Skip to main content
NCBI home page
Frontiers in Microbiology logoLink to Frontiers in Microbiology
. 2017 Mar 15;8:394. doi: 10.3389/fmicb.2017.00394

Cloning and Heterologous Expression of a Large-sized Natural Product Biosynthetic Gene Cluster in Streptomyces Species

Hee-Ju Nah 1, Hye-Rim Pyeon 1, Seung-Hoon Kang 1, Si-Sun Choi 1, Eung-Soo Kim 1,*
PMCID: PMC5350119  PMID: 28360891

Abstract

Actinomycetes family including Streptomyces species have been a major source for the discovery of novel natural products (NPs) in the last several decades thanks to their structural novelty, diversity and complexity. Moreover, recent genome mining approach has provided an attractive tool to screen potentially valuable NP biosynthetic gene clusters (BGCs) present in the actinomycetes genomes. Since many of these NP BGCs are silent or cryptic in the original actinomycetes, various techniques have been employed to activate these NP BGCs. Heterologous expression of BGCs has become a useful strategy to produce, reactivate, improve, and modify the pathways of NPs present at minute quantities in the original actinomycetes isolates. However, cloning and efficient overexpression of an entire NP BGC, often as large as over 100 kb, remain challenging due to the ineffectiveness of current genetic systems in manipulating large NP BGCs. This mini review describes examples of actinomycetes NP production through BGC heterologous expression systems as well as recent strategies specialized for the large-sized NP BGCs in Streptomyces heterologous hosts.

Keywords: Streptomyces, natural product, biosynthetic gene cluster, heterologous expression, large-sized

Introduction

Natural products (NPs) and their derivatives lead a huge pharmaceutical market share comprising 61% of anticancer drugs and 49% of anti-infection medicine in the past 30 years (Newman and Cragg, 2012). Especially, actinomycetes NPs are a major resource for drug discovery and development, mainly due to their structural novelty, diversity, and complexity (Donadio et al., 2007). Isolation and characterization of NP biosynthetic gene clusters (BGCs) have further accelerated our understanding of their molecular biosynthetic mechanisms, leading to the rational redesign of novel NPs through BGC manipulation (Fischer et al., 2003; Castro et al., 2015).

Some of these potentially valuable BGCs are, however, derived from non-culturable meta-genomes or genetically recalcitrant microorganisms. Moreover, many of these BGCs are expressed poorly or not at all under laboratory culture conditions, which makes it challenging to characterize the target NPs (Galm and Shen, 2006). Since efficient expression of actinomycetes NP BGCs present a major bottleneck for novel NP discovery, various cryptic BGC awakening strategies such as regulatory genes control, ribosome engineering, co-culture fermentation, and heterologous expression have been pursued for NP development (Tang et al., 2000; Flinspach et al., 2014; Martinez-Burgo et al., 2014; Miyamoto et al., 2014).

A traditional method for BGC cloning involves cosmid library construction by partial digestion or random shearing of chromosomal DNA. A typical size of NP BGC is usually larger than 20 kb (sometimes over 100 kb), and a cosmid vector system can only accept a relatively small BGC (up to 40 kb) or only a part of a large BGC. Therefore, cloning and efficient overexpression of an entire BGC still remains challenging due to the ineffectiveness of current host cells including the genetic and metabolic characteristics in manipulating large BGCs for heterologous expression. This mini review summarizes the list of the actinomycetes NP BGCs that have been successfully cloned and expressed in Streptomyces heterologous hosts (Table 1). In addition, three cloning and heterologous expression systems, which are quite suitable for large NP BGCs, such as transformation-associated recombination (TAR) system, integrase-mediated recombination (IR) system, and plasmid Streptomyces bacterial artificial chromosome (pSBAC) system are introduced (Figure 1).

Table 1.

Heterologous expression of NP BGCs.

NP name (Class) Original host BGC size (kb) Expression method Heterologous host WT titer (mg/L) HH titter (mg/L) References
A201A (Nucleoside) Sacchaothrix mutabilis subsp. Capreolus 34 PAC Integrative S. coelicolor S. lividans 12 8 Saugar et al., 2016
A54145 (NRPS) S. fradiae NRRL 18160 ~60 BAC Integrative S. ambofaciens S. roseosporus NR 100 ~ 385 Alexander et al., 2010
Actinorhodin (PKS II) S. coelicolor M145 33 LLHR Integrative Streptomyces NR NR Chen and Qin, 2011
Amicetin (NRPS) S. vinaceusdrappus NRRL 2363 37.3 Cosmid Replicative S. lividans NR NR Zhang et al., 2012
Ammosamides A-C (Alkaloid) S. sp. CNR-698 35 TAR Integrative S. coelicolor 4 ~ 6 17 Jordan and Moore, 2016
Anthracimycin (PKS I) S. sp. T676 53.2 PAC Integrative S. coelicolor NR 8.6 ~ 13.8 Alt and Wilkinson, 2015
Aristeromycin (Nucleoside) S. citricolor 37.5 Cosmid Replicative S. albus NR ND Kudo et al., 2016
Aureothin (PKS I) S. thioluteus HKI-227 27 Cosmid Integrative S. lividans NR NR He and Hertweck, 2003
Barbamide (PKS-NRPS) Moorea producens 26 LCHR Replicative S. venezuelae NR ND* Kim et al., 2012
Bernimamycin (Thiopeptide) S. bernensis UC5144 12.9 LLHR Integrative S. lividans S. venezuelae NR NR Malcolmson et al., 2013
Blasticidin S (Nucleoside) S. griseochromogenes 20 Cosmid Replicative S. lividans NR NR Cone et al., 2003
Cacibiocin (Aminocoumarin) Catenulispora acidiphila 20 LLHR Integrative S. coelicolor 4.9 60 Zettler et al., 2014
Caerulomycin (PKS-NRPS) Actinoalloteichus cyanogriseus WH1-2216-6 44.6 Cosmid Replicative S. coelicolor NR NR Zhu et al., 2012
Cephamycin C (NRPS) S. clavuligerus ATCC 27064 35.6 Cosmid Integrative S. flavogriseus S. coelicoor S. albus 3640 8 ~ 300# Martinez-Burgo et al., 2014
Chalcomycin (PKS I) S. bikiniensis 80 LLHR Integrative S. fradiae NR NR Ward et al., 2004
Chaxamycin (PKS I) S. leeuwenhoekii 80.2 PAC Integrative S. coelicolor NR NR Castro et al., 2015
Chloramphenicol (PKS-NRPS) S. venezuelae ATCC10712 NR Cosmid Integrative S. coelicolor NR 1.6 ~ 26.23 Gomez-Escribano and Bibb, 2011
Chlorizidine A (PKS I) S. sp. CNH-287 42.4 Fosmid Integrative S. coelicolor NR NR Mantovani and Moore, 2013
Chrysomycin (PKS II) S. albaduncus AD0819 34.65 Cosmid Replicative S. lividans NR ND Kharel et al., 2010
Clavulanic acid (β-lactam) S. clavuligerus ATCC27064 20 Cosmid Integrative S. flavogriseus S. coelicolor 164.50 0.6 Alvarez-Alvarez et al., 2013
Complestatin (Glycopeptide) S. chartreusis AN1542 54.5 LLHR Integrative S. lividans 5.57 0.24 Park et al., 2016
Congocidine (NRPS) S. ambofaciens ATCC23877 NR Cosmid Integrative S. coelicolor NR NR Gomez-Escribano and Bibb, 2011
Coumermycin A1 (Aminocoumarin) S. rishiriensis DSM40489 38.6 Cosmid Integrative S. coelicolor 0.002 ~ 0.005 0.01 Wolpert et al., 2008
Cremeonycin (Diazoquinone) S. cremeus NRRL3241 18 BAC Integrative S. lividans NR NR Waldman et al., 2015
Cyclothiazomycin (Thiopeptide) S. hygroscopicus 10-22 22.7 LLHR Integrative S. lividans NR NR Wang et al., 2010
Daptomycin (NRPS) S. roseosporus NRRL 11379 128 BAC Integrative S. lividans 900 18 Miao et al., 2005
Desotamide (NRPS) S. scopuliridis SCSIO 39 Cosmid Integrative S. coelicolor NR ND* Li et al., 2015
Epothilone (PKS-NRPS) Sorangium cellulosum SHP44 56 LLHR Replicative & Integrative S. coelicolor 0.05 ~ 0.1 20 Tang et al., 2000
FK506 (PKS I) S. sp. KCCM11116P 120 LCHR Integrative S. albus NR NR Chen et al., 2014
S. tsukubaensis 83.5 PAC Integrative S. coelicolor 1.20 5.50 Jones et al., 2013
Flustatin (PKS II) Micromonospora SCSIO N160 40 Cosmid Replicative S. coelicolor NR NR Yang et al., 2015
Fostriecin PKS (PKS I) S. pulveraceus ATCC31906 48.6 LLHR Replicative & Integrative S. coelicolor S. lividans NR ND Su et al., 2015
Galbonolide B (PKS I) S. sp. L235 12.1 LLHR Integrative S. coelicolor NR NR† Liu et al., 2015
GE2270 (Thiopeptide) Planobispora rosea ATCC53733 21.4 LLHR Integrative S. coelicolor NR 0.08 Flinspach et al., 2014
GE37468 (Thiazolyl peptide) S. ATCC 55365 17.1 LLHR Integrative S. lividans 5 ~ 7 2 ~ 3 Young and Walsh, 2011
Gilvocarcin V (PKS II) S. griseoflavus Gö 3592 32.9 Cosmid Replicative S. lividans 20 ~ 30 NR Fischer et al., 2003
Goadsporin (Azole) S. sp. TP-A0584 14 LLHR Integrative S. lividans 126.3 342.7 Haginaka et al., 2014
Gougerotin (Nucleoside) S. graminearus 28.7 LCHR Integrative S. coelicolor NR NR Niu et al., 2013
Granaticin (PKS II) S. violaceoruber Tü22 39 Cosmid Replicative S. coelicolor NR NR Ichinose et al., 1998
Grecocycline (PKS II) S. sp. Acta 1362 36 TAR Integrative S. albus NR ND* Bilyk et al., 2016
Grincamycin (PKS II) S. lusitanus SCSIO LR32 37 LCHR Integrative S. coelicolor NR ND* Zhang et al., 2013
Holomycin (NRPS) S. clavuligerus ATCC27064 24 LLHR Integrative S. coelicolor NR NR Robles-Reglero et al., 2013
Kanamycin (Aminoglycoside) S. kanamyceticus ATCC12853 32 Cosmid Replicative S. venezuelae 1.80 0.50 Thapa et al., 2007
Kinamycin (PKS II) S. murayamaensis 40 Cosmid Replicative S. lividans NR ND Gould et al., 1998
Lincomycin (Lincosamide) S. lincolnensis ATCC25466 35 Cosmid Integrative S. coelicolor 50.1 0.66 ~ 1.49 Koberska et al., 2008
Lyngbyatoxin A (NRPS) Moorea products 11.3 LLHR Replicative S. coelicolor NR NR Jones et al., 2012
Lysolipin (PKS II) S. tendae Tü 4042 43.2 Cosmid Replicative S. albus NR NR Lopez et al., 2010
Macrotetrolide (PKS II) S. griseus DSM40695 25 LLHR Integrative S. lividans 40 10 Kwon et al., 2001
Marineosin (Oligopyrrole) S. sp. CNQ-617 32 Cosmid Integrative S. venezuelae 0.5 5 Salem et al., 2014
Medermycin (PKS II) S. sp. AM7161 30 LLHR Integrative S. coelicolor S. lividans NR NR Ichinose et al., 2003
S. sp. K73 36.2 Cosmid Replicative S. coelicolor NR NR Ichinose et al., 2003
Mensacarcin (PKS II) S. bottropensis 40 Cosmid Integrative S. albus NR ND* Yan et al., 2012
Meridamycin (PKS-NRPS) S. sp. NRRL 30748 90 pSBAC Integrative S. lividans NR 0.1# Liu et al., 2009
Merochlorin A-D (PKS-terpenoid) S. sp. CNH-189 57.6 Fosmid Integrative S. coelicolor 10.0 NR Kaysser et al., 2012
Mycosperine Actinosynnema mirum DSM43827 6.3 LLHR Integrative S. avermitilis NR NR# Miyamoto et al., 2014
Naphthocyclinone (PKS II) S. arenae DSM40737 12 Cosmid Replicative S. coelicolor NR NR Brunker et al., 1999
Nataxazole (PKS I) S. sp. Tü6176 44.1 TAR Integrative S. lividans NR ND* Cano-Prieto et al., 2015
Neocarzilin (PKS I) S. carzinostaticus var. F-41 33 Cosmid Integrative S. lividans NR NR Otsuka et al., 2004
Nogalamycin (PKS II) S. nogalater 20 Cosmid Replicative S. lividans NR NR Ylihonko et al., 1996
29 LLHR Replicative S. lividans S. galilaeus S. peucetius NR NR Torkkell et al., 2001
Novobiocin (Aminocoumarin) S. spheroides 25.6 Cosmid Replicative S. lividans NR NR† Steffensky et al., 2000
Oleandomycin (PKS I) S. antibiticus 65 LLHR Replicative S. lividans NR NR† Shah et al., 2000
Oxytetracycline (PKS II) S. rimosus M4018 29 Cosmid Integrative S. venezuelae 75 431 Yin et al., 2016
S. rimosus 34 Cosmid Replicative S. lividans NR 20 Binnie et al., 1989
Phosphinothricin (NRPS) S. viridochromogenes DSM 40736 40 Fosmid Integrative S. lividans NR NR Blodgett et al., 2005
Puromycin (Nucleoside) S. alboniger 13 Cosmid Replicative S. lividans S. griseofuscus 150.00 4 ~ 15 Lacalle et al., 1992
R1128 (PKS II) S. sp. R1128 17 Cosmid Replicative S. lividans NR 1.00 Marti et al., 2000
Ravidomycin PKS II S. ravidus 33.28 Cosmid Replicative S. lividans NR NR Kharel et al., 2010
Rebeccamycin (Indolocarbazole) Saccharothrix aerocolonigenes ATCC 39243 25.6 Cosmid Replicative S. albus NR NR Sanchez et al., 2002
Resorcinomycin Streptorerticilium roseoverticillatum 11 LLHR Replicative S. lividans NR ND* Ooya et al., 2015
Rimosamide (NRPS-PKS) S. rimosus NRRL B-2659 30.5 Fosmid Integrative S. lividans NR NR McClure et al., 2016
Rishirilide A (PKS II) S. bottropensis 50 Cosmid Integrative S. albus, S. lividans NR NR Yan et al., 2012
Salinomycin (PKS I) S. albus DSM41398 106 LLHR Integrative S. coelicolor NR NR Yin et al., 2015
Sparsomycin (NRPS) S. sparsogenes 30 TAR Integrative S. lividans NR NR Rui et al., 2015
Staurosporine (Indolocarbazole) S. sanyensis FMA 34.6 Cosmid Integrative S. coelicolor NR NR Li T. et al., 2013
S. sp. TP-A0274 20 Cosmid Integrative S. lividans 10.5 2.6 Onaka et al., 2002
Streptocolin (Lanthipeptide) S. colimus Tü365 6 Cosmid Integrative S. coelicolor NR 5.4 ~ 110 Iftime et al., 2015
Streptothricin (NRPS) S. sp. TP-A0356 41 Cosmid Replicative S. coelicolor NR NR Li J. et al., 2013
Tautomycetin (PKS I) S. sp. CK4412 80 pSBAC Integrative S. coelicolor S. lividans 3.10 3.91 ~ 4.05 Nah et al., 2015
Tetracenomycin C (PKS II) S. glaucescens 24 LLHR Replicative S. lividans NR NR Motamedi and Hutchinson, 1987
Tetrangulol (PKS II) S. sp. WP4669 S. rimosus NRRL3016 40 Cosmid Replicative S. lividans NR NR Hong et al., 1997
Thioriridamide (Ribosomal peptide) S. olivoriridis NA05001 14.5 LLHR Replicative S. lividans NR NR Izawa et al., 2013
70 BAC Integrative S. avermitilis NR 2.5 Izumikawa et al., 2015
TP-1161 (Thiopeptide) Nocardiopsis sp. TFS65-07 16 Cosmid Replicative S. coelicolor NR ND Engelhardt et al., 2010
Undecylprodigiosin (NRPS) S. coelicolor M145 38 LLHR Replicative S. parvulus NR NR Malpartida et al., 1990
Validamycin (Pseudosaccharide) S. hygroscopicus var. limoneus KTCC 1715 37 Cosmid Replicative S. lividans S. albus NR NR Singh et al., 2006
Venemycin (PKS I) S. venezuelae 29.5 Cosmid Integrative S. coelicolor NR ND Thanapipatsiri et al., 2016
Versipelostatin (PKS I) S. versipellis 4083 108 BAC Integrative S. albus 1.5 21.0 Hashimoto et al., 2015
YM-216391 (NRPS) S. nobilis <40 Cosmid Replicative S. lividans NR 0.18 Jian et al., 2012
Open in a new tab

PKS, polyketide synthase; NRPS, non-ribosomal peptide synthase; S, Streptomyces; sp, species; TAR, transformation-associated recombination; PAC, phage P1 artificial chromosome; BAC, bacterial artificial chromosome; LLHR, linear-plus-linear homologous recombination; LCHR, linear-plus-circular homologous recombination; NR, not reported (but produced); ND, not detected (not produced); WT, wild type; HH, heterologous host;

*

intermediate produced only;

†

expressed part of gene cluster;

#

produced by gene cluster modification (e.g., Promoter substitution).

Figure 1.

Figure 1

Open in a new tab

Overview of large BGC cloning system (A) TAR system (B) IR system (C) pSBAC system. HR, Homologous region; RE, restriction enzyme.

Traditional method for heterologous expression of NP BGCs

We summarized about 90 actinomycetes NP BGCs that have been successfully expressed in Streptomyces heterologous hosts from the last several decades (Table 1). Relatively small BGCs encoding Type II polyketide were first to be isolated at the beginning of heterologous expression research. Many of the listed BGCs (about 83%) were isolated by cosmid/fosmid library construction and some of these BGCs were cloned into replicative or integrative vector by linear-plus-linear (recombination between two linear DNAs) or linear-plus-circular (recombination between linear and replicating circular DNA) homologous recombination. Approximately 60% of BGCs were integrated into the heterologous host chromosome and only 37% of BGCs existed in the heterologous host via replicative plasmid. Cosmid vectors such as pOJ446 and SuperCos1 were used to be replicative or integrative in the heterologous host, so the production level of the heterologously expressed NP BGC varied significantly. Some BGCs were isolated with two different vector systems, followed by heterologous expression via both integrative and replicative systems. For example, the epothilone BGC was expressed by both pSET152-based integration vector and SCP2*-based replication vectors, so that its expression level was increased from 0.1 mg/L in the original Sorangium cellulosum system to 20 mg/L in the epothilone BGC-expressing Streptomyces host (Tang et al., 2000). S. coelicolor and S. lividans were two major strains for heterologous expression, thanks to their well-characterized genetic and biochemical properties. About 12% BGCs were expressed in another popular heterologous host, S. albus, which has fast growth and an efficient genetic system (Zaburannyi et al., 2014). Comparing with the original NP producing strains, approximately 14% of NPs had a higher expression level and 12% lower when they were expressed in the heterologous hosts. When bernimamycin BGC was heterologously expressed both in S. lividans and S. venezuelae, its production yield was increased 2.4-fold in S. lividans with no production in S. venezuelae (Malcolmson et al., 2013).

Cloning systems of large NP BGCs for heterologous expression in Streptomyces

TAR system

The TAR system takes advantage of the natural in vivo homologous recombination of Saccharomyces cerevisiae (Larionov et al., 1994). It has also been applied to capture and express large biosynthetic gene clusters from environmental DNA samples (Feng et al., 2010; Kim et al., 2010). Yamanaka and colleagues designed TAR cloning vector, pCAP01, which consists of three elements, one from each of yeast, E. coli, and actinobacteria (Yamanaka et al., 2014). The target BGC can be directly captured and manipulated in yeast background, and the captured BGC can be shuttled between E. coli and actinobacteria species. It also has a pUC ori that could stably carry an over 50 kb insert in E. coli hosts. The pCAP01 vector contains oriT and attP-int that can transfer the target BGC by conjugation, and the DNA stability can be maintained by insertion into heterologous host chromosomes. To generate a capturing vector, both flanking homologous arms of the target BGC were PCR-amplified and cloned into the pCAP01. The linearized capturing vector and the restriction enzyme digested genomic DNA were co-transformed into yeast, then the target BGC was captured by yeast recombination activities (Figure 1A). The marinopyrrole BGC (30 kb) and the taromycin A BGC (67 kb) were captured by this TAR system, and functionally expressed in Streptomyces coelicolor (Yamanaka et al., 2014).

IR system

Most cloning systems to clone a large DNA fragment directly from bacterial genome are based on different site-specific recombination systems that consist of a specialized recombinase and its target sites. The IR system is based on ΦBT1 integrase-mediated site-specific recombination and simultaneous Streptomyces genome engineering (Du et al., 2015). The actinorhodin BGC, the napsamycin BGC and the daptomycin BGC were successfully isolated by the IR system (Du et al., 2015). pUC119-based suicide vector and pKC1139 carrying mutated attP or attB, respectively, and an integrative plasmid containing the ΦBT1 integrase gene were used for the system (Figure 1B). The pUC119-based plasmid carrying mutated attB and a homologous region to 5′ end of the target BGC was introduced into the chromosome by single crossover. The pKC1139 carrying mutated attP and a homologous region to 3′ end of the BGC was transferred and integrated into chromosome by conjugation and single crossover through cultivation at high temperature above 34°C. Expression of ΦBT1 integrase leads to excision of the pKC1139 containing the target BGC. The pKC1139 containing BGC from original producing Streptomyces was extracted and transferred into E. coli for recovery. The IR system was only expressed in parental strain not heterologous host, but it was presumed to be transferred and maintained by replication in heterologous host (Du et al., 2015).

pSBAC vector system

In the early 1990s, Bacterial Artificial Chromosomes (BAC) was reported to carry inserts approaching 200 kb in length emerged (Shizuya et al., 1992). Various BAC vectors have been used extensively for construction of DNA libraries to facilitate physical genomic mapping and DNA sequencing efforts (Sosio et al., 2000; Martinez et al., 2004; Fuji et al., 2014; Varshney et al., 2014). Several E. coli-Streptomyces shuttle BAC vectors have been developed to carry the large-sized NP BGCs such as pStreptoBAC V and pSBAC (Miao et al., 2005; Liu et al., 2009). The utility of pSBAC was demonstrated through the precise cloning and heterologous expression of the tautomycetin BGC and the pikromycin BGC of the type I PKS biosynthetic pathway, as well as the meridamycin BGC of the PKS-NRPS hybrid biosynthetic pathways (Liu et al., 2009; Nah et al., 2015). Unique restriction enzyme recognition sites naturally existing or artificially inserted into both flanking regions of the entire BGC were used for capturing the BGCs. The pSBAC vector was also inserted within the unique restriction enzyme site by homologous recombination. And then the entire target BGC was captured in a single pSBAC through straightforward single restriction enzyme digestion and self-ligation (Figure 1C). The pSBAC contains two replication origins, ori2 and oriV, for DNA stability in E. coli, and oriT and ΦC31 attP-int for BGC integration into the surrogate host chromosome through intergenic conjugation. The recombinant pSBAC containing the large BGCs of varied length from 40 kb to over 100 kb have been successfully cloned and conjugated from E. coli to S. coelicolor and S. lividans (Liu et al., 2009; Nah et al., 2015), implying that the pSBAC system seems to be the most suitable for large BGC cloning comparing with TAR and IR systems.

Recently, a new cloning method named CATCH (Cas9-Assisted Targeting of Chromosome) based on the in vitro application of RNA-guided Cas9 nuclease was developed (Jiang and Zhu, 2016). The Cas9 nuclease cleaves target DNA in vitro from intact bacterial chromosomes embedded in agarose plugs, which can be subsequently ligated with cloning vector through Gibson assembly. Jiang and colleagues cloned the 36-kb jadomycin BGC from S. venezuelae and the 32-kb chlortetracycline BGC from S. aureofaciens by CATCH (Jiang et al., 2015).

Streptomyces heterologous expression of NP BGCs

The Streptomyces genus is suitable for heterologous expression of large NP BGCs due to its intrinsic ability to produce various valuable secondary metabolites. Well-studied Streptomyces strains such as S. coelicolor, S. lividans, and S. albus have been mainly used as heterologous expression surrogate hosts (Table 1). The regulatory networks of secondary metabolite production have been well characterized in these strains, and thus several NP high-level producing strains have been constructed (Baltz, 2010; Gomez-Escribano and Bibb, 2011). In addition, some of these Streptomyces host genomes have been further engineered to eliminate precursor-competing biosynthetic BGCs, so that the extra precursors such as malonyl-CoA and acetyl-CoA could be funneled into the target polyketide NP biosynthesis (Gomez-Escribano and Bibb, 2011).

As shown in Table 1, most of the heterologously expressed NPs were detected as a final product, but some were detected as an intermediate due to their partial BGC expression. The NP production yield was similar to or slightly lower than that in WT. To increase the production level in heterologous hosts, it was devised to substitute with strong promoters or to increase the copy number of BGCs (Montiel et al., 2015; Nah et al., 2015). In case of pSBAC system, the tautomycetin production yield in the heterologous hosts was similar to that in the original producing strain. The selection marker on the tautomycetin BGC was changed and re-introduced into the heterologous host by tandem repeat, resulting in further yield increase from 3.05 to 13.31 mg/L in comparison with the heterologous host harboring only single copy of tautomycetin BGC. The heterologous host harboring tandem copies of tautomycetin BGC was proved to stably maintain two BGCs in the presence of appropriate antibiotic selection (Nah et al., 2015).

Meanwhile, the TAR system used yeast homologous recombination-based promoter engineering for the activation of silent natural product BGCs (Montiel et al., 2015). Bi-directional promoter cassettes were generated by PCR amplification of varied yeast selectable markers, which contains promoter-insulator-RBS combinations, and they were co-transformed with the cosmid or BAC clone harboring the target BGC into yeast. The rebeccamycin BGC was used as a model BGC. The promoter-replaced rebeccamycin BGC was transferred into S. albus by conjugation, and the production of rebeccamycin was examined in the heterologous host (Montiel et al., 2015). Using the TAR-based promoter engineering strategy, multiple promoter cassettes could be inserted simultaneously into the target BGC, thereby expediting the re-engineering process. The TAR-based promoter engineering strategy was also used to capture the silent tetarimycin BGC and the silent, cryptic pseudogene-containing, environmental DNA-derived lazarimide BGC (Montiel et al., 2015).

In conclusion, Streptomyces heterologous expression systems have been proved to be a very attractive strategy to awaken cryptic NP BGCs, and could also be applied to overexpression of a variety of large NP BGCs in actinomycetes.

Author contributions

HN, SK, SC, and EK planned, outlined, and revised the manuscript. HN, HP, and EK wrote and revised the manuscript.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This research was supported by “National Research Foundation of Korea (NRF)” (Project No. NRF-2014R1A2A1A11052236 & NRF-2016K2A9A2A10005545).

References

  1. Alexander D. C., Rock J., He X., Brian P., Miao V., Baltz R. H. (2010). Development of a genetic system for combinatorial biosynthesis of lipopeptides in Streptomyces fradiae and heterologous expression of the A54145 biosynthesis gene cluster. Appl. Environ. Microbiol. 76, 6877–6887. 10.1128/AEM.01248-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alt S., Wilkinson B. (2015). Biosynthesis of the novel macrolide antibiotic anthracimycin. ACS Chem. Biol. 10, 2468–2479. 10.1021/acschembio.5b00525 [DOI] [PubMed] [Google Scholar]
  3. Alvarez-Alvarez R., Martinez-Burgo Y., Perez-Redondo R., Brana A. F., Martin J. F., Liras P. (2013). Expression of the endogenous and heterologous clavulanic acid cluster in Streptomyces flavogriseus: why a silent cluster is sleeping. Appl. Microbiol. Biotechnol. 97, 9451–9463. 10.1007/s00253-013-5148-7 [DOI] [PubMed] [Google Scholar]
  4. Baltz R. H. (2010). Streptomyces and Saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters. J. Ind. Microbiol. Biotechnol. 37, 759–772. 10.1007/s10295-010-0730-9 [DOI] [PubMed] [Google Scholar]
  5. Bilyk O., Sekurova O. N., Zotchev S. B., Luzhetskyy A. (2016). Cloning and heterologous expression of the grecocycline biosynthetic gene cluster. PLoS ONE 11:e0158682. 10.1371/journal.pone.0158682 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Binnie C., Warren M., Butler M. J. (1989). Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis. J. Bacteriol. 171, 887–895. 10.1128/jb.171.2.887-895.1989 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blodgett J. A., Zhang J. K., Metcalf W. W. (2005). Molecular cloning, sequence analysis, and heterologous expression of the phosphinothricin tripeptide biosynthetic gene cluster from Streptomyces viridochromogenes DSM 40736. Antimicrob. Agents Chemother. 49, 230–240. 10.1128/AAC.49.1.230-240.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brunker P., McKinney K., Sterner O., Minas W., Bailey J. E. (1999). Isolation and characterization of the naphthocyclinone gene cluster from Streptomyces arenae DSM 40737 and heterologous expression of the polyketide synthase genes. Gene 227, 125–135. 10.1016/S0378-1119(98)00618-0 [DOI] [PubMed] [Google Scholar]
  9. Cano-Prieto C., Garcia-Salcedo R., Sanchez-Hidalgo M., Brana A. F., Fiedler H. P., Mendez C., et al. (2015). Genome mining of Streptomyces sp. Tu 6176: characterization of the nataxazole biosynthesis pathway. Chembiochem 16, 1461–1473. 10.1002/cbic.201500153 [DOI] [PubMed] [Google Scholar]
  10. Castro J. F., Razmilic V., Gomez-Escribano J. P., Andrews B., Asenjo J. A., Bibb M. J. (2015). Identification and heterologous expression of the chaxamycin biosynthesis gene cluster from Streptomyces leeuwenhoekii. Appl. Environ. Microbiol. 81, 5820–5831. 10.1128/AEM.01039-15 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen C., Zhao X., Jin Y., Zhao Z. K., Suh J. W. (2014). Rapid construction of a bacterial artificial chromosomal (BAC) expression vector using designer DNA fragments. Plasmid 76, 79–86. 10.1016/j.plasmid.2014.10.002 [DOI] [PubMed] [Google Scholar]
  12. Chen W., Qin Z. (2011). Development of a gene cloning system in a fast-growing and moderately thermophilic Streptomyces species and heterologous expression of Streptomyces antibiotic biosynthetic gene clusters. BMC Microbiol. 11:243. 10.1186/1471-2180-11-243 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cone M. C., Yin X., Grochowski L. L., Parker M. R., Zabriskie T. M. (2003). The blasticidin S biosynthesis gene cluster from Streptomyces griseochromogenes: sequence analysis, organization, and initial characterization. ChemBioChem 4, 821–828. 10.1002/cbic.200300583 [DOI] [PubMed] [Google Scholar]
  14. Donadio S., Monciardini P., Sosio M. (2007). Polyketide synthases and nonribosomal peptide synthetases: the emerging view from bacterial genomics. Nat. Prod. Rep. 24, 1073–1109. 10.1039/b514050c [DOI] [PubMed] [Google Scholar]
  15. Du D., Wang L., Tian Y., Liu H., Tan H., Niu G. (2015). Genome engineering and direct cloning of antibiotic gene clusters via phage ΦBT1 integrase-mediated site-specific recombination in Streptomyces. Sci. Rep. 5:8740. 10.1038/srep08740 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Engelhardt K., Degnes K. F., Zotchev S. B. (2010). Isolation and characterization of the gene cluster for biosynthesis of the thiopeptide antibiotic TP-1161. Appl. Environ. Microbiol. 76, 7093–7101. 10.1128/AEM.01442-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Feng Z., Kim J. H., Brady S. F. (2010). Fluostatins produced by the heterologous expression of a TAR reassembled environmental DNA derived type II PKS gene cluster. J. Am. Chem. Soc. 132, 11902–11903. 10.1021/ja104550p [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fischer C., Lipata F., Rohr J. (2003). The complete gene cluster of the antitumor agent gilvocarcin V and its implication for the biosynthesis of the gilvocarcins. J. Am. Chem. Soc. 125, 7818–7819. 10.1021/ja034781q [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Flinspach K., Kapitzke C., Tocchetti A., Sosio M., Apel A. K. (2014). Heterologous expression of the thiopeptide antibiotic GE2270 from Planobispora rosea ATCC 53733 in Streptomyces coelicolor requires deletion of ribosomal genes from the expression construct. PLoS ONE 9:e90499. 10.1371/journal.pone.0090499 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fuji K., Koyama T., Kai W., Kubota S., Yoshida K., Ozaki A., et al. (2014). Construction of a high-coverage bacterial artificial chromosome library and comprehensive genetic linkage map of yellowtail Seriola quinqueradiata. BMC Res. Notes 7:200. 10.1186/1756-0500-7-200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Galm U., Shen B. (2006). Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis. Expert Opin. Drug Discov. 1, 409–437. 10.1517/17460441.1.5.409 [DOI] [PubMed] [Google Scholar]
  22. Gomez-Escribano J. P., Bibb M. J. (2011). Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters. Microb. Biotechnol. 4, 207–215. 10.1111/j.1751-7915.2010.00219.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gould S. J., Hong S. T., Carney J. R. (1998). Cloning and heterologous expression of genes from the kinamycin biosynthetic pathway of Streptomyces murayamaensis. J. Antibiot. (Tokyo) 51, 50–57. 10.7164/antibiotics.51.50 [DOI] [PubMed] [Google Scholar]
  24. Haginaka K., Asamizu S., Ozaki T., Igarashi Y., Furumai T., Onaka H. (2014). Genetic approaches to generate hyper-producing strains of goadsporin: the relationships between productivity and gene duplication in secondary metabolite biosynthesis. Biosci. Biotechnol. Biochem. 78, 394–399. 10.1080/09168451.2014.885824 [DOI] [PubMed] [Google Scholar]
  25. Hashimoto T., Hashimoto J., Teruya K., Hirano T., Shin-ya K., Ikeda H., et al. (2015). Biosynthesis of versipelostatin: identification of an enzyme-catalyzed [4+2]-cycloaddition required for macrocyclization of spirotetronate-containing polyketides. J. Am. Chem. Soc. 137, 572–575. 10.1021/ja510711x [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. He J., Hertweck C. (2003). Iteration as programmed event during polyketide assembly; molecular analysis of the aureothin biosynthesis gene cluster. Chem. Biol. 10, 1225–1232. 10.1016/j.chembiol.2003.11.009 [DOI] [PubMed] [Google Scholar]
  27. Hong S. T., Carney J. R., Gould S. J. (1997). Cloning and heterologous expression of the entire gene clusters for PD 116740 from Streptomyces strain WP 4669 and tetrangulol and tetrangomycin from Streptomyces rimosus NRRL 3016. J Bacteriol. 179, 470–476. 10.1128/jb.179.2.470-476.1997 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ichinose K., Bedford D. J., Tornus D., Bechthold A., Bibb M. J., Revill W. P., et al. (1998). The granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tu22: sequence analysis and expression in a heterologous host. Chem. Biol. 5, 647–659. 10.1016/S1074-5521(98)90292-7 [DOI] [PubMed] [Google Scholar]
  29. Ichinose K., Ozawa M., Itou K., Kunieda K., Ebizuka Y. (2003). Cloning, sequencing and heterologous expression of the medermycin biosynthetic gene cluster of Streptomyces sp. AM-7161: towards comparative analysis of the benzoisochromanequinone gene clusters. Microbiology 149, 1633–1645. 10.1099/mic.0.26310-0 [DOI] [PubMed] [Google Scholar]
  30. Iftime D., Jasyk M., Kulik A., Imhoff J. F., Stegmann E., Wohlleben W., et al. (2015). Streptocollin, a Type IV lanthipeptide produced by Streptomyces collinus Tu 365. Chembiochem 16, 2615–2623. 10.1002/cbic.201500377 [DOI] [PubMed] [Google Scholar]
  31. Izawa M., Kawasaki T., Hayakawa Y. (2013). Cloning and heterologous expression of the thioviridamide biosynthesis gene cluster from Streptomyces olivoviridis. Appl. Environ. Microbiol. 79, 7110–7113. 10.1128/AEM.01978-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Izumikawa M., Kozone I., Hashimoto J., Kagaya N., Takagi M., Koiwai H., et al. (2015). Novel thioviridamide derivative–JBIR-140: heterologous expression of the gene cluster for thioviridamide biosynthesis. J. Antibiot. (Tokyo) 68, 533–536. 10.1038/ja.2015.20 [DOI] [PubMed] [Google Scholar]
  33. Jian X. H., Pan H. X., Ning T. T., Shi Y. Y., Chen Y. S., Li Y., et al. (2012). Analysis of YM-216391 biosynthetic gene cluster and improvement of the cyclopeptide production in a heterologous host. ACS Chem. Biol. 7, 646–651. 10.1021/cb200479f [DOI] [PubMed] [Google Scholar]
  34. Jiang W., Zhao X., Gabrieli T., Lou C., Ebenstein Y., Zhu T. F. (2015). Cas9-assisted targeting of chromosome segments CATCH enables one-step targeted cloning of large gene clusters. Nat. Commun. 6:9101. 10.1038/ncomms9101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Jiang W., Zhu T. F. (2016). Targeted isolation and cloning of 100-kb microbial genomic sequences by Cas9-assisted targeting of chromosome segments. Nat. Protoc. 11, 960–975. 10.1038/nprot.2016.055 [DOI] [PubMed] [Google Scholar]
  36. Jones A. C., Gust B., Kulik A., Heide L., Buttner M. J., Bibb M. J. (2013). Phage p1-derived artificial chromosomes facilitate heterologous expression of the FK506 gene cluster. PLoS ONE 8:e69319. 10.1371/journal.pone.0069319 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Jones A. C., Ottilie S., Eustaquio A. S., Edwards D. J., Gerwick L., Moore B. S., et al. (2012). Evaluation of Streptomyces coelicolor A3(2) as a heterologous expression host for the cyanobacterial protein kinase C activator lyngbyatoxin A. FEBS J. 279, 1243–1251. 10.1111/j.1742-4658.2012.08517.x [DOI] [PubMed] [Google Scholar]
  38. Jordan P. A., Moore B. S. (2016). Biosynthetic pathway connects cryptic ribosomally synthesized posttranslationally modified peptide genes with pyrroloquinoline alkaloids. Cell Chem. Biol. 23, 1504–1514. 10.1016/j.chembiol.2016.10.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kaysser L., Bernhardt P., Nam S. J., Loesgen S., Ruby J. G., Skewes-Cox P., et al. (2012). Merochlorins, A.-D., cyclic meroterpenoid antibiotics biosynthesized in divergent pathways with vanadium-dependent chloroperoxidases. J. Am. Chem. Soc. 134, 11988–11991. 10.1021/ja305665f [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kharel M. K., Nybo S. E., Shepherd M. D., Rohr J. (2010). Cloning and characterization of the ravidomycin and chrysomycin biosynthetic gene clusters. Chembiochem 11, 523–532. 10.1002/cbic.200900673 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kim E. J., Lee J. H., Choi H., Pereira A. R., Ban Y. H., Yoo Y. J., et al. (2012). Heterologous production of 4-O-demethylbarbamide, a marine cyanobacterial natural product. Org. Lett. 14, 5824–5827. 10.1021/ol302575h [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kim J. H., Feng Z., Bauer J. D., Kallifidas D., Calle P. Y., Brady S. F. (2010). Cloning large natural product gene clusters from the environment: piecing environmental DNA gene clusters back together with TAR. Biopolymers 93, 833–844. 10.1002/bip.21450 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Koberska M., Kopecky J., Olsovska J., Jelinkova M., Ulanova D., Man P., et al. (2008). Sequence analysis and heterologous expression of the lincomycin biosynthetic cluster of the type strain Streptomyces lincolnensis ATCC 25466. Folia Microbiol. (Praha) 53, 395–401. 10.1007/s12223-008-0060-8 [DOI] [PubMed] [Google Scholar]
  44. Kudo F., Tsunoda T., Takashima M., Eguchi T. (2016). Five-membered cyclitol phosphate formation by a myo-inositol phosphate synthase orthologue in the biosynthesis of the carbocyclic nucleoside antibiotic aristeromycin. Chembiochem 17, 2143–2148. 10.1002/cbic.201600348 [DOI] [PubMed] [Google Scholar]
  45. Kwon H. J., Smith W. C., Xiang L., Shen B. (2001). Cloning and heterologous expression of the macrotetrolide biosynthetic gene cluster revealed a novel polyketide synthase that lacks an acyl carrier protein. J. Am. Chem. Soc. 123, 3385–3386. 10.1021/ja0100827 [DOI] [PubMed] [Google Scholar]
  46. Lacalle R. A., Tercero J. A., Jimenez A. (1992). Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts. EMBO J. 11, 785–792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Larionov V., Kouprina N., Eldarov M., Perkins E., Porter G., Resnick M. A. (1994). Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks. Yeast 10, 93–104. 10.1002/yea.320100109 [DOI] [PubMed] [Google Scholar]
  48. Li J., Guo Z., Huang W., Meng X., Ai G., Tang G., et al. (2013). Mining of a streptothricin gene cluster from Streptomyces sp. TP-A0356 genome via heterologous expression. Sci. China Life Sci. 56, 619–627. 10.1007/s11427-013-4504-2 [DOI] [PubMed] [Google Scholar]
  49. Li Q., Song Y., Qin X., Zhang X., Sun A., Ju J. (2015). Identification of the biosynthetic gene cluster for the anti-infective desotamides and production of a new analogue in a heterologous host. J. Nat. Prod. 78, 944–948. 10.1021/acs.jnatprod.5b00009 [DOI] [PubMed] [Google Scholar]
  50. Li T., Du Y., Cui Q., Zhang J., Zhu W., Hong K., et al. (2013). Cloning, characterization and heterologous expression of the indolocarbazole biosynthetic gene cluster from marine-derived Streptomyces sanyensis FMA. Mar. Drugs 11, 466–488. 10.3390/md11020466 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Liu C., Zhang J., Lu C., Shen Y. (2015). Heterologous expression of galbonolide biosynthetic genes in Streptomyces coelicolor. Antonie Van Leeuwenhoek 107, 1359–1366. 10.1007/s10482-015-0415-5 [DOI] [PubMed] [Google Scholar]
  52. Liu H., Jiang H., Haltli B., Kulowski K., Muszynska E., Feng X., et al. (2009). Rapid cloning and heterologous expression of the meridamycin biosynthetic gene cluster using a versatile Escherichia coli-Streptomyces artificial chromosome vector, pSBAC. J. Nat. Prod. 72, 389–395. 10.1021/np8006149 [DOI] [PubMed] [Google Scholar]
  53. Lopez P., Hornung A., Welzel K., Unsin C., Wohlleben W., Weber T., et al. (2010). Isolation of the lysolipin gene cluster of Streptomyces tendae Tu. Gene 461, 5–14. 10.1016/j.gene.2010.03.016 [DOI] [PubMed] [Google Scholar]
  54. Malcolmson S. J., Young T. S., Ruby J. G., Skewes-Cox P., Walsh C. T. (2013). The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds. Proc. Natl. Acad. Sci. U.S.A. 110, 8483–8488. 10.1073/pnas.1307111110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Malpartida F., Niemi J., Navarrete R., Hopwood D. A. (1990). Cloning and expression in a heterologous host of the complete set of genes for biosynthesis of the Streptomyces coelicolor antibiotic undecylprodigiosin. Gene 93, 91–99. 10.1016/0378-1119(90)90141-D [DOI] [PubMed] [Google Scholar]
  56. Mantovani S. M., Moore B. S. (2013). Flavin-linked oxidase catalyzes pyrrolizine formation of dichloropyrrole-containing polyketide extender unit in chlorizidine A. J. Am. Chem. Soc. 135, 18032–18035. 10.1021/ja409520v [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Marti T., Hu Z., Pohl N. L., Shah A. N., Khosla C. (2000). Cloning, nucleotide sequence, and heterologous expression of the biosynthetic gene cluster for R1128, a non-steroidal estrogen receptor antagonist. Insights into an unusual priming mechanism. J. Biol. Chem. 275, 33443–33448. 10.1074/jbc.M006766200 [DOI] [PubMed] [Google Scholar]
  58. Martinez A., Kolvek S. J., Tiong Yip C. L., Hopke J., Brown K. A., MacNeil I. A., et al. (2004). Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts. Appl. Environ. Microbiol. 70, 2452–2463. 10.1128/AEM.70.4.2452-2463.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Martinez-Burgo Y., Alvarez-Alvarez R., Perez-Redondo R., Liras P. (2014). Heterologous expression of Streptomyces clavuligerus ATCC 27064 cephamycin C gene cluster. J. Biotechnol. 186, 21–29. 10.1016/j.jbiotec.2014.06.002 [DOI] [PubMed] [Google Scholar]
  60. McClure R. A., Goering A. W., Ju K. S., Baccile J. A., Schroeder F. C., Metcalf W. W., et al. (2016). Elucidating the rimosamide-detoxin natural product families and their biosynthesis using metabolite/gene cluster correlations. ACS Chem. Biol. 11, 3452–3460. 10.1021/acschembio.6b00779 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Miao V., Coeffet-LeGal M., Brian P., Brost R., Penn J., Whiting A., et al. (2005). Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry. Microbiology 151(Pt 5), 1507–1523. 10.1099/mic.0.27757-0 [DOI] [PubMed] [Google Scholar]
  62. Miyamoto K. T., Komatsu M., Ikeda H. (2014). Discovery of gene cluster for mycosporine-like amino acid biosynthesis from Actinomycetales microorganisms and production of a novel mycosporine-like amino acid by heterologous expression. Appl. Environ. Microbiol. 80, 5028–5036. 10.1128/AEM.00727-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Montiel D., Kang H. S., Chang F. Y., Charlop-Powers Z., Brady S. F. (2015). Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters. Proc. Natl. Acad. Sci. U.S.A. 112, 8953–8958. 10.1073/pnas.1507606112 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Motamedi H., Hutchinson C. R. (1987). Cloning and heterologous expression of a gene cluster for the biosynthesis of tetracenomycin C, the anthracycline antitumor antibiotic of Streptomyces glaucescens. Proc. Natl. Acad. Sci. U.S.A. 84, 4445–4449. 10.1073/pnas.84.13.4445 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Nah H. J., Woo M. W., Choi S. S., Kim E. S. (2015). Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system. Microb. Cell Fact. 14, 140. 10.1186/s12934-015-0325-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Newman D. J., Cragg G. M. (2012). Natural products as sources of new drugs over the 30 years from 1981 to 2010. J. Nat. Prod. 75, 311–335. 10.1021/np200906s [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Niu G., Li L., Wei J., Tan H. (2013). Cloning, heterologous expression, and characterization of the gene cluster required for gougerotin biosynthesis. Chem. Biol. 20, 34–44. 10.1016/j.chembiol.2012.10.017 [DOI] [PubMed] [Google Scholar]
  68. Onaka H., Taniguchi S., Igarashi Y., Furumai T. (2002). Cloning of the staurosporine biosynthetic gene cluster from Streptomyces sp. TP-A0274 and its heterologous expression in Streptomyces lividans. J. Antibiot. (Tokyo) 55, 1063–10671. 10.7164/antibiotics.55.1063 [DOI] [PubMed] [Google Scholar]
  69. Ooya K., Ogasawara Y., Noike M., Dairi T. (2015). Identification and analysis of the resorcinomycin biosynthetic gene cluster. Biosci. Biotechnol. Biochem. 79, 1833–1837. 10.1080/09168451.2015.1050992 [DOI] [PubMed] [Google Scholar]
  70. Otsuka M., Ichinose K., Fujii I., Ebizuka Y. (2004). Cloning, sequencing, and functional analysis of an iterative type I polyketide synthase gene cluster for biosynthesis of the antitumor chlorinated polyenone neocarzilin in “Streptomyces carzinostaticus.” Antimicrob. Agents Chemother. 48, 3468–3476. 10.1128/AAC.48.9.3468-3476.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Park O. K., Choi H. Y., Kim G. W., Kim W. G. (2016). Generation of new complestatin analogues by heterologous expression of the complestatin biosynthetic gene cluster from Streptomyces chartreusis AN1542. Chembiochem 17, 1725–1731. 10.1002/cbic.201600241 [DOI] [PubMed] [Google Scholar]
  72. Robles-Reglero V., Santamarta I., Alvarez-Alvarez R., Martin J. F., Liras P. (2013). Transcriptional analysis and proteomics of the holomycin gene cluster in overproducer mutants of Streptomyces clavuligerus. J. Biotechnol. 163, 69–76. 10.1016/j.jbiotec.2012.09.017 [DOI] [PubMed] [Google Scholar]
  73. Rui Z., Huang W., Xu F., Han M., Liu X., Lin S., et al. (2015). Sparsomycin biosynthesis highlights unusual module architecture and processing mechanism in non-ribosomal peptide synthetase. ACS Chem. Biol. 10, 1765–1769. 10.1021/acschembio.5b00284 [DOI] [PubMed] [Google Scholar]
  74. Salem S. M., Kancharla P., Florova G., Gupta S., Lu W., Reynolds K. A. (2014). Elucidation of final steps of the marineosins biosynthetic pathway through identification and characterization of the corresponding gene cluster. J. Am. Chem. Soc. 136, 4565–4574. 10.1021/ja411544w [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Sanchez C., Butovich I. A., Brana A. F., Rohr J., Mendez C., Salas J. A. (2002). The biosynthetic gene cluster for the antitumor rebeccamycin: characterization and generation of indolocarbazole derivatives. Chem. Biol. 9, 519–531. 10.1016/S1074-5521(02)00126-6 [DOI] [PubMed] [Google Scholar]
  76. Saugar I., Molloy B., Sanz E., Blanca Sanchez M., Fernandez-Lobato M., Jimenez A. (2016). Characterization of the biosynthetic gene cluster (ata) for the A201A aminonucleoside antibiotic from Saccharothrix mutabilis subsp. capreolus. J. Antibiot. (Tokyo: ). [Epub ahead of print]. 10.1038/ja.2016.123 [DOI] [PubMed] [Google Scholar]
  77. Shah S., Xue Q., Tang L., Carney J. R., Betlach M., McDaniel R. (2000). Cloning, characterization and heterologous expression of a polyketide synthase and P-450 oxidase involved in the biosynthesis of the antibiotic oleandomycin. J. Antibiot. (Tokyo) 53, 502–508. 10.7164/antibiotics.53.502 [DOI] [PubMed] [Google Scholar]
  78. Shizuya H., Birren B., Kim U. J., Mancino V., Slepak T., Tachiiri Y., et al. (1992). Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. U.S.A. 89, 8794–8797. 10.1073/pnas.89.18.8794 [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Singh D., Seo M. J., Kwon H. J., Rajkarnikar A., Kim K. R., Kim S. O., et al. (2006). Genetic localization and heterologous expression of validamycin biosynthetic gene cluster isolated from Streptomyces hygroscopicus var. limoneus KCCM 11405 (IFO 12704). Gene 376, 13–23. 10.1016/j.gene.2005.12.035 [DOI] [PubMed] [Google Scholar]
  80. Sosio M., Giusino F., Cappellano C., Bossi E., Puglia A. M., Donadio S. (2000). Artificial chromosomes for antibiotic-producing actinomycetes. Nat. Biotechnol. 18, 343–345. 10.1038/73810 [DOI] [PubMed] [Google Scholar]
  81. Steffensky M., Muhlenweg A., Wang Z. X., Li S. M., Heide L. (2000). Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891. Antimicrob. Agents Chemother. 44, 1214–1222. 10.1128/AAC.44.5.1214-1222.2000 [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Su C., Zhao X., Qiu R., Tang L. (2015). Construction of the co-expression plasmids of fostriecin polyketide synthases and heterologous expression in Streptomyces. Pharm. Biol. 53, 269–274. 10.3109/13880209.2014.914956 [DOI] [PubMed] [Google Scholar]
  83. Tang L., Shah S., Chung L., Carney J., Katz L., Khosla C., et al. (2000). Cloning and heterologous expression of the epothilone gene cluster. Science 287, 640–642. 10.1126/science.287.5453.640 [DOI] [PubMed] [Google Scholar]
  84. Thanapipatsiri A., Gomez-Escribano J. P., Song L., Bibb M. J., Al-Bassam M., Chandra G., et al. (2016). Discovery of unusual biaryl polyketides by activation of a silent Streptomyces venezuelae biosynthetic gene cluster. Chembiochem 17, 2189–2198. 10.1002/cbic.201600396 [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Thapa L. P., Oh T. J., Lee H. C., Liou K., Park J. W., Yoon Y. J., et al. (2007). Heterologous expression of the kanamycin biosynthetic gene cluster (pSKC2) in Streptomyces venezuelae YJ003. Appl. Microbiol. Biotechnol. 76, 1357–1364. 10.1007/s00253-007-1096-4 [DOI] [PubMed] [Google Scholar]
  86. Torkkell S., Kunnari T., Palmu K., Mantsala P., Hakala J., Ylihonko K. (2001). The entire nogalamycin biosynthetic gene cluster of Streptomyces nogalater: characterization of a 20-kb DNA region and generation of hybrid structures. Mol. Genet. Genomics 266, 276–288. 10.1007/s004380100554 [DOI] [PubMed] [Google Scholar]
  87. Varshney R. K., Mir R. R., Bhatia S., Thudi M., Hu Y., Azam S., et al. (2014). Integrated physical, genetic and genome map of chickpea (Cicer arietinum L.). Funct. Integr. Genomics 14, 59–73. 10.1007/s10142-014-0363-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Waldman A. J., Pechersky Y., Wang P., Wang J. X., Balskus E. P. (2015). The cremeomycin biosynthetic gene cluster encodes a pathway for diazo formation. Chembiochem 16, 2172–2175. 10.1002/cbic.201500407 [DOI] [PMC free article] [PubMed] [Google Scholar]
  89. Wang J., Yu Y., Tang K., Liu W., He X., Huang X., et al. (2010). Identification and analysis of the biosynthetic gene cluster encoding the thiopeptide antibiotic cyclothiazomycin in Streptomyces hygroscopicus 10-22. Appl. Environ. Microbiol. 76, 2335–2344. 10.1128/AEM.01790-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
  90. Ward S. L., Hu Z., Schirmer A., Reid R., Revill W. P., Reeves C. D., et al. (2004). Chalcomycin biosynthesis gene cluster from Streptomyces bikiniensis: novel features of an unusual ketolide produced through expression of the chm polyketide synthase in Streptomyces fradiae. Antimicrob. Agents Chemother. 48, 4703–4712. 10.1128/AAC.48.12.4703-4712.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Wolpert M., Heide L., Kammerer B., Gust B. (2008). Assembly and heterologous expression of the coumermycin A1 gene cluster and production of new derivatives by genetic engineering. Chembiochem 9, 603–612. 10.1002/cbic.200700483 [DOI] [PubMed] [Google Scholar]
  92. Yamanaka K., Reynolds K. A., Kersten R. D., Ryan K. S., Gonzalez D. J., Nizet V., et al. (2014). Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc. Natl. Acad. Sci. U.S.A. 111, 1957–1962. 10.1073/pnas.1319584111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  93. Yan X., Probst K., Linnenbrink A., Arnold M., Paululat T., Zeeck A., et al. (2012). Cloning and heterologous expression of three type II PKS gene clusters from Streptomyces bottropensis. Chembiochem 13, 224–230. 10.1002/cbic.201100574 [DOI] [PubMed] [Google Scholar]
  94. Yang C., Huang C., Zhang W., Zhu Y., Zhang C. (2015). Heterologous expression of fluostatin gene cluster leads to a bioactive heterodimer. Org. Lett. 17, 5324–5327. 10.1021/acs.orglett.5b02683 [DOI] [PubMed] [Google Scholar]
  95. Yin J., Hoffmann M., Bian X., Tu Q., Yan F., Xia L., et al. (2015). Direct cloning and heterologous expression of the salinomycin biosynthetic gene cluster from Streptomyces albus DSM41398 in Streptomyces coelicolor A3(2). Sci. Rep. 5:15081. 10.1038/srep15081 [DOI] [PMC free article] [PubMed] [Google Scholar]
  96. Yin S., Li Z., Wang X., Wang H., Jia X., Ai G., et al. (2016). Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae WVR2006 to improve production level and to alter fermentation process. Appl. Microbiol. Biotechnol. 100, 10563–10572. 10.1007/s00253-016-7873-1 [DOI] [PubMed] [Google Scholar]
  97. Ylihonko K., Hakala J., Kunnari T., Mantsala P. (1996). Production of hybrid anthracycline antibiotics by heterologous expression of Streptomyces nogalater nogalamycin biosynthesis genes. Microbiology 142(Pt 8), 1965–1972. 10.1099/13500872-142-8-1965 [DOI] [PubMed] [Google Scholar]
  98. Young T. S., Walsh C. T. (2011). Identification of the thiazolyl peptide GE37468 gene cluster from Streptomyces ATCC 55365 and heterologous expression in Streptomyces lividans. Proc. Natl. Acad. Sci. U.S.A. 108, 13053–13058. 10.1073/pnas.1110435108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  99. Zaburannyi N., Rabyk M., Ostash B., Fedorenko V., Luzhetskyy A. (2014). Insights into naturally minimised Streptomyces albus J1074 genome. BMC Genomics 15:97. 10.1186/1471-2164-15-97 [DOI] [PMC free article] [PubMed] [Google Scholar]
  100. Zettler J., Xia H., Burkard N., Kulik A., Grond S., Heide L., et al. (2014). New aminocoumarins from the rare actinomycete Catenulispora acidiphila DSM 44928: identification, structure elucidation, and heterologous production. Chembiochem 15, 612–621. 10.1002/cbic.201300712 [DOI] [PubMed] [Google Scholar]
  101. Zhang G., Zhang H., Li S., Xiao J., Zhang G., Zhu Y., et al. (2012). Characterization of the amicetin biosynthesis gene cluster from Streptomyces vinaceusdrappus NRRL 2363 implicates two alternative strategies for amide bond formation. Appl. Environ. Microbiol. 78, 2393–2401. 10.1128/AEM.07185-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
  102. Zhang Y., Huang H., Chen Q., Luo M., Sun A., Song Y., et al. (2013). Identification of the grincamycin gene cluster unveils divergent roles for GcnQ in different hosts, tailoring the L-rhodinose moiety. Org. Lett. 15, 3254–3257. 10.1021/ol401253p [DOI] [PubMed] [Google Scholar]
  103. Zhu Y., Fu P., Lin Q., Zhang G., Zhang H., Li S., et al. (2012). Identification of caerulomycin A gene cluster implicates a tailoring amidohydrolase. Org. Lett. 14, 2666–2669. 10.1021/ol300589r [DOI] [PubMed] [Google Scholar]

Articles from Frontiers in Microbiology are provided here courtesy of Frontiers Media SA

RESOURCES