76 CP023703 PRJNA412292
4 Streptomyces nitrosporeus 6,364 18 74 CP023702 PRJNA412292
5 Streptomyces subrutilus 6,431 21 68 CP023701 PRJNA412292
6 Streptomyces viridosporus T7A 6,211 18 70 CP023700 PRJNA412292
7 Streptomyces kanamyceticus 8,384 18 66 CP023699 PRJNA412292
8 Streptomyces aureofaciens 6,453 33 71 CP023698 PRJNA412292
9 Streptomyces prasinus 6,263 18 68 CP023697 PRJNA412292
10 Streptomyces fradiae 5,465 18 65 CP023696 PRJNA412292
11 Streptomyces alboniger 6,613 18 67 CP023695 PRJNA412292
12 Streptomyces coeruleorubidus 8,058 18 67 CP023694 PRJNA412292
13 Streptomyces cinereoruber 6,392 18 69 CP023693 PRJNA412292
14 Streptomyces nodosus 6,491 18 68 CP023747 PRJNA412292
15 Streptomyces vinaceus 6,603 21 68 CP023692 PRJNA412292
16 Streptomyces platensis 7,032 21 67 CP023691 PRJNA412292
17 Streptomyces spectabilis 8,212 18 65 CP023690 PRJNA412292
18 Streptomyces chartreusis 8,396 18 71 CP023689 PRJNA412292
19 Stretpomyces rimosus 7,756 21 68 CP023688 PRJNA412292
20 Streptomyces albofaciens 7,520 21 67 PDCM00000000 PRJNA412292
21 Streptomyces filamentosus 6,832 24 70 PDCL00000000 PRJNA412292
22 Streptomyces venezuelae ATCC 10712 7,377 21 67 CP029197 PRJNA454547
23 Streptomyces venezuelae ATCC 21113 6,987 21 67 CP029196 PRJNA454547
24 Streptomyces venezuelae ATCC 10595 6,942 21 67 CP029195 PRJNA454547
25 Streptomyces venezuelae ATCC 15068 7,700 21 69 CP029194 PRJNA454547
26 Streptomyces venezuelae ATCC 14583 7,154 18 66 CP029193 PRJNA454547
27 Streptomyces venezuelae ATCC 14584 7,832 18 65 CP029192 PRJNA454547
28 Streptomyces venezuelae ATCC 14585 7,096 18 66 CP029191 PRJNA454547
29 Streptomyces venezuelae ATCC 21782 6,655 18 69 CP029190 PRJNA454547
30 Streptomyces venezuelae ATCC 21018 6,769 21 71 CP029189 PRJNA454547
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Technical Validation

Streptomyces have drawn considerable attention because of their ability to produce various clinically important secondary metabolites. Total 30 streptomycetes genomes were sequenced by using both PacBio and Illumina sequencing methods to elucidate their biosynthetic potential. After cleaning the reads, on average 98,380 PacBio reads with 11,725 bp length and 18,223,235 Illumina reads with 100 bp length (50 bp for S. tsukubaensis) were generated (Fig. 1a,b and Online-only Table 1). Through the assembly of reads from two sequencing platforms using HGAP, CLC workbench, MAUVE, and GAP5 programs, single linear scaffolds ranging from 6.7 to 10.1 Mbp in length with 72% G + C contents were obtained for 27 streptomycetes, whereas two scaffolds were finally constructed for three remaining streptomycetes, S. clavuligerus (6.7 and 1.8 Mbp), S. albofaciens (4.8 and 4.5 Mbp), and S. filamentosus (5.7 and 2.1 Mbp) (Table 1). S. clavuligerus has been reported to have a large linear plasmid with a length of 1.8 Mbp, so the genome was correctly assembled into a single chromosome, while the S. albofaciens and S. filamentosus genomes appear to be assembled into two divided scaffolds11,57. To increase the accuracy of the assembled genome sequences, Illumina sequences showing more than 80% coverage at the conflict sites were taken as the corrected ones (Table 1). Approximately, 96.32% of Illumina sequencing reads were successfully mapped to the corresponding genomes (Table 1 and Fig. 2b). The completeness of the genomes were assessed using the BUSCO approach with a total of 352 orthologue groups from the Actinobacteria Dataset21. Results showed that 29 genomes have more than 99.1% gene space completeness and the S. clavuligerus genome has 97.4% gene space completeness (Table 2). Following NCBI PGAP, 30 genomes were annotated with 7,000 protein coding genes, 20 rRNAs, and 68 tRNAs on average (Table 3). Finally, based on the annotation, a total of 922 smBGCs were predicted in 30 streptomycetes genomes (Fig. 3). Detailed information, such as genomic positions, types, and putative products of each smBGC are publicly available in Figshare56.

Fig. 3.

Fig. 3

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Secondary metabolite biosynthetic gene clusters in 30 complete streptomycetes genomes.

Acknowledgements

This work was supported by a grant from the Novo Nordisk Foundation (grant number NNF10CC1016517). This work was also supported by the Bio & Medical Technology Development Program (2018M3A9F3079664 to B.-K.C.) through the National Research Foundation (NRF) funded by the Ministry of Science and ICT (MSIT).

Online-only Table

Author contributions

B.-K.C. conceived and supervised the study. N.L. and B.-K.C. designed the experiments. N.L., W.K., S.H. and Y.L. performed the experiments. N.L., W.K., S.H., Y.L., S.C., B.P. and B.-K.C. analyzed the data. N.L., W.K., S.C., B.P. and B.-K.C. wrote the manuscript.

Code availability

The version and parameter of all bioinformatics tools used in this work are described in the Methods section.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Namil Lee and Woori Kim.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

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Data Availability Statement

The version and parameter of all bioinformatics tools used in this work are described in the Methods section.

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