Genome-based analysis of non-ribosomal peptide synthetase and type-I polyketide synthase gene clusters in all type strains of the genus Herbidospora.

BACKGROUND: The genus Herbidospora comprises actinomycetes belonging to the family Streptosporangiaceae and currently contains five recognized species. Although other genera of this family often produce bioactive secondary metabolites, Herbidospora strains have not yet been reported to produce secondary metabolites. In the present study, to assess their potential as secondary metabolite producers, we sequenced the whole genomes of the five type strains and searched for the presence of their non-ribosomal peptide synthetase (NRPS) and type-I polyketide synthase (PKS) gene clusters. These clusters are involved in the major secondary metabolite-synthetic pathways in actinomycetes.
RESULTS: The genome sizes of Herbidospora cretacea NBRC 15474(T), Herbidospora mongoliensis NBRC 105882(T), Herbidospora yilanensis NBRC 106371(T), Herbidospora daliensis NBRC 106372(T) and Herbidospora sakaeratensis NBRC 102641(T) were 8.3, 9.0, 7.9, 8.5 and 8.6 Mb, respectively. They contained 15-18 modular NRPS and PKS gene clusters. Thirty-two NRPS and PKS pathways were identified, among which 9 pathways were conserved in all 5 strains, 8 were shared in 2-4 strains, and the remaining 15 were strain-specific. We predicted the chemical backbone structures of non-ribosomal peptides and polyketides synthesized by these gene clusters, based on module number and domain organization of NRPSs and PKSs. The relationship between 16S rRNA gene sequence-based phylogeny of the five strains and the distribution of their NRPS and PKS gene clusters were also discussed.
CONCLUSIONS: The genomes of Herbidospora strains carry as many NRPS and PKS gene clusters, whose products are yet to be isolated, as those of Streptomyces. Herbidospora members should synthesize large and diverse metabolites, many of whose chemical structures are yet to be reported. In addition to those conserved within this genus, each strain possesses many strain-specific gene clusters, suggesting the diversity of these pathways. This diversity could be accounted for by genus-level vertical inheritance and recent acquisition of these gene clusters during evolution. This genome analysis suggested that Herbidospora strains are an untapped and attractive source of novel secondary metabolites.

Background

Actinomycetes are rich sources for bioactive secondary metabolites. In particular, members of the genus Streptomyces have attracted attention as the most useful screening sources for new drug leads. Since the discovery of streptomycin from Streptomyces griseus, a large number of antibiotics have been identified from cultures of this genus [1, 2]. Consequently, the chance of finding novel secondary metabolites from Streptomyces members has recently dwindled. Thus, the focus of screening has moved to less exploited genera of rare actinomycetes. For example, members of the family Streptosporangiaceae are reported to be a promising source, and many novel compounds have been isolated from genera such as Streptosporangium in this family [3].

The genus Herbidospora was established as a new genus of the family Streptosporangiaceae in 1993 and currently contains five species: Herbidospora cretacea, Herbidospora yilanensis, Herbidospora daliensis,Herbidospora sakaeratensis and Herbidospora mongoliensis [4-7]. Although this genus belongs to the family Streptosporangiaceae, no secondary metabolites have been reported from Herbidospora strains in over 20 years, which motivated us to assess the potential of Herbidospora members as secondary metabolite producers.

Recent genome projects of actinomycetes revealed that each actinomycete genome encodes various biosynthetic pathways, and half to three quarters are associated with non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) pathways [8]. This suggested that non-ribosomal peptide and polyketide compounds are the major secondary metabolites of actinomycetes [8]. Non-ribosomal peptides, polyketides and their hybrid compounds often show pharmaceutically useful bioactivities, many of which have been developed into various drugs, such as antibiotics, anticancer agents and immunosuppressants. Therefore, NRPS and PKS genes in actinomycete strains are often assessed to screen potential secondary metabolite producers [9, 10].

Genes for each non-ribosomal peptide and/or polyketide synthesis are generally organized into a gene cluster, in which NRPS and PKS genes play main roles to synthesize non-ribosomal peptides and polyketide chains, respectively. NRPSs and type-I PKSs are mega-synthases, containing multiple catalytic domains organized into modules, where each module carries out a cycle of chain elongation. Typically, each module contains at least three domains: a condensation (C) domain, an adenylation (A) domain and a thiolation (T) domain in NRPS modules; and a ketosynthase (KS) domain, an acyltransferase (AT) domain and an acyl carrier protein (ACP) in type-I PKS modules. Optional domains may also be present in each module to chemically modify elongating chains. The products are synthesized from simple building blocks such as acyl-CoA and amino-acid units based on an accepted theory called the assembly line rule [11]; therefore, the chemical structures of synthesized peptides and/or polyketide backbones can be predicted from domain organizations of the NRPS and/or PKS gene clusters, respectively.

In this study, we sequenced the whole genomes of all type strains of the genus Herbidospora because no Herbidospora genome sequence was registered in public databases when we began this study. We then examined the NRPS and type-I PKS gene clusters in the genome sequences and predicted the chemical backbone structures of these metabolites to assess the potential of the genus as secondary metabolite producers, and to provide information on the novelty and diversity of NRPS and PKS pathways. We also discussed how diversity was acquired during the evolution of Herbidospora species, based on the relationship between the distribution of these pathways and the taxonomic position of each strain.

Methods

Whole-genome sequencing

Genomic DNAs of H. cretacea NBRC 15474T, H. mongoliensis NBRC 105882T, H. yilanensis NBRC 106371T, H. daliensis NBRC 106372T and H. sakaeratensis NBRC 102641T were prepared from liquid-dried cells in ampoules provided from the NBRC culture collection, using a Qiagen EZ1 tissue kit and an EZ1 advanced instrument (Qiagen), and sequenced using paired-end sequencing with MiSeq (Illumina). The sequence redundancy for the five draft genomes ranged from 61.7 to 70.6. The sequence reads were assembled using Newbler version 2.6 software and subsequently assessed using GenoFinisher software [12].

Analysis of NRPS and type-I PKS gene clusters

Coding sequences in the draft genome sequences were predicted using Prodigal version 2.6 [13]. NRPS and type-I PKS gene clusters were determined as previously reported [9, 10]. PKS and NRPS genes having only a single domain were excluded from the present analysis, because we considered them atypical; we focused on multi-domain genes.

Searches for orthologous gene clusters among strains

A BLASTP search was performed using the NCBI Protein BLAST program against the non-redundant protein sequence database. We considered genes of distinct strains to be orthologous when their closest homologs in the BLASTP search were the same, and also when their domain organizations were identical or almost the same.

Prediction of metabolites derived from NRPS and/or type-I PKS gene clusters

We used antiSMASH [14], a website for antibiotics and secondary metabolite analysis, to predict substrates for A domains and AT domains. Based on the substrates and the assembly line rule [11], we predicted the amino acid combinations of peptide chains and chemical structures of polyketide chains synthesized by NRPS and type-I PKS gene clusters, respectively.

Phylogenetic tree based on 16S rRNA gene sequences

16S rRNA gene sequences were downloaded from ‘Sequence Information’ of the NBRC Culture Catalogue [15], and aligned using ClustalX2 [16]. A phylogenetic tree was reconstructed by the neighbor-joining method [17]. The resultant tree topologies were evaluated by bootstrap analysis [18]. The 16S rRNA gene sequence of Acrocarpospora corrugata NBRC 13972T was used as the outgroup.

Results and discussion

We sequenced the whole genomes of all the type strains in the genus Herbidospora. The genome sizes ranged from 7.9 to 9.0 Mb, showing medium size compared with those of Streptomyces strains (5.0–11.9 Mb) and of strains in the family Streptosporangiaceae (5.5–13 Mb). The five strains each possessed 15–18 gene clusters for NRPS, PKS/NRPS hybrid and type-I PKS pathways, which were similar to the numbers found in Streptomyces [8, 10, 19–22]. The numbers of the three types of gene clusters in each strain are listed in Table 1. Table 2 shows details of all the clusters found in each genome. Orthologous genes and gene clusters are aligned in the same row of the table. These orthologous genes showed the same domain organization; therefore, their gene clusters should synthesize the same products, as shown in the ‘Presumable product’ column of Table 2. Among the 32 gene clusters (nrps-1 to -16, pks/nrps-1 to -4, pks-1 to -12) identified from the 5 strains, 9 were conserved in all strains, 8 were shared in 2–4 strains, and 15 were strain-specific. During this study, the draft genome sequence of H. cretacea NRRL B-16917 was published in GenBank/EMBL/DDBJ databases (accession no., JODQ00000000.1). However, it is questionable whether strain NRRL B-16917 is H. cretacea, because its 16S rRNA gene showed higher sequence similarity to those of type strains of H. yilanensis (99.1 %), H. sakaeratensis (98.8 %), H. daliensis (98.3 %) than to the type strain of H. cretacea (98.0 %), and its phylogenetic position was not close to the type strain of H. cretacea in the phylogenetic tree based on 16S rRNA gene sequences (data not shown). The scientific name of strain NRRL B-16917 is unclear; therefore, we did not analyze its NRPS and PKS gene clusters and focused on those of the five type strains in the present study.

Table 1

Genome sequencing and numbers of modular non-ribosomal peptide synthetase (NRPS) and type-I polyketide synthase (PKS) gene clusters in Herbidospora strains

Strain	Reads (Mb)	No. of scaffolds	Genome size (bp)	G + C content (%)	Accession no.	Number of gene clusters
						NRPS	PKS/NRPS hybrid	PKS	Total
H. cretacea NBRC 15474T	582.1	36	82,82,092	70.7	BBXG01000000	9	1	5	15
H. mongoliensis NBRC 105882T	556.2	47	90,71,776	69.4	BBXD01000000	9	2	6	17
H. yilanensis NBRC 106371T	546.7	67	78,57,004	70.7	BBXE01000000	9	2	4	15
H. daliensis NBRC 106372T	586.2	18	85,23,669	70.8	BBXF01000000	10	1	4	15
H. sakaeratensis NBRC 102641T	590.3	35	83,49,170	70.9	BBXC01000000	12	2	4	18

Table 2

Open reading frames encoding multidomain NRPSs and PKSs in modular NRPS and PKS gene clusters of Herbidospora strains

Gene cluster	Scaffold-orf no. (size, % identity/similarity to closest homolog)					Domain organization	Presumed product	Closest homolog
	H. cretacea NBRC 15474T	H. mongoliensis NBRC 105882T	H. yilanensis NBRC 106371T	H. daliensis NBRC 106372T	H. sakaeratensis NBRC 102641T			Accession no.	Origin
nrps-1	s01-orf204 (6699aa, 88/92)	s02-orf3 (6749aa, 85/90)	s34-orf56 (6711aa, 92/94)	s03-orf732 (6697aa, 89/93)	s01-orf733 (6657aa, 91/94)	A/MT/T-C/Aser/T-C/Aorn/MT/T-C/A/T-C/Aser/T-C/A/T/E	x-Ser-mOrn-x-Ser-x (albachelin-like siderophore)	WP_034384656	Herbidospora cretacea NRRL B-16917
nrps-2	s17-orf53* (1026aa, 91/93)	s03-orf338* (988aa, 89/92)	s13-orf130* (1010aa, 93/94)	s05-orf276* (1051aa, 91/94)	s15-orf51* (974aa, 91/94)	A/T-C	x-Gly-Lys-x	WP_030456013	Herbidospora cretacea NRRL B-16917
	s17-orf54 (973aa, 88/92)	s03-orf339 (990aa, 86/91)	s13-orf129 (1015aa, 90/92)	s05-orf275 (974aa, 90/93)	s15-orf52 (975aa, 91/94)	C/Agly/T		WP_030456012	Herbidospora cretacea NRRL B-16917
	s17-orf55 (979aa, 88/90)	s03-orf340 (991aa, 85/89)	s13-orf128 (972aa, 90/92)	s05-orf274 (961aa, 91/93)	s15-orf53 (961aa, 91/93)	C/A(lys)/T		WP_034385955	Herbidospora cretacea NRRL B-16917
	s17-orf58 (1351aa, 88/92)	s03-orf343 (1341aa, 89/93)	s13-orf125 (1348aa, 94/96)	s05-orf271 (1343aa, 92/95)	s15-orf56 (1350aa, 92/95)	C/A/T-TE		WP_030456008	Herbidospora cretacea NRRL B-16917
nrps-3	s08-orf344 (2105aa, 86/90)	s17-orf93 (2104aa, 85/90)	s12-orf143 (2199aa, 90/91)	s01-orf374 (2125aa, 89/92)	s17-orf87 (2150aa, 90/92)	Agly/T-C/T-C/Agly/T	Gly-?-Gly-Asp	WP_034384991	Herbidospora cretacea NRRL B-16917
	s08-orf343 (1775aa, 91/95)	s17-orf92 (1766aa, 86/92)	s12-orf144 (1766aa, 95/97)	s01-orf373 (1776aa, 92/96)	s17-orf86 (1777aa, 92/95)	C/Aasp/T-TE		WP_030453964	Herbidospora cretacea NRRL B-16917
nrps-4	s01-orf4* (1934aa, 88/91)	s02-orf218* (1932aa, 88/93)	s08-orf109* (1933aa, 95/97)	s03-orf522* (1932aa, 93/95)	s01-orf545* (1932aa, 94/95)	C/A/T-C/Alys/T	x-Lys-Asp	WP_030453192	Herbidospora cretacea NRRL B-16917
	s01-orf5 (1721aa, 93/96)	s02-orf217 (1722aa, 90/94)	s08-orf108 (1721aa, 96/98)	s03-orf523 (1721aa, 94/96)	s01-orf546 (1721aa, 95/96)	C/Aasp/T-TE		WP_030453193	Herbidospora cretacea NRRL B-16917
nrps-5	s03-orf142 (1742aa, 87/91)	s04-orf515 (1751aa, 86/91)	s21-orf82 (1738aa, 95/97)	s01-orf929 (1731aa, 91/93)	s02-orf479 (1729aa, 91/94)	C/Aasp/T-TE	Gly-Asp	WP_030454483	Herbidospora cretacea NRRL B-16917
	s03-orf139 (1011aa, 93/95)	s04-orf518 (1011aa, 93/95)	s21-orf85 (1011aa, 96/97)	s01-orf932 (1011aa, 93/96)	s02-orf482 (1011aa, 94/96)	C/Agly/T		WP_034384898	Herbidospora cretacea NRRL B-16917
nrps-6	s06-orf42* (474aa, 87/91)	s05-orf174* (470aa, 86/90)	s06-orf123* (471aa, 92/94)	s14-orf189* (471aa, 92/93)	s12-orf45* (471aa, 92/94)	C/T	?-Asn	WP_030450820	Herbidospora cretacea NRRL B-16917
	s06-orf40 (1678aa, 57/68)	s05-orf172 (1716aa, 55/66)	s06-orf125 (1684aa, 57/68)	s14-orf187 (1677aa, 57/67)	s12-orf47 (1677aa, 57/67)	C/Aasn/T-TE		WP_031172905	Streptosporangium roseum NRRL B-2638
nrps-7	s05-orf29 (1709aa, 91/94)	s02-orf251 (1777aa, 90/94)	s08-orf140 (1776aa, 96/97)	s03-orf492 (1789aa, 94/96)	s01-orf513 (1812aa, 94/95)	C/Aasn/T-TE	(Asn)	WP_030453154	Herbidospora cretacea NRRL B-16917
nrps-8	–	–	s05-orf295 (1018aa, 92/94)	s03-orf778 (1021aa, 89/92)	s01-orf772 (1019aa, 89/91)	A/T-TE	x–x	WP_030453437	Herbidospora cretacea NRRL B-16917
			s05-orf293 (1097aa, 95/97)	s03-orf780 (1097aa, 93/96)	s01-orf774 (1097aa, 94/96)	C/A/T		WP_030453439	Herbidospora cretacea NRRL B-16917
nrps-9	–	–	s02-orf346 (3681aa, 56/63)	–	s02-orf28 (3673aa, 53/63)	Agly/T-C/Aasn/T-C/A/T-C/A(lys)/T	Gly-Asn-x-Lys-Thr-x	WP_026126874	Nocardiopsis xinjiangensis YIM 90004
			s02-orf347 (2407aa, 59/70)		s02-orf27 (2385aa, 59/70)	C/Athr/T-C/A/T-TE		WP_040918882	Saccharomonospora glauca K62
nrps-10	s05-orf197* (1110aa, 58/67)	s02-orf401* (1110aa, 58/68)	–	–	–	T-C/Aser/T	x-Ser-?-x–x-Ser	WP_037800301	Streptomyces sp. Mg1
	s05-orf195* (1679aa, 37/48)	s02-orf399* (1679aa, 37/48)				C/T-C/A/T		AGC43421	Myxococcus stipitatus DSM 14675
	s05-orf184 (540aa, 39/52)	s02-orf388 (527aa, 38/50)				A/T		WP_017558240	Nocardiopsis baichengensis YIM 90130
	s05-orf182 (1693aa, 48/57)	s02-orf386 (1700aa, 46/57)				C/T-C/Aser/T-TE		ACU38342	Actinosynnema mirum DSM 43827
nrps-11	s08-orf166 (1237aa, 63/71)	s13-orf80 (1230aa, 65/73)	–	–	–	C/A/T-TE	x	ACU75141	Catenulispora acidiphila DSM 44928
nrps - 12	–	–	–	s01-orf155 (3126aa, 96/97)	–	C/Aphe/T-C/Aasp/T-C/Aasp/T	Phe-Asp-Asp-x-Asp-Ser-x–x-Phe-x-Val-x-Tyr-Asp-Asn-Tyr-x-Tyr-Asp–Asp-x-Asp	WP_034385663	Herbidospora cretacea NRRL B-16917
				s01-orf140 (11331aa, 55/65)	–	C/A/T-C/Aasp/T-C/Aser/T-C/A/T-C/A/T-C/Aphe/T-C/A/T-C/Aval/T-C/A/T-C/Atyr/T-C/Aasp/T		EIF87998	Streptomyces tsukubensis NRRL 18488
				s01-orf139 (8503aa, 54/64)	–	C/Aasn/T-C/Atyr/T-C/A/T-C/Atyr/T-C/Aasp/T-C/Aasp/T-C/A/T-C/Aasp/T-TE		EIF87998	Streptomyces tsukubensis NRRL 18488
nrps - 13	–	–	–	s02-orf353 (1637aa, 51/59)	–	C/Agly-C/Aala/T	Gly-Ala	WP_026213748	Nonomuraea coxensis DSM 45129
nrps - 14	–	–	–	–	s01-orf286 (2965aa, 39/50)	C/A/T-C/A/T-C/Aser/T	Val-x-x-Ser-Asn–Asn	ERK92233	Myxococcus sp. (contaminant ex DSM 436)
					s01-orf285 (2462aa, 56/67)	C/Aasn/T-C/Aasn/T-C		WP_039739900	Saccharomonospora halophila 8
					s01-orf277* (620aa, 52/63)	Aval/T		WP_033666206	Salinispora pacifica CNS055
nrps - 15	–	–	–	–	s13-orf204 (1086aa, 58/66)	Agly/C/T	?-Gly	ETK35217	Microbispora sp. ATCC PTA-5024
nrps - 16	–	–	–	–	s14-orf131 (1233aa, 66/75)	C/A/T	x	ELS56639	Streptomyces viridochromogenes Tue57
pks/nrps-1	s04-orf226 (3534aa, 82/87)	s07-orf8 (3458aa, 82/87)	s05-orf58 (3534aa, 90/92)	s06-orf132 (3570aa, 84/88)	s01-orf1038 (3515aa, 86/89)	CoL/ACP-KS/AT/KR/ACP-C/A/T-C	?-pk-x	WP_034384725	Herbidospora cretacea NRRL B-16917
pks/nrps - 2	–	s11-orf48 (1776aa, 52/66)	–	–	–	KS/AT/DH/KR/ACP	pk-x-x–x-Val-x-x-Ser-Thr-Asn-Asn–Asn-Thr-Asn	EPH45771	Streptomyces aurantiacus JA 4570
		s11-orf45 (1637aa, 50/59)				A/T-C/A/T		EPH45774	Streptomyces aurantiacus JA 4570
		s11-orf44 (1073aa, 71/76)				C/A/T		CAC01623	Planobispora rosea ATCC 53733
		s11-orf43 (2007aa, 50/60)				C/Aval/T-C/A/T		EPH43046	Streptomyces aurantiacus JA 4570
		s11-orf41 (2145aa, 47/57)				C/A/T-C/Aser/T		EPH43048	Streptomyces aurantiacus JA 4570
		s11-orf40 (3193aa, 41/54)				C/Athr/T-C/Aasn/T-C/Aasn/T		WP_018350842	Longispora albida DSM 44784
		s11-orf39 (1064aa, 45/58)				C/Aasn/T		WP_030327232	Streptomyces sp. NRRL B-3229
		s11-orf38 (2516aa, 44/55)				C/Athr/T-C/Aasn/T-TE		EPH43049	Streptomyces aurantiacus JA 4570
pks/nrps - 3	–	–	s05-orf134 (1316aa, 44/56)	–	–	KS/AT/ACP-TE	Leu-Val-Leu-Ser-pk	EWC62839	Actinokineospora sp. EG49
			s05-orf130 (1006aa, 42/59)			Aleu/T/E		BAH43926	Brevibacillus brevis NBRC 100599
			s05-orf129 (3118aa, 46/60)			C/Aval/T-C/Aleu/T-C/Aser/T		AGC43421	Myxococcus stipitatus DSM 14675
pks/nrps - 4	–	–	–	–	s08-orf1 (> 346aa, 55/61)	…ACP	See Fig. 1h	WP_037075741	Pseudonocardia spinosispora DSM 44797
					s08-orf2 (6590aa, 72/79)	KS/ATm/DH/KR/ACP-KS/ATm/DH/KR/ACP-KS/ATm/DH/KR/ACP-KS/AT/DH		WP_042407435	Streptacidiphilus carbonis NBRC 100919
					s08-orf2_1 (379aa, 83/88)	KR/ACP		WP_042407435	Streptacidiphilus carbonis NBRC 100919
					s08-orf3 (4716aa, 57/67)	KS/AT/KR/ACP-KS/AT/KR/ACP-KS/AT/KR/ACP-TE		WP_037075679	Pseudonocardia spinosispora DSM 44797
					s08-orf8 (7045aa, 69/77)	KS/ATm/ACP-KS/AT/DH/ER/KR/ACP-KS/AT/DH/ER/KR/ACP-KS/AT/DH/ER/KR/ACP		WP_037075740	Pseudonocardia spinosispora DSM 44797
					s08-orf9 (5636aa, 51/62)	KS/AT/DH/ER/KR/ACP-KS/AT/DH/ER/KR/ACP-KS/AT/DH/KR/ACP		ADL46003	Micromonospora aurantiaca ATCC 27029
					s08-orf10* (997aa, 65/76)	A/T		WP_042397191	Streptacidiphilus carbonis NBRC 100919
pks-1	s14-orf123 (2788aa, 90/92)	s10-orf265 (2815aa, 89/92)	s18-orf5 (2796aa, 90/92)	s03-orf254 (2800aa, 89/91)	s01-orf250 (2782aa, 91/94)	KS/ATm/ACP-KS/ATm/DH/KR/ACP	See Fig. 1a	WP_034382778	Herbidospora cretacea NRRL B-16917
	s14-orf124 (1807aa, 93/95)	s10-orf264 (1806aa, 93/96)	s18-orf6 (1800aa, 94/96)	s03-orf255 (1794aa, 94/96)	s01-orf251 (1793aa, 94/95)	KS/AT/DH/KR/ACP		WP_030450097	Herbidospora cretacea NRRL B-16917
	s14-orf125 (1800aa, 91/93)	s10-orf263 (1801aa, 92/93)	s18-orf7 (1795aa, 92/94)	s03-orf256 (1795aa, 90/93)	s01-orf252 (1798aa, 92/93)	KS/ATm/DH/KR/ACP		WP_030450096	Herbidospora cretacea NRRL B-16917
	s14-orf126 (1567aa, 91/94)	s10-orf262 (1578aa, 92/94)	s18-orf8 (1573aa, 91/94)	s03-orf257 (1567aa, 91/94)	s01-orf253 (1595aa, 90/92)	KS/AT/KR/ACP		WP_030450095	Herbidospora cretacea NRRL B-16917
	s14-orf127 (3057aa, 92/)94	s10-orf261 (3070aa, 91/94)	s18-orf9 (3062aa, 92/94)	s03-orf258 (3031aa, 92/94)	s01-orf254 (3033aa, 92/94)	KS/AT/KR/ACP-KS/AT/KR/ACP		WP_030450094	Herbidospora cretacea NRRL B-16917
	s14-orf135* (1265aa, 94/95)	s10-orf253* (1264aa, 95/96)	s18-orf17* (1261aa, 94/96)	s03-orf266* (1264aa, 94/96)	s01-orf262* (1261aa, 95/96)	KS/KR/ACP		WP_034382545	Herbidospora cretacea NRRL B-16917
	s14-orf136* (1573aa, 95/97)	s10-orf252* (1575aa, 95/97)	s18-orf18* (1573aa, 95/97)	s03-orf267* (1573aa, 94/96)	s01-orf263* (1567aa, 96/97)	KS/AT/KR/ACP		WP_034382775	Herbidospora cretacea NRRL B-16917
pks-2	–	s04-orf63 (6103aa, 52/62)	s12-orf1 (> 2049aa, 54/63)	s01-orf485 (6202aa, 50/60)	s26-orf2 (5982aa, 56/65)	(KS)/AT/ACP-KS/AT/DH/KR/ACP-KS/AT/DH/KR/ACP-KS/AT/DH/KR/ACP	See Fig. 1b	AEB44393	Verrucosispora maris AB-18-032
			s47-orf2 (> 3456aa, 53/62)
		s04-orf64 (3854aa, 58/66)	s47-orf1 (> 566aa, 71/78)	s01-orf486 (3774aa, 57/64)	s26-orf1 (> 1579aa, 53/60)	KS/AT(m,e)/DH/KR/ACP-KS/AT(m)/DH/ER/KR/ACP		ABW12874	Frankia sp. EAN1pec
			s02-orf383 (> 1384aa, 66/72)		s02-orf1 (> 2192aa, 63/70)
		s04-orf65 (1070aa, 81/86)	s02-orf382 (997aa, 91/93)	s01-orf487 (1019aa, 87/90)	s02-orf2 (996aa, 89/91)	KS/AT/ACP		WP_030454081	Herbidospora cretacea NRRL B-16917
pks-3	s12-orf80 (1259aa, 88/92)	–	s09-orf200 (1258aa, 95/96)	s01-orf1205 (1258aa, 94/96)	–	KS/AT?/DH/ACP	?	WP_030454772	Herbidospora cretacea NRRL B-16917
pks-4	–	–	s27-orf91	s06-orf349 (1795aa, 53/64)	–	KS/AT/ACP/KR/DH	Enediyne	AAP92148	Actinomadura verrucosospora ATCC 39334
pks-5	s04-orf1 (> 901aa, 70/79)	s28-orf45 (> 660aa, 64/73)	–	–	–	KS/AT…	See Fig. 1c	WP_018514184	Streptomyces sp. ScaeMP-e10
	s36-orf55 (> 3542aa, 58/67)	s11-orf1 (> 405aa, 54/66)				…ACP-KS/AT/DH/KR/ACP-KS/AT/DH/KR/ACP		WP_032771883	Streptomyces cyaneofuscatus NRRL B-2570
		s11-orf2 (3315aa, 50/61)
	s36-orf54 (2142aa, 66/75)	s11-orf3 (2345aa, 50/62)				KS/AT(m)/DH/KR/ACP		WP_032771870	Streptomyces cyaneofuscatus NRRL B-2570
pks - 6	s01-orf132 (1020aa, 52/63)	–	–	–	–	KS/ATm/ACP	See Fig. 1d	EJJ02887	Streptomyces auratus AGR0001
	s01-orf133 (7608aa, 50/61)					KS/AT/KR/ACP-KS/AT/DH/ER/KR/ACP-KS/AT/KR/ACP-KS/ATm/KR/ACP-KS/AT/KR/ACP		WP_035304435	Actinokineospora inagensis DSM 44258
	s01-orf134 (4820aa, 54/64)					KS/AT/KR/ACP-KS/AT/DH/ER/KR/ACP-KS/AT/KR/ACP		EHY88974	Saccharomonospora azurea NA-128
	s01-orf135 (6313aa, 52/63)					KS/ATm/KR/ACP-KS/AT/DH/KR/ACP-KS/AT/DH/KR/ACP-KS/AT/DH/KR/ACP		ACU36619	Actinosynnema mirum DSM 43827
	s01-orf136 (4533aa, 55/65)					KS/AT/DH/KR/ACP-KS/AT/KR/ACP-KS/AT/KR/ACP		AAX98184	Streptomyces aizunensis NRRL B-11277
	s01-orf137 (4803aa, 55/65)					KS/ATe(m)/DH/KR/ACP-KS/ATe(m)/KR/ACP-KS/AT/DH/KR/ACP		WP_033261216	Amycolatopsis vancoresmycina NRRL B-24208
	s01-orf138 (5850aa, 54/65)					KS/AT/KR/ACP-KS/ATm/KR/ACP-KS/ATm/KR/ACP-KS/AT/KR/ACP		ABC87511	Streptomyces sp. NRRL 30748
	s01-orf139 (4995aa, 52/64)					KS/ATm/KR/ACP-KS/AT/DH/ER/KR/ACP-KS/AT/DH/KR/ACP		AHH99925	Kutzneria albida DSM 43870
	s01-orf140 (5337aa, 54/64)					KS/AT/KR/ACP-KS/AT/DH/ER/KR/ACP-KS/AT/DH/ER/KR/ACP		AEP40936	Nocardiopsis sp. FU40
	s01-orf141 (3460aa, 57/68)					KS/AT/DH/KR/ACP-KS/AT/DH/KR/ACP		WP_035796302	Kitasatospora mediocidica KCTC 9733
	s01-orf142 (4383aa, 54/65)					KS/AT/KR/ACP-KS/ATm/KR/ACP-KS/AT/KR/ACP		AAX98186	Streptomyces aizunensis NRRL B-11277
	s01-orf143 (1247aa, 53/64)					KS/AT/ACP-TE		KIR65900	Micromonospora carbonacea JXNU-1
pks - 7	s20-orf48 (2065aa, 59/68)	–	–	–	–	KS/ATe(m)/ACP-KS/AT/DH/ACP	See Fig. 1e	WP_042439448	Streptacidiphilus albus NBRC 100918
	s20-orf49 (1530aa, 53/63)					KS/AT/KR/ACP		WP_042494385	Streptomyces avermitilis MA-4680 = NBRC 14893
	s20-orf50 (2820aa, 62/70)					KS/AT/DH/KR/ACP-KS/ATm/ACP		CCH32016	Saccharothrix espanaensis DSM 44229
	s20-orf51 (2064aa, 57/66)					KS/AT/DH/KR/ACP-TE		WP_041313683	Saccharothrix espanaensis DSM 44229
pks - 8	–	s08-orf244 (5375aa, 59/69)	–	–	–	KS/AT/DH/ER/KR/ACP-KS/ATe(m)/KR/ACP-KS/ATm/DH/KR/ACP	See Fig. 1f	WP_033660827	Salinispora pacifica CNS237
		s08-orf245 (1559aa, 55/66)				KS/AT/KR/ACP		EXU62139	Streptomyces sp. PRh5
		s08-orf246 (3222aa, 53/65)				KS/ATe(m)/KR/ACP-KS/AT/DH/KR/ACP		EGX61520	Streptomyces zinciresistens K42
		s08-orf251 (10965aa, 51/63)				KS/AT/ACP-KS/ATe(m)/DH/KR/ACP-KS/ATm/KR/ACP-KS/AT/DH/KR/ACP-KS/ATe(m)/DH/KR/ACP-KS/ATe(m)/KR/ACP-KS/AT/KR/ACP		ADI03772	Streptomyces bingchenggensis BCW-1
		s08-orf252 (1565aa, 64/73)				KS/ATm/KR/ACP		WP_033775512	Salinispora pacifica DSM 45546
		s08-orf253 (3638aa, 59/69)				KS/AT/DH/ER/KR/ACP-KS/ATm/KR/ACP		CAJ88176	Streptomyces ambofaciens ATCC 23877
		s08-orf254 (7933aa, 67/76)				KS/AT/DH/KR/ACP-KS/ATm/DH/ER/KR/ACP-KS/AT/KR/ACP-KS/AT/KR/ACP-KS/AT/KR/ACP		CAJ88175	Streptomyces ambofaciens ATCC 23877
pks - 9	–	s11-orf206 (7923aa, 54/66)	–	–	–	KS/AT/KR/ACP-KS/AT/KR/ACP-KS/AT/DH/KR/ACP-KS/AT/DH/KR/ACP-KS/AT/DH/KR/ACP	See Fig. 1g	BAC68129	Streptomyces avermitilis MA-4680 = NBRC 14893
		s11-orf207 (1582aa, 54/65)				KS/AT/KR/ACP		WP_032769932	Streptomyces sp. CNS654
		s11-orf208 (4703aa, 59/69)				KS/ATm/KR/ACP-KS/AT/KR/ACP-KS/AT/KR/ACP-TE		CAJ88175	Streptomyces ambofaciens ATCC 23877
pks - 10	–	s26-orf59 (1260aa, 87/92)	–	–	–	KS/AT/ACP	?	WP_030454772	Herbidospora cretacea NRRL B-16917
pks - 11	–	–	–	–	s22-orf6 (4492aa, 74/80)	KS/AT/DH/KR/ACP-KS/ATm/KR/ACP-KS/AT/KR/ACP	See Fig. 1i	WP_042397188	Streptacidiphilus carbonis NBRC 100919
					s22-orf5 (6067aa, 75/82)	KS/ATm/KR/ACP-KS/AT/KR/ACP-KS/ATm/KR/ACP-KS/AT/KR/ACP		WP_042397185	Streptacidiphilus carbonis NBRC 100919
					s22-orf4 (3381aa, 56/65)	KS/ATm/DH/KR/ACP-KS/ATm/KR/ACP		WP_037075673	Pseudonocardia spinosispora DSM 44797
					s22-orf3 (6184aa, 71/78)	KA/ATm/KR/ACP-KS/ATm/KR/ACP-KS/ATm/KR/ACP-KS/ATm/KR/ACP		WP_037075676	Pseudonocardia spinosispora DSM 44797
					s22-orf2 (6426aa, 76/83)	KS/ATm/KR/ACP-KS/ATm/KR/ACP-KS/AT/KR/ACP-KS/ATm/DH/KR/ACP		WP_042408959	Streptacidiphilus carbonis NBRC 100919
					s22-orf1 (> 4929aa, 77/83)	KS/ATm/KR/ACP-KS/ATm/DH/ER/KR/ACP-KS/ATm/DH…		WP_042408956	Streptacidiphilus carbonis NBRC 100919
pks - 12	–	–	–	–	s02-orf765 (1258aa, 94/95)	KS/AT?/DH/ACP	?	WP_030454772	Herbidospora cretacea NRRL B-16917

Strain-specific gene clusters are in boldface. NRPS and PKS genes not completely sequenced are shown in italics. C, condensation; A, adenylation; T, thiolation; E, epimerization; MT, methyltransferase; CoL, CoA ligase; KS, ketosynthase; AT, acyltransferase incorporating malonyl-CoA, ATm, acyltransferase incorporating methylmalonyl-CoA; ATe, acyltransferase incorporating ethylmalonyl-CoA; AT?, acyltransferase whose substrate has not been determined by antiSMASH; DH, dehydratase; ER, enoylreductase; KR, ketoreductase; ACP, acyl carrier protein; TE, thioesterase; x, unpredicted amino-acid; mOrn, methyl ornithine; ?, lack of A domain. A domain amino-acid substrate predicted by antiSMASH is shown in subscripted letters

Gene clusters conserved in all the five strains

Table 2 suggested that nine presumable products (nrps-1 to -7, pks/nrps-1, pks-1) are common among all five type strains belonging to the genus Herbidospora. Nrps-1 is assumed to be involved in the synthesis of a siderophore similar to albachelin [23], because the module numbers of albachelin NRPS and nrps-1 are the same, and their domain organizations and amino-acid substrates of their A domains are quite similar (albachelin, C/A/T-C/ASer/T/E-C/AOrn/MT/T-C/ASer/T-C/ASer/T-C/A/T/E; NRPS-1, A/MT/T-C/ASer/T-C/AOrn/MT/T-C/A/T-C/ASer/T-C/A/T/E. Distinct domains between them are underlined). Nrps-2 to -6 are predicted to synthesize non-ribosomal peptides comprising 4, 4, 3, 2 and 2 amino acids, respectively, based on their module numbers. Nrps-7 had only a single NRPS module; therefore, we were not able to predict the chemical structure of the product as a peptide. Pks/nrps-1 is a PKS/NRPS hybrid gene encoding a protein comprising three modules for the synthesis of a starter unit, a polyketide unit and an amino-acid unit, respectively. Pks-1 gene clusters contained seven PKS genes, whose assembly line was composed of nine modules. According to the assembly line rule and the substrates of their AT domains, the gene clusters were assumed to synthesize the polyketide chain shown in Fig. 1a. The structure has similar characteristics to those of antifungal polyene compounds, containing multiple carbon–carbon conjugated double bonds and multiple hydroxyl groups.

Fig. 1

Presumed chemical structures of polyketide backbones synthesized by Herbidospora type-I polyketide synthase (PKS) gene clusters. R=H or methyl. The chemical structures are shown attached to the ACP of the last module by thioester bonds

Gene clusters shared between/among two to four strains

Nrps-8 gene clusters were present in three strains (H. yilanensis NBRC 106371T, H. daliensis NBRC 106372T and H. sakaeratensis NBRC 102641T). The nrps-8 gene clusters had two modules; therefore, the products were predicted to be dipeptides. Nrps-9 gene clusters present in H. yilanensis NBRC 106371T and H. sakaeratensis NBRC 102641T possessed five modules. According to the predicted substrates of the A domains in each module, the products would be hexapeptides including glycine (Gly), asparagine acid (Asp), lysine and threonine (Thr) as the building blocks. Nrps-10 and nrps-11 gene clusters were present in H. cretacea NBRC 15474T and H. mongoliensis NBRC 105882T. The nrps-10 gene clusters contained six modules and two A domains were predicted to incorporate serine (Ser) as the substrates; therefore, the products were predicted to be hexapeptides including two Ser molecules. In contrast, nrps-11 had only a single module, and we were not able to predict the peptide product.

Pks-2 gene clusters were present in four strains, with the exception of H. cretacea NBRC 15474T, although those of H. yilanensis NBRC 106371T and H. sakaeratensis NBRC 102641T were not completely sequenced. The gene clusters contained seven modules, suggesting the products would be molecules derived from C14 polyketide chains. Substrates of AT domains in modules 5 and 6 were predicted to be methylmalonyl-CoA or ethylmalonyl-CoA, and those of all the remaining modules were malonyl-CoA. Four pairs of dehydratase (DH)-ketoreductase (KR) and one trio of DH-enoylreductase (ER)-KR were present as the optional domains in the gene clusters; therefore, four keto groups would be reduced to four conjugated double bonds and one keto group would be completely reduced, respectively. Hence, we predicted the chemical structure of the polyketide backbones shown in Fig. 1b. Pks-3 genes were present in H. cretacea NBRC 15474T, H. yilanensis NBRC 106371T and H. daliensis NBRC 106372T. They contained only a single module and showed low sequence similarities to characterized PKS genes (data not shown); therefore, we were not able to predict the metabolites. Pks-4 genes were present in H. yilanensis NBRC 106371T and H. daliensis NBRC 106372T. They were predicted to be iterative type-I PKSs for enediyne synthesis, called PksE, because they showed higher sequence similarities to PksEs than to normal modular type-I PKSs (data not shown) and included a pair of KR-DH domains, specific for PksE, after the ACP. Their products would be polyketide compounds with an enediyne core [24]. Pks-5 gene clusters were present in H. cretacea NBRC 15474T and H. mongoliensis NBRC 105882T; however, they were not completely sequenced. Hence, we were not able to predict whole chemical structure of the polyketide chain.

Strain-specific clusters

We found 2, 4, 1, 2 and 6 strain-specific NRPS and/or PKS gene clusters in H. cretacea NBRC 15474T, H. mongoliensis NBRC 105882T, H. yilanensis NBRC 106371T, H. daliensis NBRC 106372T and H. sakaeratensis NBRC 102641T, respectively.

H. cretacea NBRC 15474T possessed 2 specific PKS gene clusters, named pks-6 and pks-7. The pks-6 gene cluster contained 35 modules encoded by 12 PKS genes. To the best of our knowledge, this is the largest type-I PKS gene cluster ever reported. We predicted the chemical structure of the polyketide backbone synthesized by pks-6, as shown in Fig. 1d, which is most likely a novel compound because no similar compounds were found in our database searches. The pks-7 gene cluster contained 6 modules encoded by 4 PKS genes and we predicted the metabolites to be hexaketide compounds with 2 C–C double bonds and one hydroxyl group.

H. mongoliensis NBRC 105882T possessed a specific PKS/NRPS gene cluster and 3 specific PKS gene clusters, named pks/nrps-2, pks-8, pks-9 and pks10. Pks/nrps-2 contained 1 PKS module and 13 NRPS modules, encoded by a PKS gene and seven NRPS genes, respectively. According to the module numbers and the A domain substrates, the product was predicted to be large polyketide-non-ribosomal peptide hybrid compound including Ser, Thr and asparagine (Asn). The pks-8 gene cluster contained seven PKS genes encoding 21 modules. As shown in Fig. 1f, its products would be large polyketide compounds with 6 C–C double bonds and 11 hydroxyl groups. The pks-9 contained 3 PKS genes encoding 9 modules. Its products were predicted to be nonaketide compounds with 3 conjugated double bonds and 5 hydroxyl groups (Fig. 1g). The pks-10 gene encoded only a single module; therefore, we were not able to predict the chemical structure of its metabolite.

H. yilanensis NBRC 106371T possessed a specific PKS/NRPS hybrid gene cluster named pks/nrps-3. The products were predicted to be polyketide-non-ribosomal peptide hybrid compounds whose backbone is leucine (Leu)-valine (Val)-Leu-Ser-a polyketide unit.

H. daliensis NBRC 106372T possessed 2 specific NRPS gene clusters, named nrps-12 and nrps-13. The nrps-12 gene cluster encoded 22 modules, the products of which were predicted to be peptide compounds comprising 22 amino-acids, including 2 phenylalanine (Phe), 7 Asp, 1 Ser, 1 Val, 3 tyrosine (Tyr), and 1 Asn molecules. In contrast, nrps-13 contained only two modules, whose A domains were predicted to incorporate Gly and Ala, respectively. Hence, the products will be dipeptides including Gly and Ala molecules.

H. sakaeratensis NBRC 102641T possessed 3 specific NRPS gene clusters, one specific PKS/NRPS hybrid gene cluster and two specific PKS gene clusters named nrps-14 to -16, pks/nrps-4, pks-11 and pks-12. In the nrps-14 gene cluster, three NRPS genes were present encoding six modules. The A domains of four modules were predicted to incorporate Val, Ser, Asn and Asn, suggesting that nrps-14 would produce hexapeptides including Val, Ser and two Asn molecules. Nrps-15 had only one module, but the domain organization (A-C-T) was different from that of normal NRPS (C-A-T). Because a CoA-ligase, an ACP, and an NRPS comprising only one C domain were also encoded adjacent to nrps-15 gene (data not shown), this gene cluster might synthesize compounds composed of a starter molecule and a Gly molecule loaded by the unusual NRPS. Nrps-16 also had only a single module; therefore, we were not able to predict its peptide products. The pks/nrps-4 gene cluster encoded at least 15 PKS modules and one NRPS module, but it was not completely sequenced because the sequence of a PKS gene named s08-orf1 was partial and the adjacent genes remain unclear. Although we were not able to predict the whole chemical structure synthesized by this gene cluster, the product will include a C28 or longer polyketide chain. The pks-11 gene cluster encoded at least twenty modules, although s22-orf1 was not completely sequenced and the adjacent genes were unclear. This product was predicted to be a large compound including a polyhydroxyl polyketide chain, as shown in Fig. 1i. The pks-12 gene cluster encoded only a single module; therefore, we were not able to predict chemical structures of its products.

Distribution and evolutionary history of NRPS and PKS gene clusters

We constructed a phylogenetic tree of the type strains of the genus Herbidospora based on 16S rRNA gene sequences. By mapping the inferred ancestral nodes of the individual gene clusters onto the tree, we traced the evolutionary histories of these pathways (Fig. 2). Nine gene clusters, underlined in Fig. 2, appeared to have been acquired early in the evolution of the genus Herbidospora, because they are conserved in all the type strains. By contrast, 15 gene clusters, indicated by asterisks in Fig. 2, would have been acquired relatively recently, appearing toward the branch terminals in the tree. Gene clusters shared between/among 2–4 strains are in boldface in Fig. 2. Pks-2 gene clusters are present in 4 strains, except for H. cretacea NBRC 15474T, suggesting they were acquired early, and inherited vertically; however, they were lost just before evolution to H. cretacea. The nrps-10, nrps-11 and pks-5 clusters are present in H. mongoliensis NBRC 105882T and H. cretacea NBRC 15474T, but not in the other 3 strains, we speculated that these 3 gene clusters were acquired early, but lost just before evolution to H. yilanensis, H. sakaeratensis and H. daliensis. The pks-3 gene clusters are present in three strains except for H. mongoliensis NBRC 105882T and H. sakaeratensis NBRC 102641T, suggesting that they were acquired just branching off from H. mongoliensis and lost just before evolution to H. sakaeratensis. Nrps-8 gene clusters are present in H. yilanensis NBRC 106371T, H. sakaeratensis NBRC 102641T and H. daliensis NBRC 106372T, suggesting acquisition just before evolution to these three species. Similarly, the nrps-9 and pks-4 gene clusters would also have been acquired at the same point; however, these clusters seemed to have been lost during evolution to H. daliensis and H. sakaeratensis, respectively. To confirm the hypothesis, we conducted phylogenetic analysis of NRPSs and PKSs in gene clusters conserved among more than 4 strains. Except for pks-1, all the phylogenetic trees showed the same topology (Fig. 3) as that based on 16S rDNA sequences (Fig. 2). This supports that nrps-1 to -7, pks/nrps-1 and pks-2 were actually acquired early in the evolution and inherited vertically. In contrast, pks-1 may not be inherited in the same manner as these gene clusters, because the topology of pks-1 phylogenetic tree differed from those of other gene clusters and 16S rDNA sequence.

Fig. 2

Phylogenetic tree of the type strains of the genus Herbidospora, based on 16S rRNA gene sequences, depicting the inferred ancestry of NRPS and PKS gene clusters. Bootstrap values (>50 %) from 1000 replicates are shown at branch nodes. Arrows indicate acquisitions and losses of NRPS and PKS gene clusters. Gene clusters conserved all the five strains, shared between/among two to four strains, and specific to each strain are underlined, boldfaced, and asterisked, respectively

Fig. 3

Phylogenetic tree of the type strains of the genus Herbidospora, based on NRPS and PKS sequences. Bootstrap values (>50 %) from 1000 replicates are shown at branch nodes. The proteins used in this analysis are shown in parentheses by open reading frame numbers listed in Table 2

Conclusions

We concluded the following: (1) The genomes of Herbidospora strains carry as many NRPS and PKS gene clusters as those of other actinomycetes such as Streptomyces; however, their products are yet to be isolated; (2) members of the genus Herbidospora can synthesize large and diverse metabolites, many of whose chemical structures are yet to be reported; (3) each strain possesses 1–6 strain-specific NRPS and/or PKS gene clusters, in addition to those conserved within this genus, suggesting diversity of these pathways; and (4) the diversity of NRPS and PKS pathways in each strain has increased by genus-level vertical inheritance and relatively recent acquisitions of these gene clusters during evolution of this genus.

To summarize, in this study, we sequenced whole genomes of all the five type strains belonging to the genus Herbidospora and examined their NRPS and PKS gene clusters. Each strain harbored 15–18 modular NRPS and PKS gene clusters. Through the comparison of these gene clusters, 32 NRPS and PKS pathways were identified from the 5 strains. Among them, 9 pathways were conserved in all 5 strains, 8 were shared in 2–4 strains, and the remaining 15 were strain-specific suggesting the strain diversity of these pathways. We revealed that these strains harbor a wealth of NRPS and PKS pathways, many of whose products are large and have yet to be discovered. This study also provided useful information about the inferred numbers and molecular structures of secondary metabolites, such as non-ribosomal peptides and polyketides, potentially produced by these strains, suggesting that Herbidospora strains are an untapped and attractive source of novel secondary metabolites.