C H A P T E R

T W E N T Y- O N E

Synthetic Biology in Streptomyces Bacteria

Marnix H. Medema,*, Rainer Breitling,, and Eriko Takano* Contents
1. Synthetic Biology for Novel Compound Discovery in Streptomyces 2. Practical Considerations for Synthetic Biology in Streptomyces 2.1. Choice of host organism 3. Iterative Reengineering of Secondary Metabolite Gene Clusters 3.1. Transcriptional control engineering in Streptomyces 3.2. Translational control engineering in Streptomyces 4. The Molecular Toolbox for Streptomyces Synthetic Biology 5. Transcriptional Control 5.1. Inducible promoters 5.2. Constitutive promoters 5.3. Terminators 6. Translational Control 6.1. Positive translational control 6.2. Negative translational control 7. Vectors 7.1. Low copy number vectors Acknowledgments References 486 488 488 489 490 491 491 492 492 493 494 494 494 494 494 495 497 497

Abstract
Actinomycete bacteria of the genus Streptomyces are major producers of bioactive compounds for the biotechnology industry. They are the source of most clinically used antibiotics, as well as of several widely used drugs against common diseases, including cancer . Genome sequencing has revealed that the potential of Streptomyces species for the production of valuable secondary metabolites is even larger than previously realized. Accessing this rich genomic
* Department of Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Groningen Bioinformatics Centre, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands { Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow, United Kingdom
{

Methods in Enzymology, Volume 497 ISSN 0076-6879, DOI: 10.1016/B978-0-12-385075-1.00021-4

2011 Elsevier Inc. All rights reserved.

485

486

Marnix H. Medema et al.

resource to discover new compounds by activating cryptic pathways is an interesting challenge for synthetic biology. This approach is facilitated by the inherent natural modularity of secondary metabolite biosynthetic pathways, at the level of individual enzymes (such as modular polyketide synthases), but also of gene cassettes/operons and entire biosynthetic gene clusters. It also benefits from a long tradition of molecular biology in Streptomyces, which provides a number of specific tools, ranging from cloning vectors to inducible promoters and translational control elements. In this chapter, we first provide an overview of the synthetic biology challenges in Streptomyces and then present the existing toolbox of molecular methods that can be employed in this organism.

1. Synthetic Biology for Novel Compound Discovery in Streptomyces

The rapidly decreasing costs of genome sequencing have made genome mining the most promising source of raw material for drug discovery: a great number of putative secondary metabolite biosynthesis pathways have been discovered in silico (Medema et al., 2010). In genome-sequenced Streptomyces species (Dyson, 2011; Hopwood, 2007), the number of gene clusters encoding pathways for secondary metabolite biosynthesis has been found to range from about 20 to 50 per genome (Bentley et al., 2002; Ikeda et al., 2003; Medema et al., 2010; Ohnishi et al., 2008; Wang et al., 2010). Intriguingly, a large number of these pathways are cryptic: they are not expressed under standard laboratory conditions, and their products are therefore unknown. Exciting proof-of-principle successes have already been achieved in awakening cryptic secondary metabolites. Untargeted approaches have been used to randomly awaken some clusters (Tala et al., 2009), and recently a targeted approach has resulted in the specific awakening of a cryptic gene cluster in Streptomyces coelicolor by inactivating a pathway-specific repressor within the cluster (Gottelt et al., 2010). However, a key limitation to these approaches is the difficulty of implementing a high-throughput screening of the hundreds of cryptic gene clusters that are available in the databases. Synthetic biology may offer the possibility of achieving the large-scale reengineering of the native regulation of these clusters, as well as the directed modification of their metabolic network context that is necessary for successful novel compound identification at a rapid rate (Medema et al., 2011). One advantage of a synthetic biology approach would be the option to completely remove the native regulation of the cryptic clusters, replacing it by a completely synthetic regulation that is predictable, easy to manipulate, as specifically tuned to the function of the pathway. To achieve this, the

Streptomyces Synthetic Biology

487

cryptic gene clusters need to be completely redesigned in silico in terms of promoters, composition of the transcriptional units, ribosome-binding sites (RBSs), and possibly even their codon usage (Bayer et al., 2009; Salis et al., 2009; Widmaier et al., 2009). Streptomyces biology is in a good position for achieving this ambitious aim, given the existing body of research into regulatory mechanisms (Martin and Liras, 2010), as will be further discussed in the methods section below. As the gene clusters encoding secondary metabolite biosynthetic pathways of many compound classes are often very similar to one another and largely consist of the same biosynthetic modules combined in different ways (Fischbach et al., 2008), a synthetic version of one optimized model gene cluster could serve as a chassis for an entire class of cryptic gene clusters, which could be inserted into it in their entirety instead of being modified piece-by-piece. This would enable the rapid and versatile activation of new biosynthetic pathways from a variety of sources. It has the advantage that it achieves high throughput, while at the same time being focused on evolutionarily optimized designs present in naturally occurring cryptic gene clusters. As these have been selected by evolution to exhibit a specific and stable bioactivity, the probability of finding novel antibacterial activities in the resulting compound libraries is much higher than in those produced by more random approaches (Zerikly and Challis, 2009). This approach can be complemented by other forms of (semi-)synthetic biology in Streptomyces, which aim at exploiting the highly modular assembly line mechanism of secondary metabolite biosynthetic pathways (Fischbach and Walsh, 2006) to create new chemical diversity from wellcharacterized gene clusters (Gokhale et al., 1999; Khosla and Zawada, 1996; Menzella et al., 2005, 2007) or to modify the chemistry of the end product for increased efficacy or novel functionality (Baltz, 2008; Caffrey et al., 2008; Donadio and Sosio, 2008; Heide et al., 2008). These combinatorial biosynthesis and biosynthetic engineering methods can be used quite independently from the ambitious redesign (and synthesis) of entire gene clusters described in this chapter. They have been reviewed before (Luzhetska et al., 2010; Menzella and Reeves, 2007; Walsh, 2002; Zhang and Tang, 2008) and are not further discussed here. A sensible target for pioneering this approach is a class of antibiotic compounds called polyketides that are synthesized by so-called type II polyketide synthases (PKSs) (Hertweck et al., 2007). Among the polyketides produced by type II PKSs are chemically very diverse bioactive compounds, including tetracycline antibiotics, anthracycline chemotherapeutics (e.g., daunomycin and doxorubicin), angucyclines with a wide range of antibiotic and antitumor activities (e.g., landomycin), and the benzoisochromanequinones, which include the widely studied antibiotic actinorhodin from S. coelicolor (Fig. 21.1) (Hertweck et al., 2007).

488

Marnix H. Medema et al.

B A 1

2 VI

2 3 VA

6 R

A II

III

1 I

3 VII IV VB

Minimal PKS Aromatase/cyclase Ketoreductase

Regulation/export SARP transcriptional activator Tailoring enzymes

Figure 21.1 The actinorhodin gene cluster, an example of a well-studied type II PKS polyketide biosynthetic gene cluster. Genes are annotated using different patterns as indicated.

The exclusively bacterial type II PKSs are single-domain proteins that form a complex that acts in an iterative fashion to produce the polyketide scaffold that can afterward be modified by a variety of accessory enzymes (Hertweck et al., 2007). The gene clusters containing type II PKSs are smaller than most other secondary metabolite biosynthetic clusters, making a synthetic approach as outlined above particularly feasible. Moreover, the genetic parts of type II PKS systems, including the post-PKS tailoring reactions, have been shown to be generally interchangeable (Ichinose et al., 2001) and combinable (Hopwood et al., 1985; Khosla and Zawada, 1996; McDaniel et al., 1995) to produce functional compounds. Finally, more than one hundred cryptic gene clusters of this type are currently present in the databases, and this number is still increasing rapidly. Consequently, these gene clusters offer great potential for drug discovery.

2. Practical Considerations for Synthetic Biology in Streptomyces

2.1. Choice of host organism
Certain biosynthetic enzymes, among which bacterial type II PKSs, are not functional in Escherichia coli (Zhang et al., 2008), while the metabolism of Streptomyces bacteria is already optimized for the production of secondary metabolites at high rates. Therefore, the host organism of choice for most synthetic biology projects on genome-based drug discovery would be a Streptomyces species, such as the model streptomycete S. coelicolor, or a closely related actinomycete bacterium used in biotechnological applications. Useful host strains for the heterologous expression of different types of secondary metabolite gene clusters have been reviewed in detail by Baltz (2010). Starting from the model organism S. coelicolor not only has the advantage of being able to use a wide range of existing molecular tools, but additionally

Streptomyces Synthetic Biology

489

a strain of this species is available in which all four highly active (noncryptic) antibiotic biosynthesis gene clusters (actinorhodin: act; undecylprodigiosin: red; calcium dependent antibiotic: cda; coelicolorpolyketide: cpk) have been deleted from the chromosome, freeing metabolic resources for the synthetic pathways that are inserted (Gottelt et al., 2010; GomezEscribano et al., 2011). Alternatively, comprehensively genome-minimized strains would be interesting hosts, such as the recently published genomeminimized Streptomyces avermitilis strains (Komatsu et al., 2010) or a plasmidcured strain of Streptomyces clavuligerus, as was recently suggested (Medema et al., 2010).

3. Iterative Reengineering of Secondary Metabolite Gene Clusters

As our understanding of gene regulation in Streptomyces is not yet detailed enough to perfectly predict the functioning of all components in an integrated pathway, synthesizing and inserting a whole gene cluster at once is very likely to result in problems that cannot easily be traced to a particular gene. It is, therefore, more promising to use an iterative strategy in which the target gene cluster is subdivided into independent transcriptional units that are individually optimized. After the first design and synthesis of a transcriptional unit, it can be tested for complementation of a deletion mutant of the same genes in the native pathway, by inserting it into the S. coelicolor chromosome at the phiC31 or phiBT1 sites (see below). If this step is unsuccessful, the problem can be traced and the design adapted until a successful complementation of pathway functionality is achieved. Initial proof-of-concept studies can focus on pigmented compounds, such as the actinorhodin antibiotic of S. coelicolor. In that way, the debugging or troubleshooting process can be aided by photospectrometry to rapidly identify eventual blocks in the biosynthetic pathway, as many intermediates or shunt products produced by knock-out mutants of tailoring genes show absorption spectra distinct from the end product. For instance, in the case of actinorhodin, which has a dark blue color (Fig. 21.2), actVI-orf1 or ActVI-orf2 mutants produce a brown pigment (Taguchi et al., 2000), actVIorf3 mutants produce a reddish pigment (Taguchi et al., 2000), and actVAorf5,6 mutants produce a yellowish brown pigment (Okamoto et al., 2009). A second blue pigment, gamma-actinorhodin, is also known to be produced by the same gene cluster (Bystrykh et al., 1996). When photospectrometry is not informative, as will be the case for most bioactive compounds targeted in high-throughput genome mining approaches, it will still be possible to predict the structures of most pathway intermediates based on genome annotation (Aoki-Kinoshita and Kanehisa,

490
A
ORF1

Marnix H. Medema et al.

ORF3

wt

actVI-orf1

actVI-orf3

actVA -orf5,6

actVAorf6

Figure 21.2 (A) Brown pigment produced by actVI-orf1 mutant (K. Ichinose, personal communication). (B) Red pigment produced by actVI-orf3 mutant (K. Ichinose personal communication). (C) Yellowish brown pigment produced by the actVA-orf5,6 mutant compared to the blue actinorhodin produced by wt and the actVA-orf6 mutant (taken from Okamoto et al., 2009 with permission from Elsevier Limited).

2009). In these cases, high-accuracy liquid chromatography mass-spectrometry can be a promising tool for identifying metabolic signatures that characterize bottlenecks at specific steps in the biosynthetic pathway (Kol et al., 2010).

3.1. Transcriptional control engineering in Streptomyces

While the individual transcriptional units are optimized independently, the synthetic operons can all be controlled by thiostrepton-inducible tipA promoters (Takano et al., 1995) (see below), and phage fd bidirectional terminators which are functional both in E. coli and Streptomyces can be used for transcription termination (Ward et al., 1986) (see below). However, once the complete synthetic gene cluster is assembled, one would most likely need to control the timing and expression rate of some operons separately. For instance, in our example of type II PKS engineering, we might want to control the transcriptional unit encoding the core PKS proteins independently of those encoding the tailoring steps. This would especially be advantageous when genes from cryptic pathways will be inserted in these units later on, which may well need a different mRNA expression stoichiometry compared to the initial model cluster. In this case, at least two promoters with different timing and strength would be required. The use of inducible promoters would have the advantage that one can start at low induction rates to avoid build-up of toxic intermediates, and increase induction later. Concomitant expression of the cluster-specific secondary metabolite transporters will also be required for toxic compounds. In the wild-type gene cluster, expression of the transporter genes is often governed by an intricate system: in our example of the actinorhodin gene cluster, repression of actinorhodin transporter expression by ActR is abolished by binding of ActR to intermediates in the biosynthetic pathway. In this way the

Streptomyces Synthetic Biology

491

transporters will be produced just in time to avoid bacterial suicide (Tahlan et al., 2007, 2008; Willems et al., 2008). It is expected that by simultaneously expressing the tailoring genes and the transporters, toxic effects to the cell can be avoided in a similar fashion. Yet, if toxicity problems arise due to lack of transport capacity, it can also be appropriate to insert a strong constitutive promoter, such as ermE (see below), in front of the transporter genes.

3.2. Translational control engineering in Streptomyces

As the translational efficiency of redesigned synthetic genes will be different from those of the wild-type genes if the wild-type RBSs are usedRBS functionality is context-dependent (Salis et al., 2009) and codon usage also affects translational efficiencynew synthetic RBSs have to be designed to restore the wild-type stoichiometry of the enzymes. This requires an accurate estimate of the relative wild-type translation rates in each operon. This can be obtained, for example, by fusing the relevant RBS-containing sequence of the wild-type gene to a GFP or RFP reporter in a high-copy number plasmid, such as pTONA5 (Hatanaka et al., 2008) or pIJ8630 (de Jong et al., 2009), with a constitutive promoter in front of it; screening for activity is then easily done by measuring the resulting fluorescence. The necessary synthetic RBSs can be identified in the same way, using a library of Streptomyces RBSs in the context of each synthetic gene based on oligonucleotides randomized around known RBS sequences. Subsequently, the translation rates of all proteins in the synthetic cluster can be balanced by inserting RBSs from this library that closely match the wild-type RBS strength. Using this methodology, RBSs can even be constructed to match translation efficiencies of those unconventional genes in Streptomyces that do not have RBSs/UTRs and for which translation starts at the far 50 end of the transcript at the transcription start site itself (Fernandez-Moreno et al., 1994; Strohl, 1992).

4. The Molecular Toolbox for Streptomyces Synthetic Biology

Fifty years of genetics and molecular biology in Streptomyces have yielded a large and versatile collection of molecular tools, many of which will be useful for synthetic biology applications. The list below provides concise descriptions of some important sets of tools, including the various components discussed in the text above. For detailed protocols on the stateof-art of Streptomyces molecular biology and secondary metabolite biosynthesis see the books by Kieser et al. (2000), Dyson (2011), and Hopwood (2009a,b).

492

Marnix H. Medema et al.

5. Transcriptional Control
The transcriptional control of gene expression in Streptomyces is different from most organisms. Consequently, commercially available tools and kits cannot be utilized readily. Particularly, the high genomic GC content of streptomycetes and the presence of multiple unusual enzymes hinder their use. For example, lacZ promoters cannot be used efficiently for screening, although a modified lacZ-based system was developed and used for some time (King and Chater, 1986). The reason for the incompatibility is that there are multiple endogenous beta-galactosidase homologues (five in total) encoded in the genome of S. coelicolor. Furthermore, IPTG was not transported into the cell; instead, methylumbelliferyl B galactoside had to be used (King and Chater, 1986). Recently a modified T7 expression system in Streptomyces has been reported. However, this system requires a T7 expression strain harbouring the T7 RNA polymerase under the control of a tipA promoter (Lussier et al., 2010) (see below). This expression strain is not favorable for use due to the T7 polymerase induction using thiostrepton, but a derivative may be useful in the future. Severalinducible and constitutivepromoters are available and some are listed below.

5.1. Inducible promoters

tipA: induced by thiostrepton. Most commonly used promoter. Very strong promoter, induced with minimum of 5 mg/ml of thiostrepton (Takano et al., 1995). It has several disadvantages: (1) Thiostrepton is only soluble in DMSO, which can result in inaccurate concentration of thiostrepton in the medium, but thiostrepton can potentially be replaced by water soluble derivates (Schoof et al., 2010). (2) The repression of the promoter is weak and it can therefore be expressed without any induction (due to the induction mechanism of the tipA protein; Chiu et al., 1999). The strain used needs to contain a thiostrepton resistance gene. cpkO: Induced by Streptomyces coelicolor gamma-butyrolactones (SCBs). Tightly controlled and relatively strong promoter. Promoter activity responds well to the inducer concentration (Fig. 21.3). Only nanomolar concentrations of inducer needed for induction. Inducer diffuses into the cell and is non-toxic. Hosts mutated in the inducer biosynthesis pathway are available. No need to introduce extra resistant gene cassettes (Takano et al., 2005). tetR: Induced by tetracycline (Tc) and anhydrotetracycline (aTc). Promoter was synthesized by combining the 10 and 35 regions of the strong ermEp1 promoter (Bibb et al., 1985) with the tetO1 and tetO2 operator sequences from the E. coli transposon Tn10. Promoter activity

Streptomyces Synthetic Biology

493

chemically synthesized SCB1 6.25 1.25 0.25 0.05 0.025 0.001 mg

Figure 21.3 The strength of the cpkO promoter shown by resistance to kanamycin resulting in the growth of the indicator strain (adapted from Hsiao et al., 2009 with permission from Elsevier Limited).

A
50.0 35.0 30.0 25.0 20.0

B
2.2 2.0 1.8

Induction factor

log10 (OD 200)

0.001 0.01 0.1 1.0 2.0

15.0 10.0 6.0

1.6 1.4 1.2 1.0 0.8 0.6 0.4

3.0

1.5 1.0

0.2 0.0 0 4 8 12 16 20 24 28 32 36

aTc concentration (mg/ml)

Growth period (h)

Figure 21.4 (A) Increase of promoter strength in relation to the concentration of aTc. (B) Streptomyces growth in the presence of different amounts of inducer. Inducer was added at OD492 0.04. Solid line, cultures without inducer; long dashed line, 1 mg/ml aTc; dotted line, 1 mg/ml Tc; dot and dashed line 1 mg/ml doxycyline (adapted from Rodrguez-Garca et al. (2005), with permission from Oxford University Press).

has a good response to the concentration of the inducer (Fig. 21.4). aTc is not toxic to the cell (Dangel et al., 2010; Rodriguez-Garcia et al., 2005).

5.2. Constitutive promoters

ermE*: The promoter of the erythromycin resistance gene (ermE) which is reported to be constitutive. Commonly used promoter for strong expression. One base pair mutation was introduced which enhanced promoter activity (Bibb et al., 1985).

494

Marnix H. Medema et al.

5.3. Terminators
Terminators from other organisms are used often in Streptomyces and work efficiently. The following are the most commonly used: Fd: the major terminator from E. coli phage fd, bidirectional (Ward et al., 1986), and lambda phage T0 terminator (Scholtissek and Grosse, 1987).

6. Translational Control
6.1. Positive translational control
Not much engineering of RBSs has been done in Streptomyces compared to the extensive redesigning employed in E. coli (Salis et al., 2009). However, RBSs from proteins that are known to be highly expressed, for example, ribosomal proteins, are used (Takano et al., 1995). A synthetic RBS with the sequence AAGGAGG has also been implemented successfully (Horinouchi et al., 1987).

6.2. Negative translational control

Antisense RNA (asRNA) is a recently developed addition to the Streptomyces regulatory toolbox. Applications use 50100 bp of sequence antisense to coding sequence of the gene which needs to be regulated. The antisense sequence can be cloned into a multicopy plasmid under the control of an inducible promoter, for example, tipA, for regulated repression (DAlia et al., 2010).

7. Vectors
The most widely used vectors in Streptomyces molecular biology are self-replicating plasmids, but integrating vectors are also available and can be particularly suitable for synthetic biology applications. The major advantage of the integrating vectors is their stability, especially when introducing large inserts. There are two attachment sites that can be used for the integration, either the phiC31 or the phiBT1 attachment site. These sites have been shown to be at different locations of the chromosome, phiC31 inside SCO3798 (putative chromosome condensation protein) and phiBT1 inside SCO4848 (putative membrane protein) (Combes et al., 2002; Gregory et al., 2003). This is convenient when it is

Streptomyces Synthetic Biology

495

desired to introduce multiple genetic constructs by integration into Streptomyces. Another important technique is the introduction of plasmids via conjugation. Earlier, chemical transformation using protoplasts was the main method of introducing DNA. However, the transformation frequency was often very low (hindering cloning of very large inserts), and the process often encouraged recombination within the chromosome causing unwanted mutations to the host. The conjugation method developed by Flett et al. (1997) allows easy cloning in E. coli. Finished constructs can then be transferred in a dam/ dcm E. coli strain (i.e., ET12567) that lacks a restriction modification system and carries a plasmid, pUZ8002, for conjugal transfer to Streptomyces by just mixing the E. coli with spores of Streptomyces. For cloning of very large fragments, especially the antibiotic biosynthetic clusters, which are often more than 100 kb in length, cosmids or artificial bacterial chromosomes (BACs) are used. These large vectors are nowadays all introduced via conjugation. Some vectors introduced to Streptomyces by both methods are listed below.

7.1. Low copy number vectors

Self-replicating vectors: Derivatives of SCP2*, the low copy number vector isolated from S. coelicolor. Inserts can be very large (>10 kb), for example, pRM5, an E. coli bifunctional vector, and can be introduced into Streptomyces by conjugation from E. coli (McDaniel et al., 1993). Integrative vectors: In theory, the insertion of the plasmid is thought to be as a single copy; however, often multiple copies can integrate into the same site (Takano et al., unpublished observations). To reduce multiple integration, the conjugation protocol has been revised so that the E. coli is grown with the Streptomyces for only 68 h. The following integrative vectors are used for medium insert sizes (< 8 kb) (Fig. 21.5). pSET152: Integrates into phiC31 attachment site (attP), apramycin resistant (aac(3)IV), has E. coli ori (Bierman et al., 1992). GenBank: AJ414670. pMS82: Integrates into phiBT1 attachment site (attP), uses hygromycin resistance (hyg) (Gregory et al., 2003). pIJ10257: Integrates into phiBT1 attachment site (attP), constitutive ermE* promoter in front of the cloning site (Hong et al., 2005). Based on pMS82 (Gregory et al., 2003). pIJ6902: Integrates into phiC31 attachment site, apramycin resistant (aac(3) IV) and thiostrepton resistant (thio), tipA inducible promoter in front of

496

Marnix H. Medema et al.

A
XbaI BamHI EcoRV EcoRI

B
AgeI

SpeI

EcoRV PvuII

AlwNI

lacZa 6000 int 5000 1000 int 5000 1000 BlnI BamHI HindIII BfrBI NsiI Acc65I KpnI Bsu36I

pMS82
6108 bps
2000

Van91I NruI

pSET152
4000

5715 bps
2000 attp NcoI SphI aac(3)IV oriT 3000

4000 3000

hyg

attp

oriT SphI XhoI MscI PmlI

SpeI

EcoRV PvuII

D
AlwNI

BglII EcoRI Acc65I KpnI BamHI XbaI NdeI DraIII

int 6000 1000 5000 PciI

pIJ10257
6672 bps
2000 hyg 3000

BsaI 7000 int 1000 6000

Alw44I AlwNI

NcoI SphI

4000 oriT

pIJ6902
7340 bps
2000 aac (3)IV

5000 3000

MscI BlnI BamHI HindIII PacI XhoI NdeI AleI Bsu36I Acc65I KpnI AsiSI PvuI

NruI Van91I

AhdI

4000

tsr SgrAI ApaI PspOMI MscI

Figure 21.5 Map of (A) pSET152 (GenBank: AJ414670), (B) pMS82 (M. C. Smith personal communication), (C) pIJ10257 (H.J. Hong personal communication), and (D) pIJ6902 (GenBank: AJ937361).

cloning site flanked with to/fd termintors (Huang et al., 2005). GenBank: AJ937361. The following vectors are suitable for large insertion sizes (<100 kb) (Fig. 21.6): BACs: pSBAC, integrates into phiBT1 attachment site (attP-int). Has the ori2 to replicate in E. coli in a single copy to maintain stability of the large insert and the copy number can be induced by L-arabinose for isolation of DNA. This plasmid has been used to clone the 90 kb meridamycin biosynthetic gene cluster (Liu et al., 2009).

Streptomyces Synthetic Biology

497

SphI KpnI KpnI EcoRI SacI

PvuII

rep

10,000 AmR SacI PstI oriT 6000 PstI DraI DraI 4000 cos 8000 pOJ446 10400 bps 2000

SmaI NdeI AatII NheI ClaI XbaI EcoRV BamHI SpeI

rep cos cos HpaI DraI

DraI PvuII BglII

DraI

Figure 21.6

Map of pOJ446 (adapted from Bierman et al., 1992).

Cosmids: pOJ446, can be used in both E. coli and Streptomyces (Bierman et al., 1992). There are many examples of cloning large fragments of 4070 kb (Kharel et al., 2010). pKC505, a useful cosmid-based shuttle vector for cloning of large constructs (Richardson et al., 1987), which can be conjugally transferred between Streptomyces strains.

ACKNOWLEDGMENTS
We thank D. Hopwood for critical reading of a draft of the manuscript and K. Ichinose, M.C.M. Smith, and H.J. Hong for providing figures and sequence information. This work was supported by the Dutch Technology Foundation STW, which is the applied science division of NWO, and the Technology Programme of the Ministry of Economic Affairs [STW 10463]. R. B. is supported by an NWO-Vidi fellowship, and E. T. by a Rosalind Franklin Fellowship, University of Groningen.

REFERENCES
Aoki-Kinoshita, K. F., and Kanehisa, M. (2009). Using KEGG in the transition from genomics to chemical genomics. In Bioinformatics for Systems Biology, (S. Krawetz, ed.), pp. 437452. Humana Press, Heidelberg.

498

Marnix H. Medema et al.

Baltz, R. H. (2008). Biosynthesis and genetic engineering of lipopeptide antibiotics related to daptomycin. Curr. Top. Med. Chem. 8, 618638. Baltz, R. H. (2010). Streptomyces and Saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters. J. Ind. Microbiol. Biotechnol. 37, 759772. Bayer, T. S., Widmaier, D. M., Temme, K., Mirsky, E. A., Santi, D. V., and Voigt, C. A. (2009). Synthesis of methyl halides from biomass using engineered microbes. J. Am. Chem. Soc. 131, 65086515. Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M., Challis, G. L., Thomson, N. R., James, K. D., Harris, D. E., Quail, M. A., Kieser, H., and Harper, D. (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3 (2). Nature 417, 141147. Bibb, M. J., Janssen, G. R., and Ward, J. M. (1985). Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus. Gene 38, 215226. Bierman, M., Logan, R., OBrien, K., Seno, E. T., Rao, R. N., and Schoner, B. E. (1992). Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.. Gene 116, 4349. Bystrykh, L. V., Fernandez-Moreno, M. A., Herrema, J. K., Malpartida, F., Hopwood, D. A., and Dijkhuizen, L. (1996). Production of actinorhodin-related blue pigments by Streptomyces coelicolor A3(2). J. Bacteriol. 178, 22382244. Caffrey, P., Aparicio, J. F., Malpartida, F., and Zotchev, S. B. (2008). Biosynthetic engineering of polyene macrolides towards generation of improved antifungal and antiparasitic agents. Curr. Top. Med. Chem. 8, 639653. Chiu, M. L., Folcher, M., Katoh, T., Puglia, A. M., Vohradsky, J., Yun, B. S., Seto, H., and Thompson, C. J. (1999). Broad spectrum thiopeptide recognition specificity of the Streptomyces lividans TipAL protein and its role in regulating gene expression. J. Biol. Chem. 274, 2057820586. Combes, P., Till, R., Bee, S., and Smith, M. C. (2002). The Streptomyces genome contains multiple pseudo-attB sites for the (phi)C31-encoded site-specific recombination system. J. Bacteriol. 184, 57465752. DAlia, D., Nieselt, K., Steigele, S., Muller, J., Verburg, I., and Takano, E. (2010). Noncoding RNA of glutamine synthetase I modulates antibiotic production in Streptomyces coelicolor A3(2). J. Bacteriol. 192, 11601164. Dangel, V., Westrich, L., Smith, M. C., Heide, L., and Gust, B. (2010). Use of an inducible promoter for antibiotic production in a heterologous host. Appl. Microbiol. Biotechnol. 87, 261269. de Jong, W., Manteca, A., Sanchez, J., Bucca, G., Smith, C. P., Dijkhuizen, L., Claessen, D., and Wosten, H. A. (2009). NepA is a structural cell wall protein involved in maintenance of spore dormancy in Streptomyces coelicolor. Mol. Microbiol. 71, 15911603. Donadio, S., and Sosio, M. (2008). Biosynthesis of glycopeptides: Prospects for improved antibacterials. Curr. Top. Med. Chem. 8, 654666. Dyson, P, ed. (2011). Streptomyces: Molecular Biology and Biotechnology. Caister Academic Press, Norwich. Fernandez-Moreno, M. A., Martinez, E., Caballero, J. L., Ichinose, K., Hopwood, D. A., and Malpartida, F. (1994). DNA sequence and functions of the actVI region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(2). J. Biol. Chem. 269, 2485424863. Fischbach, M. A., and Walsh, C. T. (2006). Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: Logic, machinery, and mechanisms. Chem. Rev. 106, 34683496.

Streptomyces Synthetic Biology

499

Fischbach, M. A., Walsh, C. T., and Clardy, J. (2008). The evolution of gene collectives: How natural selection drives chemical innovation. Proc. Natl. Acad. Sci. USA 105, 46014608. Flett, F., Mersinias, V., and Smith, C. P. (1997). High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol. Lett. 155, 223229. Gokhale, R. S., Tsuji, S. Y., Cane, D. E., and Khosla, C. (1999). Dissecting and exploiting intermodular communication in polyketide synthases. Science 284, 482485. Gomez-Escribano, J. P., and Bibb, M. J. (2011). Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters. Microb. Biotechnol. 4, 207215. Gottelt, M., Kol, S., Gomez-Escribano, J. P., Bibb, M., and Takano, E. (2010). Deletion of a regulatory gene within the cpk gene cluster reveals novel antibacterial activity in Streptomyces coelicolor A3(2). Microbiology 156, 23432353. Gregory, M. A., Till, R., and Smith, M. C. (2003). Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors. J. Bacteriol. 185, 53205323. Hatanaka, T., Onaka, H., Arima, J., Uraji, M., Uesugi, Y., Usuki, H., Nishimoto, Y., and Iwabuchi, M. (2008). pTONA5: A hyperexpression vector in streptomycetes. Protein Expr. Purif. 62, 244248. Heide, L., Gust, B., Anderle, C., and Li, S. M. (2008). Combinatorial biosynthesis, metabolic engineering and mutasynthesis for the generation of new aminocoumarin antibiotics. Curr. Top. Med. Chem. 8, 667679. Hertweck, C., Luzhetskyy, A., Rebets, Y., and Bechthold, A. (2007). Type II polyketide synthases: Gaining a deeper insight into enzymatic teamwork. Nat. Prod. Rep. 24, 162190. Hong, H. J., Hutchings, M. I., Hill, L. M., and Buttner, M. J. (2005). The role of the novel Fem protein VanK in vancomycin resistance in Streptomyces coelicolor. J. Biol. Chem. 280, 1305513061. Hopwood, D. A. (2007). Streptomyces in Nature and Medicine: The Antibiotic Makers. Oxford University Press, New York. Hopwood, D. A, ed. (2009a). Complex enzymes in microbial natural product biosynthesis, part A: Overview Articles and Peptides. Methods Enzymol. 458. Hopwood, D. A, ed. (2009b). Complex enzymes in microbial natural product biosynthesis, part B: Polyketides, aminocoumarins and carbohydrates. Methods Enzymol. 459. Hopwood, D. A., Malpartida, F., Kieser, H. M., Ikeda, H., Duncan, J., Fujii, I., Rudd, B. A., Floss, H. G., and Omura, S. (1985). Production of hybrid antibiotics by genetic engineering. Nature 314, 642644. Horinouchi, S., Furuya, K., Nishiyama, M., Suzuki, H., and Beppu, T. (1987). Nucleotide sequence of the streptothricin acetyltransferase gene from Streptomyces lavendulae and its expression in heterologous hosts. J. Bacteriol. 169, 19291937. Hsiao, N. H., Nakayama, S., Merlo, M. E., de Vries, M., Bunet, R., Kitani, S., Nihira, T., and Takano, E. (2009). Analysis of two additional signalling molecules in Streptomyces coelicolor and development of a butyrolactone-specific reporter system. Chem. Biol. 16, 951960. Huang, J., Shi, J., Molle, V., Sohlberg, B., Weaver, D., Bibb, M. J., Karoonuthaisiri, N., Lih, C. J., Kao, C. M., Buttner, M. J., and Cohen, S. N. (2005). Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor. Mol. Microbiol. 58, 12761287. Ichinose, K., Taguchi, T., Bedford, D. J., Ebizuka, Y., and Hopwood, D. A. (2001). Functional complementation of pyran ring formation in actinorhodin biosynthesis in Streptomyces coelicolor A3(2) by ketoreductase genes for granaticin biosynthesis. J. Bacteriol. 183, 32473250. Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, Y., Hattori, M., and Omura, S. (2003). Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat. Biotechnol. 21, 526531.

500

Marnix H. Medema et al.

Kharel, M. K., Nybo, S. E., Shepherd, M. D., and Rohr, J. (2010). Cloning and characterization of the ravidomycin and chrysomycin biosynthetic gene clusters. Chembiochem. 11, 523532. Khosla, C., and Zawada, R. J. (1996). Generation of polyketide libraries via combinatorial biosynthesis. Trends Biotechnol. 14, 335341. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A. (2000). Practical Streptomyces Genetics. The John Innes Foundation, Norwich. King, A. A., and Chater, K. F. (1986). The expression of the Escherichia coli lacZ gene in Streptomyces. J. Gen. Microbiol. 132, 17391752. Kol, S., Merlo, M. E., Scheltema, R. A., De, V. M., Vonk, R. J., Kikkert, N. A., Dijkhuizen, L., Breitling, R., and Takano, E. (2010). Metabolomic characterization of the salt stress response in Streptomyces coelicolor. Appl. Environ. Microbiol. 76, 25742581. Komatsu, M., Uchiyama, T., Omura, S., Cane, D. E., and Ikeda, H. (2010). Genomeminimized Streptomyces host for the heterologous expression of secondary metabolism. Proc. Natl. Acad. Sci. USA 107, 26462651. Liu, H., Jiang, H., Haltli, B., Kulowski, K., Muszynska, E., Feng, X., Summers, M., Young, M., Graziani, E., Koehn, F., Carter, G. T., and He, M. (2009). Rapid cloning and heterologous expression of the meridamycin biosynthetic gene cluster using a versatile Escherichia coliStreptomyces artificial chromosome vector, pSBAC. J. Nat. Prod. 72, 389395. Lussier, F. X., Denis, F., and Shareck, F. (2010). Adaptation of the highly productive T7 expression system to Streptomyces lividans. Appl. Environ. Microbiol. 76, 967970. Luzhetska, M., Harle, J., and Bechthold, A. (2010). Combinatorial and synthetic biosynthesis in actinomycetes. Fortschr. Chem. Org. Naturst. 93, 211237. Martin, J. F., and Liras, P. (2010). Engineering of regulatory cascades and networks controlling antibiotic biosynthesis in Streptomyces. Curr. Opin. Microbiol. 13, 263273. McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., and Khosla, C. (1993). Engineered biosynthesis of novel polyketides. Science 262, 15461550. McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., and Khosla, C. (1995). Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits. Nature 375, 549554. Medema, M. H., Trefzer, A., Kovalchuk, A., van den Berg, M., Muller, U., Heijne, W., Wu, L., Alam, M. T., Ronning, C. M., Nierman, W. C., Bovenberg, R. A. L., Breitling, R., et al. (2010). The sequence of a 1.8-Mb bacterial linear plasmid reveals a rich evolutionary reservoir of secondary metabolic pathways. Genome Biol. Evol. 2, 212224. Medema, M. H., Breitling, R., Bovenberg, R., and Takano, E. (2011). Exploiting plugand-play synthetic biology for drug discovery and production in microorganisms. Nat. Rev. Microbiol. 9, 131137. Menzella, H. G., and Reeves, C. D. (2007). Combinatorial biosynthesis for drug development. Curr. Opin. Microbiol. 10, 238245. Menzella, H. G., Reid, R., Carney, J. R., Chandran, S. S., Reisinger, S. J., Patel, K. G., Hopwood, D. A., and Santi, D. V. (2005). Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat. Biotechnol. 23, 11711176. Menzella, H. G., Carney, J. R., and Santi, D. V. (2007). Rational design and assembly of synthetic trimodular polyketide synthases. Chem. Biol. 14, 143151. Ohnishi, Y., Ishikawa, J., Hara, H., Suzuki, H., Ikenoya, M., Ikeda, H., Yamashita, A., Hattori, M., and Horinouchi, S. (2008). Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350. J. Bacteriol. 190, 40504060. Okamoto, S., Taguchi, T., Ochi, K., and Ichinose, K. (2009). Biosynthesis of actinorhodin and related antibiotics: Discovery of alternative routes for quinone formation encoded in the act gene cluster. Chem. Biol. 16, 226236.

Streptomyces Synthetic Biology

501

Richardson, M. A., Kuhstoss, S., Solenberg, P., Schaus, N. A., and Rao, R. N. (1987). A new shuttle cosmid vector, pKC505, for streptomycetes: Its use in the cloning of three different spiramycin-resistance genes from a Streptomyces ambofaciens library. Gene 61, 231241. Rodriguez-Garcia, A., Combes, P., Perez-Redondo, R., Smith, M. C., and Smith, M. C. (2005). Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces. Nucleic Acids Res. 33, e87. Salis, H. M., Mirsky, E. A., and Voigt, C. A. (2009). Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946950. Scholtissek, S., and Grosse, F. (1987). A cloning cartridge of lambda t(o) terminator. Nucleic Acids Res. 15, 3185. Schoof, S., Pradel, G., Aminake, M. N., Ellinger, B., Baumann, S., Potowski, M., Najajreh, Y., Kirschner, M., and Arndt, H. D. (2010). Antiplasmodial thiostrepton derivatives: Proteasome inhibitors with a dual mode of action. Angew. Chem. Int. Ed. Engl. 49, 33173321. Strohl, W. R. (1992). Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res. 20, 961974. Taguchi, T., Itou, K., Ebizuka, Y., Malpartida, F., Hopwood, D. A., Surti, C. M., BookerMilburn, K. I., Stephenson, G. R., and Ichinose, K. (2000). Chemical characterisation of disruptants of the Streptomyces coelicolor A3(2) actVI genes involved in actinorhodin biosynthesis. J. Antibiot. (Tokyo) 53, 144152. Tahlan, K., Ahn, S. K., Sing, A., Bodnaruk, T. D., Willems, A. R., Davidson, A. R., and Nodwell, J. R. (2007). Initiation of actinorhodin export in Streptomyces coelicolor. Mol. Microbiol. 63, 951961. Tahlan, K., Yu, Z., Xu, Y., Davidson, A. R., and Nodwell, J. R. (2008). Ligand recognition by ActR, a TetR-like regulator of actinorhodin export. J. Mol. Biol. 383, 753761. Takano, E., White, J., Thompson, C. J., and Bibb, M. J. (1995). Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp. Gene 166, 133137. Takano, E., Kinoshita, H., Mersinias, V., Bucca, G., Hotchkiss, G., Nihira, T., Smith, C. P., Bibb, M., Wohlleben, W., and Chater, K. (2005). A bacterial hormone (the SCB1) directly controls the expression of a pathway-specific regulatory gene in the cryptic type I polyketide biosynthetic gene cluster of Streptomyces coelicolor. Mol. Microbiol. 56, 465479. Tala, A., Wang, G., Zemanova, M., Okamoto, S., Ochi, K., and Alifano, P. (2009). Activation of dormant bacterial genes by Nonomuraea sp. strain ATCC 39727 mutanttype RNA polymerase. J. Bacteriol. 191, 805814. Walsh, C. T. (2002). Combinatorial biosynthesis of antibiotics: Challenges and opportunities. Chembiochem. 3, 125134. Wang, X. J., Yan, Y. J., Zhang, B., An, J., Wang, J. J., Tian, J., Jiang, L., Chen, Y. H., Huang, S. X., Yin, M., Zhang, J., Gao, A. L., et al. (2010). Genome sequence of the milbemycin-producing bacterium Streptomyces bingchenggensis. J. Bacteriol. 192, 45264527. Ward, J. M., Janssen, G. R., Kieser, T., Bibb, M. J., Buttner, M. J., and Bibb, M. J. (1986). Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol. Gen. Genet. 203, 468478. Widmaier, D. M., Tullman-Ercek, D., Mirsky, E. A., Hill, R., Govindarajan, S., Minshull, J., and Voigt, C. A. (2009). Engineering the Salmonella type III secretion system to export spider silk monomers. Mol. Syst. Biol. 5, 309. Willems, A. R., Tahlan, K., Taguchi, T., Zhang, K., Lee, Z. Z., Ichinose, K., Junop, M. S., and Nodwell, J. R. (2008). Crystal structures of the Streptomyces coelicolor TetR-like protein ActR alone and in complex with actinorhodin or the actinorhodin biosynthetic precursor (S)-DNPA. J. Mol. Biol. 376, 13771387.

502

Marnix H. Medema et al.

Zerikly, M., and Challis, G. L. (2009). Strategies for the discovery of new natural products by genome mining. Chembiochem. 10, 625633. Zhang, W., and Tang, Y. (2008). Combinatorial biosynthesis of natural products. J. Med. Chem. 51, 26292633. Zhang, W., Li, Y., and Tang, Y. (2008). Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli. Proc. Natl. Acad. Sci. USA 105, 2068320688.