Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

Alessandra S Eustáquio; Bertolt Gust; Ute Galm; Shu-Ming Li; Keith F Chater; Lutz Heide

doi:10.1128/AEM.71.5.2452-2459.2005

. 2005 May;71(5):2452–2459. doi: 10.1128/AEM.71.5.2452-2459.2005

Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

Alessandra S Eustáquio ¹, Bertolt Gust ^1,2, Ute Galm ¹, Shu-Ming Li ¹, Keith F Chater ², Lutz Heide ^1,^*

PMCID: PMC1087579 PMID: 15870333

Abstract

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage φC31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.

The development of new antimicrobial agents is crucial to reduce the future clinical impact of resistant pathogens (35). Natural products play a dominant role in antimicrobial drug discovery (19), and streptomycetes produce two-thirds of the clinically useful antibiotics of natural origin (12). Structural modification of these natural products is often necessary in drug development, e.g., for improvements in efficacy and pharmacokinetics (32). The manipulation of genes which encode enzymes of the biosynthetic pathways represents a promising approach for introducing structural changes (33). The functional analysis of biosynthetic genes is, however, a prerequisite for such approaches. Moreover, a principal limitation is often presented by the lack of suitable protocols for the genetic manipulation of natural producers (11).

The aminocoumarin antibiotics novobiocin (Albamycin; Pharmacia & Upjohn) and clorobiocin (chlorobiocin) (Fig. 1) are very potent inhibitors of DNA gyrase and are produced by different Streptomyces strains (17). Novobiocin was licensed in the United States for the treatment of human infections with multiresistant gram-positive bacteria such as Staphylococcus aureus and S. epidermidis (21, 22, 34). Novobiocin and its derivatives have also been investigated as potential anticancer drugs (16, 23, 30). The aminocoumarin antibiotics are closely related in structure and their biosynthetic gene clusters have been cloned and sequenced, and therefore, they offer excellent possibilities for the generation of new antibiotics via genetic engineering (20, 28).

However, the development of efficient protocols for the genetic manipulation of aminocoumarin antibiotic-producing Streptomyces strains is time consuming and, e.g., for the novobiocin producer S. spheroides, gives unsatisfactory results. This problem presents a severe limitation for genetic engineering and strain improvement.

In order to overcome this problem, we have developed a method which quickly allowed the heterologous expression of the entire novobiocin and clorobiocin biosynthetic gene clusters in the well-studied Streptomyces coelicolor and S. lividans strains (1, 12). By means of λ-Red-mediated recombination (4, 9), we introduced the integrase gene (int) and the attachment site (attP) of phage φC31 into cosmids containing the biosynthetic gene clusters of novobiocin or clorobiocin. The modified cosmids were site-specifically integrated into the chromosome of the heterologous hosts (31).

Prior to heterologous expression, the DNA sequence of the biosynthetic gene clusters could be easily modified in Escherichia coli by gene replacements or gene deletions, allowing functional investigations. As examples, we have removed several nonessential sequences from the cosmid inserts.

MATERIALS AND METHODS

Bacterial strains, cosmids, and culture conditions.

Streptomyces coelicolor M512 (ΔredD ΔactII-ORF4 SCP1⁻ SCP2⁻) (7) was kindly provided by E. Takano (Tübingen, Germany) and Janet White (Norwich, United Kingdom), S. lividans TK24 (str-6 SLP2⁻ SLP3⁻) was provided by D. A. Hopwood (Norwich, United Kingdom), S. roseochromogenes var. oscitans DS 12.976 was provided by Aventis, and S. spheroides NCIMB 11891 was provided by E. Cundliffe (Leicester, United Kingdom). The strains were cultured as described previously (5, 12, 28).

Escherichia coli XL1-Blue MRF′ (Stratagene, Heidelberg, Germany) was used for cloning experiments and grown as described previously (24).

The REDIRECT technology kit for PCR targeting (9) was obtained from Plant Bioscience Limited (Norwich, United Kingdom).

Kanamycin (15 μg/ml in liquid medium and 50 μg/ml in solid medium for Streptomyces and 50 μg/ml for E. coli), chloramphenicol (25 to 50 μg/ml), apramycin (50 μg/ml), carbenicillin (50 to 100 μg/ml), and thiostrepton (15 μg/ml in liquid medium and 50 μg/ml in solid medium) were used for selection of recombinant strains.

Cosmids 10-9C and D1A8 contained the novobiocin and the clorobiocin biosynthetic gene cluster in the SuperCos1 vector, respectively (20, 28).

Construction of pIJ787.

The tetracycline resistance gene (tet) from pBR328 was amplified using primers pIJ782forw (5′-CTA TGA TCG ACT GAT GTC ATC AGC GGT GGA GTG CAA TGT CAT GAA ATC TAA CAA TGC GC-3′) and pIJ782rev (5′-GAA CTT CAT GAG CTC AGC CAA TCG ACT GGC GAG CGG CAT CTC AGG TCG AGG TGG CCC GG-3′) (the beginning and the end of the coding region of the tet sequence are underlined). This fragment was used to replace the apramycin resistance gene in pIJ773 using λ-Red-mediated recombination (4, 9), generating pIJ782. The structural gene tet was thereby placed under control of the apramycin resistance promoter, resulting in slightly lower resistance levels (5 μg/ml instead of 10 μg/ml tetracycline). tet together with the apramycin resistance promoter was then amplified from pIJ782 using primers TetAatIIforw (5′-AAA AAA AGA CGT CTT GAA TGG GTT CAT GTG-3′) and TetAatII rev (5′-AAA AAA AGA CGT CTC AGG TCG AGG TGG CCC-3′) (the AatII restriction site is underlined). After digestion of the PCR product with AatII, it was cloned into the same site of pSET152. A 4,590-bp MscI-PvuI fragment obtained from these clones was ligated into the ScaI-PvuI sites (i.e., into the ampicillin resistance gene bla) of SuperCos1 to give pIJ787.

Construction of pUG019.

pUG019, containing an apramycin resistance cassette and flanked by XbaI and SpeI recognition sites, was generated by PCR amplification of two fragments from pIJ773 (REDIRECT technology kit). The first fragment of 97 bp was amplified using primers FRT_P01f (5′-CTG CAG GAA TTC GAT ATT CCG GGG ATC TCT AGA TCT-3′) (the EcoRI, XbaI, and BglII restriction sites are underlined) and FRT_P01r (5′-TGG CGG GGA TAT CGA AGT TCC-3′) (the EcoRV restriction site is underlined). After digestion with EcoRI and EcoRV, this fragment was ligated into the same sites of pBluescript SK(−) (Stratagene, Heidelberg, Germany) to give pUG017. The second fragment of about 1 kb containing the apramycin resistance gene aac(3)IV was amplified using primers apra_P03f (5′-GGG GAT GAT ATC TTT ATC ACC ACC GAC TAT TTG-3′) (the EcoRV restriction site is underlined) and apra_P02r (5′-TCG ATA AGC TTG ATG ACT AGT CTG GAG CTG GAG CTG CTT CGA-3′) (the HindIII and SpeI restriction sites are underlined). After digestion with EcoRV and HindIII, this fragment was ligated into the same sites of pUG017 to give pUG019.

DNA isolation, manipulation, and cloning.

Standard procedures for DNA isolation and manipulation were performed as described previously by Sambrook and Russell (24) and Kieser et al. (12). Isolation of DNA fragments from agarose gel and purification of PCR products were carried out with the NucleoSpin 2 in 1 Extract kit (Macherey-Nagel, Düren, Germany). Isolation of cosmids and plasmids was carried out with ion-exchange columns (Nucleobond AX kits; Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. Genomic DNA was isolated from Streptomyces strains using the Kirby mix procedure (12).

Southern blot analysis was performed on Hybond-N nylon membranes (Amersham, Braunschweig, Germany) with a digoxigenin (DIG)-labeled probe by using the DIG High Prime DNA labeling and detection starter kit II (Roche Molecular Biochemicals).

PCRs were carried out using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) according to the manufacturer's instructions.

Heterologous expression.

The DraI-BsaI fragment of pIJ787, containing the integrase cassette and flanked by about 100 bp of bla sequence on one site and about 300 bp of bla sequence on the other side, was used to replace the respective bla gene in the SuperCos1 backbone of cosmids 10-9C and D1A8, via λ-Red-mediated recombination (4, 9), generating nov-BG1 and clo-BG1, respectively.

Because of the potent methylation restriction system of S. coelicolor, cosmid DNA had to be passed through a nonmethylating host. We used E. coli ET12567 for this purpose (14). The modified cosmids nov-BG1 and clo-BG1, still carrying the kanamycin resistance gene neo, were then introduced into S. coelicolor M512 and S. lividans TK24 via polyethylene glycol-mediated protoplast transformation (12). Kanamycin-resistant clones were checked for site-specific integration into the genome by Southern blot analysis.

Protocol for single or multiple deletions within the cosmids.

The apramycin resistance cassette (approximately 1 kb) was excised from pUG019 by digestion with EcoRI and HindIII and amplified by PCR using the forward primer 5′-(N)₃₉ ATT CCG GGG ATC TCT AGA TCT-3′ and the reverse primer 5′-(N)₃₉ ACT AGT CTG GAG CTG CTT C-3′ [(N)₃₉ represents 39 nucleotide extensions for λ-Red-mediated recombination, homologous to the regions upstream and downstream of the DNA fragment to be deleted; underlined are the XbaI and SpeI restriction sites]. Amplification was performed using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) as described previously (9), with the following modification: annealing temperatures were 45°C and 48°C, respectively. The PCR product was used for gene replacement in cosmids via λ-Red-mediated recombination as described previously (9). For excision of the resistance cassette, cosmid DNA was isolated from E. coli ET12567 and digested with XbaI and SpeI, and 100 ng of DNA was religated overnight at 4°C. E. coli XL1- Blue MRF′ cells were transformed with the ligation reaction. Apramycin-sensitive kanamycin-resistant clones were analyzed by restriction enzyme digestion and gel electrophoresis. For subsequent gene deletions, the identical procedure was used.

Using this procedure, e.g., for the generation of cosmids nov-AE6 and nov-AE4, first, the region downstream of the gyrase B resistance gene (Fig. 2) was removed from nov-BG1 using primers PnovgyrB_f (5′-GGT TCC TCC AGC GTG GCC ACG ACC ATG ACC GGG AGG TCG ATT CCG GGG ATC TCT AGA TC-3′) and PT3SC1_r (5′-GTC TTC AAG AAT TCG CGG CCG CAA TTA ACC CTC ACT AAA ACT AGT CTG GAG CTG CTT C-3′) (underlined letters represent the 39 nucleotide extensions homologous to the region downstream of the gyrase B resistance gene and to the T3 promoter of SuperCos1, respectively), generating cosmid nov-AE2. Subsequently, ORF21 was deleted from nov-AE2 using primers PT7SC1_f (5′-ACA TGA GAA TTC GCG GCC GCA TAA TAC GAC TCA CTA TAG ATT CCG GGG ATC TCT AGA TC-3′) and Pnov20_r (5′-CTT CCC GAG GTT CAA TTC CGC CGC GCA CGT CAG CTC CTC ACT AGT CTG GAG CTG CTT C-3′) (sequences homologous to the T7 promoter region of SuperCos1 and to the region downstream of ORF20, respectively, are underlined), generating cosmid nov-AE6. Alternatively, the entire region upstream of novE was deleted from nov-AE2 using primers PT7SC1_f (see above) and PnovE_r (5′-GCT GGA ATG CGC GGC TGC CGT CGC CGG GAC GGT CCC GGC ACT AGT CTG GAG CTG CTT C-3′) (the sequence homologous to the region directly downstream of novD is underlined), generating cosmid nov-AE4. The insert of nov-AE6 comprises the DNA sequence from positions 5619 to 6339 of GenBank accession number AY227005, all of the sequence from GenBank accession number AF170880 (positions 1 to 25617), and from positions 1 to 2490 of GenBank accession number AF205854. The insert of nov-AE4 comprises the sequence from positions 4628 to 25617 of Gene Bank accession number AF170880 and from positions 1 to 2490 of GenBank accession number AF205854.

clo-AE2, containing genes from cloE to the gyrase B resistance gene, was generated from clo-BG1 in the same way using primers Pcloorf9_f (5′-TAG TAT GGC GAA ATT GGG TGA TCT GCT TGC CGC CGT CGA ATT CCG GGG ATC TCT AGA TC-3′) (the sequence homologous to the region directly downstream of ORF9 is underlined) and PT3SC1_r (see above). The insert of clo-AE2 comprises the DNA sequence from positions 9200 to 40573 of GenBank accession number AF329398 and from positions 1 to 2238 of GenBank accession number AY136281.

Production and analysis of secondary metabolites.

Transformants and parental strains of S. coelicolor and S. lividans, as well as S. spheroides and S. roseochromogenes, were cultured and assayed for novobiocin or clorobiocin production by high-performance liquid chromatography (HPLC) as described previously (5, 6).

Negative-ion fast atom bombardment mass spectra were recorded with a TSQ70 spectrometer (Finnigan, Bremen, Germany) using diethanolamine as matrix. ¹H nuclear magnetic resonance (NMR) spectra were measured with either an AC 250 or an AMX 400 spectrometer (Bruker, Karlsruhe, Germany) using CD₃OD as a solvent.

In the case of strains harboring nov-BG1, nov-AE6, and nov-AE4 (comprising the novobiocin cluster), the isolated compound showed a molecular ion [M-H]⁻ at m/z 611 (novobiocin, C₃₁H₃₆N₂O₁₁; molecular weight, 612). The isolated substance gave ¹H NMR signals identical to those obtained from authentic novobiocin (6).

Strains harboring clo-BG1 and clo-AE2 (comprising the clorobiocin cluster) showed four peaks in the HPLC analysis (see Fig. 4d). The first peak (peak 1), with the shortest retention time, showed a molecular ion [M-H]⁻ at m/z 661 and the following isotopic pattern [mass (percent intensity)]: 661 (100.0%), 662 (39.9%), 663 (11.9%). Furthermore, this compound gave ¹H NMR signals identical to those obtained for C-8′-deschloro-clorobiocin (novclobiocin 101; described in reference 5). The second peak (peak 2) showed a molecular ion [M-H]⁻ at m/z 695 and the following typical isotopic pattern caused by the chlorine isotopes ³⁵Cl and ³⁷Cl [mass (percent intensity)]: 695 (100.0%), 696 (36.1%), 697 (39.5%), 698 (14.6%). This compound gave ¹H NMR signals identical to those obtained for clorobiocin (5). The third peak (peak 3) had the same mass as the first one, molecular ion [M-H]⁻ at m/z 661, and showed the same isotopic pattern (mass [percent intensity], 661 [100.0%], 662 [30.2%], 663 [17.5%]), indicating the absence of a chlorine atom. The fourth peak (peak 4) had the same mass as clorobiocin, molecular ion [M-H]⁻ at m/z 695, and the isotopic pattern caused by the chlorine isotopes ³⁵Cl and ³⁷Cl (mass [percent intensity]: 695 [100.0%], 696 [37.6%], 697 [36.0%], and 698 [14.6%]). Substances 3 and 4 are therefore likely to represent structural isomers of substances 1 and 2, which carry the acyl group at 2-OH rather than 3-OH of the deoxysugar, as identified previously (8).

RESULTS

Heterologous expression.

Cosmids containing the entire biosynthetic gene clusters of novobiocin and clorobiocin had been obtained previously (20, 28) by employing the widely used cosmid vector SuperCos1 (Stratagene), which contains an ampicillin (bla) and a neomycin/kanamycin (neo) resistance gene.

We now replaced the bla gene within the SuperCos1 backbone of cosmids 10-9C and D1A8 (containing the novobiocin and clorobiocin cluster, respectively) with a cassette containing the integrase gene (int) and the attachment site (attP) of phage φC31, as well as a selectable marker (tetracycline resistance), using λ-Red-mediated recombination in E. coli (see Materials and Methods). This one-step procedure readily yielded the desired modified cosmids which were termed nov-BG1 and clo-BG1, respectively. They were introduced into S. coelicolor M512 (ΔredD ΔactII-ORF4 SCP1⁻ SCP2⁻) and S. lividans TK24 (str-6 SLP2⁻ SLP3⁻) by protoplast transformation. Selection for kanamycin resistance resulted in the desired integration mutants (approximately 10³ mutants per microgram cosmid DNA for S. coelicolor and 10⁵ mutants for S. lividans). Southern blot analysis showed that the entire cosmids had integrated site specifically into the attB site of the chromosome in both Streptomyces strains (Fig. 2c and 3c; only results for S. coelicolor strains are shown).

FIG. 3. — Cosmid constructs containing the clorobiocin biosynthetic gene cluster and their integration into the *S. coelicolor* chromosome. (a) Cosmid constructs clo-BG1 and clo-AE2. B, BglII restriction site; T3 and T7, T3 and T7 promoter of the SuperCos1 vector; gyrB^R, gyrase B resistance gene; parY^R, topoisomerase IV resistance gene. Fragment sizes resulting from digestion with BglII are indicated. The cosmid backbone is out of scale. (b) Schematic representation of site-specific integration of constructs clo-BG1 and clo-AE2 (see reference 31 for details of the integration mechanism). (c) Southern blot analysis of *S. coelicolor* (S. c.) M512 parental strain, of *S. coelicolor* M512 integration mutants harboring clo-BG1 or clo-AE2, and of the respective cosmid constructs. M, DIG-labeled DNA Molecular Weight Marker VII (Roche). Genomic and cosmid DNA were digested with BglII. The DIG-labeled cosmid clo-BG1 was used as a probe.

Integration mutants and parental host strains were cultured in production media. The analysis of secondary metabolites by HPLC (Fig. 4) showed that, in contrast to the untransformed host strains, the integration mutants accumulated novobiocin and clorobiocin, respectively. The identity of these substances was confirmed, after preparative isolation, by negative-ion fast atom bombardment mass spectrometry and ¹H NMR analysis (see Materials and Methods). S. coelicolor strains expressing the clorobiocin cluster additionally accumulated three clorobiocin analogs (Fig. 4d) identical to those observed in the natural clorobiocin producer S. roseochromogenes (5) (see Materials and Methods).

The productivity of the integration mutants of S. coelicolor was comparable to that of the original producers S. spheroides and S. roseochromogenes (Table 1). S. lividans, as a host, was much less productive. Therefore, S. coelicolor was used in the subsequent experiments.

TABLE 1.

Novobiocin and clorobiocin production by S. coelicolor and S. lividans

Strain	Production (mg/liter)
Strain	Novobiocin^a	Clorobiocin^a^,^b
Parental strains
S. spheroides	35 (30-40)
S. roseochromogenes		25 (20-30)
S. coelicolor M512
S. lividans TK24
Strains carrying full-length cosmids
S. coelicolor (nov-BG1)	31 (20-42)
S. lividans (nov-BG1)	<1
S. coelicolor (clo-BG1)		26 (18-34)
S. lividans (clo-BG1)		5 (4-6)
Strains carrying shortened cosmids
S. coelicolor (nov-AE6)	24 (23-25)
S. coelicolor (nov-AE4)	1.5 (0.8-2.2)
S. coelicolor (clo-AE2)		14 (8-19)

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Mean values (range) from at least two independent experiments.

Total amount of clorobiocin and the three major analogs (Fig. 4d).

Removal of nonessential DNA regions from the cosmid inserts.

Based on the λ-Red recombination system, we developed a two-step procedure which allowed us to readily produce single or multiple deletions at any desired site within the cosmid insert and which can be utilized for functional investigations.

The apramycin resistance cassette from the previously described plasmid pIJ773 (9) is flanked by FLP recombinase recognition targets (FRT). After gene replacement, this cassette can be conveniently removed by action of FLP recombinase, leading to the excision of the DNA region in between the FRT sites and leaving an 81-bp “scar” sequence. However, the presence of this scar sequence makes further knockouts in the same cosmid difficult, because it represents a functional FRT site as well as a target for λ-Red-mediated recombination.

To overcome this problem, an apramycin resistance cassette (Fig. 5) was generated by PCR using primers with either an XbaI or an SpeI recognition site between the apramycin resistance marker and the 39-bp flanking sequence for λ-Red-mediated recombination (9). XbaI and SpeI sites are rare in the GC-rich Streptomyces genome. After gene replacement, this cassette can be removed by digestion with XbaI and SpeI and religation of the resulting compatible ends (Fig. 5). This procedure leaves a minimal in-frame “scar” of 18 nucleotides which is not recognized by XbaI or SpeI and which does not interfere with further gene deletions or replacements.

The biosynthetic gene cluster of clorobiocin is flanked on one side by the gyrase B resistance gene and the topoisomerase IV resistance gene (26). Of the latter 2.1-kb gene, only 123 bp are contained in cosmid clo-BG1 (Fig. 3a, right-hand side of the insert). On the opposite end, the gene cloE may represent the border of the cluster (6, 26). We now deleted all genes upstream of cloE from cosmid clo-BG1 using the two-step procedure explained above (see Materials and Methods). The insert of the resulting cosmid, clo-AE2, starts 148 bp upstream of the start codon of cloE (Fig. 3).

This cosmid was transformed into S. coelicolor M512, and integration mutants were selected. Site-specific integration of the entire cosmid was confirmed by Southern blot analysis (Fig. 3c, lanes 5 and 6). When these mutants were cultured in production medium, HPLC analysis clearly showed that clorobiocin was still produced in significant amounts (Table 1), proving that the 34-kb sequence from cloE to the gyrase B resistance gene contains all genes required for clorobiocin biosynthesis.

A similar experiment was performed with the novobiocin cluster contained in cosmid nov-BG1. It has been suggested previously that novE and the gyrase B resistance gene (Fig. 2) may delineate the left and right border of this cluster, respectively (6, 26). However, novA, which is located outside of this region, shows sequence similarity to ABC transporters and has been suggested to be involved in novobiocin transport (18, 26). Therefore, we decided to produce two different shortened cosmids (see Materials and Methods): nov-AE4, containing only the genes from novE to the gyrase B resistance gene, and nov-AE6, additionally containing the genes novABCD and ORF20 (Fig. 2). ORF20 may encode a regulator of novA, as suggested by sequence comparison with database entries.

Cosmids nov-AE4 and nov-AE6 were transformed into S. coelicolor M512, and site-specific integration of the entire cosmids was confirmed by Southern blot analysis (Fig. 2). Cultivation in production medium and HPLC analysis revealed that integration mutants containing the larger cosmid, i.e., nov-AE6, still produced two-thirds of the novobiocin amount found in the natural producer, S. spheroides. In contrast, mutants containing the smaller cosmid nov-AE4 produced only 6% of the amount accumulated by S. coelicolor (nov-AE6). This proves that the DNA region from ORF20 to novD contains elements which are required for a high productivity of novobiocin.

DISCUSSION

The present work describes an efficient method for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains. λ-Red-mediated recombination in E. coli was used for the introduction of integration functions into the cosmid backbones, which allowed subsequent site-specific integration into the genome of the heterologous host. Single or multiple gene replacements and gene deletions could be created at any chosen site within the gene clusters.

During our procedure, the stability of the cosmids containing the gene clusters was quite satisfactory. Southern blot analysis of the integration mutants, using the entire cosmids as probes, proved that in more than 80% of the mutants, no rearrangements or deletions had taken place within the integrated sequence, and this was confirmed by the functional studies, i.e., by the formation of the respective antibiotics. We cannot exclude, however, that in certain other clusters, instability may present a more significant problem, e.g., in clusters containing type I polyketide synthase genes with repeated DNA sequences.

The complete genome sequence of strain M145 of S. coelicolor has been published previously (1). S. coelicolor M145 is able to produce three antibiotics, i.e., prodiginines (Red), actinorhodin (Act), and calcium-dependent antibiotic. To avoid interference in the chemical and biological analysis of antibiotic formation, we expressed the novobiocin and clorobiocin biosynthetic gene clusters in S. coelicolor M512, which is derived from M145 but which is unable to produce Red and Act (7).

Historically, S. lividans has been distinguished phenotypically from its close relative S. coelicolor by the inability of the former to produce significant amounts of antibiotics. Yet functional biosynthetic Red and Act gene clusters are present in S. lividans, suggesting different regulation of the onset of secondary metabolism in the two species (13, 15, 27). A strain of S. lividans, TK24 (str-6), was found to overproduce antibiotics in comparison to the parent strain TK21. Shima et al. (27) proposed that the str-6 mutation (point mutation in the rpsL gene encoding ribosomal protein S12) leads to a change in the ribosomal structure which gives rise to initiation of the onset of secondary metabolism. Therefore, to test S. lividans as a host, strain TK24 was chosen. However, S. coelicolor M512 showed much higher productivity than S. lividans TK24 and was therefore used for subsequent experiments.

It remains unclear whether the higher productivity observed for S. coelicolor M512-derived strains is due to the Act and Red mutations or whether S. coelicolor in general is a better producer strain than S. lividans.

After testing different strains for heterologous expression, optimization of the fermentation conditions will next be an important task in order to achieve improvement in productivity.

Using the method developed in this study, we proved, for the first time, that the DNA region from cloE to the gyrase B resistance gene (Fig. 3) contains all genes necessary for clorobiocin production. This finding is of special interest since no candidate gene has been identified which may code for the enzyme responsible for the introduction of the ring oxygen of the coumarin moiety of the aminocoumarin antibiotics (2, 6, 10). Unless this oxygenation is carried out by primary metabolic enzymes of S. coelicolor (a rather unlikely scenario), this catalytic function will have to be identified in one of the proteins encoded in cosmid clo-AE2.

The principal resistance gene of the novobiocin producer S. spheroides is the gyrase B resistance gene, encoding for an aminocoumarin-resistant gyrase B subunit (25, 26, 29). Since this gene is contained within the novobiocin cluster (Fig. 2), the S. coelicolor mutants generated in this study were able to tolerate the accumulation of the antibiotic.

The clorobiocin producer S. roseochromogenes contains, besides the gyrase B resistance gene, an additional resistance gene, that for topoisomerase IV, which encodes an aminocoumarin-resistant topoisomerase IV subunit (25, 26). Only 123 bp of the 2.1-kb topoisomerase IV resistance gene was contained in the cosmids clo-BG1 and clo-AE2 (Fig. 3). Nevertheless, clorobiocin was produced in good yield by the mutants expressing these cosmids, consistent with our previous finding that the gyrase B resistance gene alone is sufficient to provide resistance against aminocoumarin antibiotics (26).

Vectors containing the integrase gene and the attP site of φC31 can integrate not only as a single copy but also in tandem (3). Tandem integration of cosmids was observed in approximately 90% of all integration mutants obtained in this study, as identified in Southern blot analysis by a hybridizing fragment containing the unchanged attP site (e.g., 3.9 kb in Fig. 2c). Mutants with tandem integration showed somewhat higher antibiotic production than mutants with single-copy integration, but the difference, and the number of available strains, was not large enough to draw unequivocal conclusions. In contrast, the 16-fold-higher productivity of strains bearing cosmid nov-AE6, compared to strains harboring nov-AE4 (Table 1 and Fig. 2), was observed in more than 10 independent mutants each and clearly proves that the DNA sequence from ORF20 to novD contains elements required for a high novobiocin production. The gene novA, which codes for an ABC transporter, may be responsible for this effect.

The heterologous expression experiments described here have given rise to strains which, in contrast to the natural producers, are highly amenable for genetic manipulation and strain improvement. These methods may be useful both for basic research on microbial secondary metabolism and for drug discovery programs from streptomycetes, allowing, e.g., the exploitation of biosynthetic gene clusters from organisms which are difficult to cultivate.

Acknowledgments

We thank E. Takano and J. White for kindly providing S. coelicolor M512, Aventis for the generous gift of S. roseochromogenes and authentic clorobiocin, and H.-P. Trefzer for helpful technical assistance.

This work was supported by a grant from the European Community (no. 503466 to L.H.) and by grant 208/IGF12432 from the Biotechnological and Biological Research Council (to K.F.C.).

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4.Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Eustáquio, A. S., B. Gust, T. Luft, S.-M. Li, K. F. Chater, and L. Heide. 2003. Clorobiocin biosynthesis in Streptomyces: identification of the halogenase and generation of structural analogs. Chem. Biol. 10:279-288. [DOI] [PubMed] [Google Scholar]
6.Eustáquio, A. S., T. Luft, Z.-X. Wang, B. Gust, K. F. Chater, S.-M. Li, and L. Heide. 2003. Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation. Arch. Microbiol. 180:25-32. [DOI] [PubMed] [Google Scholar]
7.Floriano, B., and M. Bibb. 1996. afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2). Mol. Microbiol. 21:385-396. [DOI] [PubMed] [Google Scholar]
8.Galm, U., M. A. Dessoy, J. Schmidt, L. A. Wessjohann, and L. Heide. 2004. In vitro and in vivo production of new aminocoumarins by a combined biochemical, genetic and synthetic approach. Chem. Biol. 11:173-183. [DOI] [PubMed] [Google Scholar]
9.Gust, B., G. L. Challis, K. Fowler, T. Kieser, and K. F. Chater. 2003. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. USA 100:1541-1546. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Holzenkämpfer, M., and A. Zeeck. 2002. Biosynthesis of simocyclinone D8 in an ¹⁸O₂-rich atmosphere. J. Antibiotics 55:341-342. [DOI] [PubMed] [Google Scholar]
11.Khosla, C. 1998. Combinatorial biosynthesis of “unnatural” natural products, p. 401-417. In E. M. Gordon and J. F. Kerwin, Jr. (ed.), Combinatorial chemistry and molecular diversity in drug discovery. John Wiley & Sons, New York, N.Y.
12.Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. Practical Streptomyces genetics. John Innes Foundation, Norwich, United Kingdom.
13.Kim, E.-S., H.-J. Hong, C.-Y. Choi, and S. N. Cohen. 2001. Modulation of actinorhodin biosynthesis in Streptomyces lividans by glucose repression of afsR2 gene transcription. J. Bacteriol. 183:2198-2203. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.MacNeil, D. J., K. M. Gewain, C. L. Ruby, G. Dezeny, P. H. Gibbons, and T. MacNeil. 1992. Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111:61-68. [DOI] [PubMed] [Google Scholar]
15.Malpartida, F., J. Niemi, R. Navarrete, and D. A. Hopwood. 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. [DOI] [PubMed] [Google Scholar]
16.Marcu, M. G., T. W. Schulte, and L. Neckers. 2000. Novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins. J. Natl. Cancer Inst. 92:242-248. [DOI] [PubMed] [Google Scholar]
17.Maxwell, A. 1993. The interaction between coumarin drugs and DNA gyrase. Mol. Microbiol. 9:681-686. [DOI] [PubMed] [Google Scholar]
18.Méndez, C., and J. A. Salas. 2001. The role of ABC transporters in antibiotic-producing organisms: drug secretion and resistance mechanisms. Res. Microbiol. 152:341-350. [DOI] [PubMed] [Google Scholar]
19.Newman, D. J., G. M. Cragg, and K. M. Snader. 2003. Natural products as sources of new drugs over the period 1981-2002. J. Nat. Prod. 66:1022-1037. [DOI] [PubMed] [Google Scholar]
20.Pojer, F., S.-M. Li, and L. Heide. 2002. Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics. Microbiology 148:3901-3911. [DOI] [PubMed] [Google Scholar]
21.Raad, I., R. Darouiche, R. Hachem, M. Sacilowski, and G. P. Bodey. 1995. Antibiotics and prevention of microbial colonization of catheters. Antimicrob. Agents Chemother. 39:2397-2400. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Raad, I. I., R. Y. Hachem, D. Abi-Said, K. V. I. Rolston, E. Whimbey, A. C. Buzaid, and S. Legha. 1998. A prospective crossover randomized trial of novobiocin and rifampin prophylaxis for the prevention of intravascular catheter infections in cancer patients treated with interleukin-2. Cancer 82:403-411. [DOI] [PubMed] [Google Scholar]
23.Rappa, G., K. Shyam, A. Lorico, O. Fodstad, and A. C. Sartorelli. 2000. Structure-activity studies of novobiocin analogs as modulators of the cytotoxicity of etoposide (VP-16). Oncol. Res. 12:113-119. [DOI] [PubMed] [Google Scholar]
24.Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
25.Schmutz, E., S. Hennig, S.-M. Li, and L. Heide. 2004. Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParY^R and GyrB^R from the coumermycin A₁ producer Streptomyces rishiriensis DSM 40489. Microbiology 150:641-647. [DOI] [PubMed] [Google Scholar]
26.Schmutz, E., A. Mühlenweg, S.-M. Li, and L. Heide. 2003. Resistance genes of aminocoumarin producers: two type II topoisomerase genes confer resistance against coumermycin A₁ and clorobiocin. Antimicrob. Agents Chemother. 47:869-877. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Shima, J., A. Hesketh, S. Okamoto, S. Kawamoto, and K. Ochi. 1996. Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2). J. Bacteriol. 178:7276-7284. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Steffensky, M., A. Mühlenweg, Z.-X. Wang, S.-M. Li, and L. Heide. 2000. Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891. Antimicrob. Agents Chemother. 44:1214-1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Thiara, A. S., and E. Cundliffe. 1988. Cloning and characterization of a DNA gyrase B gene from Streptomyces sphaeroides that confers resistance to novobiocin. EMBO J. 7:2255-2259. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Thiele, A., M. Pfister, M. Erbes, M. Cross, M. Hänsch, and S. Hauschildt. 2002. Novobiocin is a novel inducer of CD38 on cells of the myelomonocytic lineage. Biochim. Biophys. Acta 1542:32-40. [DOI] [PubMed] [Google Scholar]
31.Thorpe, H. M., S. E. Wilson, and M. C. Smith. 2000. Control of directionality in the site-specific recombination system of the Streptomyces phage φC31. Mol. Microbiol. 38:232-241. [DOI] [PubMed] [Google Scholar]
32.Walsh, C. 2003. Where will new antibiotics come from? Nat. Rev. Microbiol. 1:65-70. [DOI] [PubMed] [Google Scholar]
33.Walsh, C. T. 2002. Combinatorial biosynthesis of antibiotics: challenges and opportunities. Chembiochem 3:124-134. [DOI] [PubMed] [Google Scholar]
34.Walsh, T. J., H. C. Standiford, A. C. Reboli, J. F. John, M. E. Mulligan, B. S. Ribner, J. Z. Montgomerie, M. B. Goetz, C. G. Mayhall, D. Rimland, D. A. Stevens, S. L. Hansen, G. C. Gerard, and R. J. Ragual. 1993. Randomized double-blinded trial of rifampin with either novobiocin or trimethoprim-sulfamethoxazole against methicillin-resistant Staphylococcus aureus colonization: prevention of antimicrobial resistance and effect of host factors on outcome. Antimicrob. Agents Chemother. 37:1334-1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.World Health Organization. 2001. WHO global strategy for containment of antimicrobial resistance. [Online.] http://www.who.int/emc/amrpdfs/WHO_Global_Strategy_English.pdf.

[r1] 1.Bentley, S. D., K. F. Chater, A. M. Cerdeño-Tárraga, G. L. Challis, N. R. Thomson, K. D. James, D. E. Harris, M. A. Quail, H. Kieser, D. Harper, A. Bateman, S. Brown, G. Chandra, C. W. Chen, M. Collins, A. Cronin, A. Fraser, A. Goble, J. Hidalgo, T. Hornsby, S. Howarth, C. H. Huang, T. Kieser, L. Larke, L. Murphy, K. Oliver, S. O'Neil, E. Rabbinowitsch, M. A. Rajandream, K. Rutherford, S. Rutter, K. Seeger, D. Saunders, S. Sharp, R. Squares, S. Squares, K. Taylor, T. Warren, A. Wietzorrek, J. Woodward, B. G. Barrell, J. Parkhill, and D. A. Hopwood. 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417:141-147. [DOI] [PubMed] [Google Scholar]

[r2] 2.Chen, H., and C. T. Walsh. 2001. Coumarin formation in novobiocin biosynthesis: β-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI. Chem. Biol. 8:301-312. [DOI] [PubMed] [Google Scholar]

[r3] 3.Combes, P., R. Till, S. Bee, and M. C. M. Smith. 2002. The Streptomyces genome contains multiple pseudo-attB sites for the φC31-encoded site-specific recombination system. J. Bacteriol. 184:5746-5752. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r4] 4.Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r5] 5.Eustáquio, A. S., B. Gust, T. Luft, S.-M. Li, K. F. Chater, and L. Heide. 2003. Clorobiocin biosynthesis in Streptomyces: identification of the halogenase and generation of structural analogs. Chem. Biol. 10:279-288. [DOI] [PubMed] [Google Scholar]

[r6] 6.Eustáquio, A. S., T. Luft, Z.-X. Wang, B. Gust, K. F. Chater, S.-M. Li, and L. Heide. 2003. Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation. Arch. Microbiol. 180:25-32. [DOI] [PubMed] [Google Scholar]

[r7] 7.Floriano, B., and M. Bibb. 1996. afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2). Mol. Microbiol. 21:385-396. [DOI] [PubMed] [Google Scholar]

[r8] 8.Galm, U., M. A. Dessoy, J. Schmidt, L. A. Wessjohann, and L. Heide. 2004. In vitro and in vivo production of new aminocoumarins by a combined biochemical, genetic and synthetic approach. Chem. Biol. 11:173-183. [DOI] [PubMed] [Google Scholar]

[r9] 9.Gust, B., G. L. Challis, K. Fowler, T. Kieser, and K. F. Chater. 2003. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. USA 100:1541-1546. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r10] 10.Holzenkämpfer, M., and A. Zeeck. 2002. Biosynthesis of simocyclinone D8 in an ¹⁸O₂-rich atmosphere. J. Antibiotics 55:341-342. [DOI] [PubMed] [Google Scholar]

[r11] 11.Khosla, C. 1998. Combinatorial biosynthesis of “unnatural” natural products, p. 401-417. In E. M. Gordon and J. F. Kerwin, Jr. (ed.), Combinatorial chemistry and molecular diversity in drug discovery. John Wiley & Sons, New York, N.Y.

[r12] 12.Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. Practical Streptomyces genetics. John Innes Foundation, Norwich, United Kingdom.

[r13] 13.Kim, E.-S., H.-J. Hong, C.-Y. Choi, and S. N. Cohen. 2001. Modulation of actinorhodin biosynthesis in Streptomyces lividans by glucose repression of afsR2 gene transcription. J. Bacteriol. 183:2198-2203. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r14] 14.MacNeil, D. J., K. M. Gewain, C. L. Ruby, G. Dezeny, P. H. Gibbons, and T. MacNeil. 1992. Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111:61-68. [DOI] [PubMed] [Google Scholar]

[r15] 15.Malpartida, F., J. Niemi, R. Navarrete, and D. A. Hopwood. 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. [DOI] [PubMed] [Google Scholar]

[r16] 16.Marcu, M. G., T. W. Schulte, and L. Neckers. 2000. Novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins. J. Natl. Cancer Inst. 92:242-248. [DOI] [PubMed] [Google Scholar]

[r17] 17.Maxwell, A. 1993. The interaction between coumarin drugs and DNA gyrase. Mol. Microbiol. 9:681-686. [DOI] [PubMed] [Google Scholar]

[r18] 18.Méndez, C., and J. A. Salas. 2001. The role of ABC transporters in antibiotic-producing organisms: drug secretion and resistance mechanisms. Res. Microbiol. 152:341-350. [DOI] [PubMed] [Google Scholar]

[r19] 19.Newman, D. J., G. M. Cragg, and K. M. Snader. 2003. Natural products as sources of new drugs over the period 1981-2002. J. Nat. Prod. 66:1022-1037. [DOI] [PubMed] [Google Scholar]

[r20] 20.Pojer, F., S.-M. Li, and L. Heide. 2002. Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics. Microbiology 148:3901-3911. [DOI] [PubMed] [Google Scholar]

[r21] 21.Raad, I., R. Darouiche, R. Hachem, M. Sacilowski, and G. P. Bodey. 1995. Antibiotics and prevention of microbial colonization of catheters. Antimicrob. Agents Chemother. 39:2397-2400. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r22] 22.Raad, I. I., R. Y. Hachem, D. Abi-Said, K. V. I. Rolston, E. Whimbey, A. C. Buzaid, and S. Legha. 1998. A prospective crossover randomized trial of novobiocin and rifampin prophylaxis for the prevention of intravascular catheter infections in cancer patients treated with interleukin-2. Cancer 82:403-411. [DOI] [PubMed] [Google Scholar]

[r23] 23.Rappa, G., K. Shyam, A. Lorico, O. Fodstad, and A. C. Sartorelli. 2000. Structure-activity studies of novobiocin analogs as modulators of the cytotoxicity of etoposide (VP-16). Oncol. Res. 12:113-119. [DOI] [PubMed] [Google Scholar]

[r24] 24.Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[r25] 25.Schmutz, E., S. Hennig, S.-M. Li, and L. Heide. 2004. Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParY^R and GyrB^R from the coumermycin A₁ producer Streptomyces rishiriensis DSM 40489. Microbiology 150:641-647. [DOI] [PubMed] [Google Scholar]

[r26] 26.Schmutz, E., A. Mühlenweg, S.-M. Li, and L. Heide. 2003. Resistance genes of aminocoumarin producers: two type II topoisomerase genes confer resistance against coumermycin A₁ and clorobiocin. Antimicrob. Agents Chemother. 47:869-877. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r27] 27.Shima, J., A. Hesketh, S. Okamoto, S. Kawamoto, and K. Ochi. 1996. Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2). J. Bacteriol. 178:7276-7284. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r28] 28.Steffensky, M., A. Mühlenweg, Z.-X. Wang, S.-M. Li, and L. Heide. 2000. Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891. Antimicrob. Agents Chemother. 44:1214-1222. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r29] 29.Thiara, A. S., and E. Cundliffe. 1988. Cloning and characterization of a DNA gyrase B gene from Streptomyces sphaeroides that confers resistance to novobiocin. EMBO J. 7:2255-2259. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r30] 30.Thiele, A., M. Pfister, M. Erbes, M. Cross, M. Hänsch, and S. Hauschildt. 2002. Novobiocin is a novel inducer of CD38 on cells of the myelomonocytic lineage. Biochim. Biophys. Acta 1542:32-40. [DOI] [PubMed] [Google Scholar]

[r31] 31.Thorpe, H. M., S. E. Wilson, and M. C. Smith. 2000. Control of directionality in the site-specific recombination system of the Streptomyces phage φC31. Mol. Microbiol. 38:232-241. [DOI] [PubMed] [Google Scholar]

[r32] 32.Walsh, C. 2003. Where will new antibiotics come from? Nat. Rev. Microbiol. 1:65-70. [DOI] [PubMed] [Google Scholar]

[r33] 33.Walsh, C. T. 2002. Combinatorial biosynthesis of antibiotics: challenges and opportunities. Chembiochem 3:124-134. [DOI] [PubMed] [Google Scholar]

[r34] 34.Walsh, T. J., H. C. Standiford, A. C. Reboli, J. F. John, M. E. Mulligan, B. S. Ribner, J. Z. Montgomerie, M. B. Goetz, C. G. Mayhall, D. Rimland, D. A. Stevens, S. L. Hansen, G. C. Gerard, and R. J. Ragual. 1993. Randomized double-blinded trial of rifampin with either novobiocin or trimethoprim-sulfamethoxazole against methicillin-resistant Staphylococcus aureus colonization: prevention of antimicrobial resistance and effect of host factors on outcome. Antimicrob. Agents Chemother. 37:1334-1342. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r35] 35.World Health Organization. 2001. WHO global strategy for containment of antimicrobial resistance. [Online.] http://www.who.int/emc/amrpdfs/WHO_Global_Strategy_English.pdf.

PERMALINK

Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

Alessandra S Eustáquio

Bertolt Gust

Ute Galm

Shu-Ming Li

Keith F Chater

Lutz Heide

Abstract

FIG. 1.

MATERIALS AND METHODS