Construction of new vectors for high-level expression in actinomycetes

Gene 216 (1998) 215–223

Construction of new vectors for high-level expression in actinomycetes Christine J. Rowe a, Jesu´s Corte´s a, Sabine Gaisser a, James Staunton b, Peter F. Leadlay a,* a Cambridge Centre for Molecular Recognition and Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK b Cambridge Centre for Molecular Recognition and Department of Organic Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK Received 3 February 1998; received in revised form 29 April 1998; accepted 30 April 1998; Received by M. Salas

Abstract A new integrative vector (pCJR24) was constructed for use in the erythromycin producer Saccharopolyspora erythraea and in other actinomycetes. It includes the pathway-specific activator gene actII–ORF4 from the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. The actI promoter and the associated ribosome binding site are located upstream of an NdeI site (5∞-CATATG-3∞) which encompasses the actI start codon allowing protein(s) to be produced at high levels in response to nutritional signals if these signals are faithfully mediated by the ActII–ORF4 activator. Several polyketide synthase genes were cloned in pCJR24 and overexpressed in S. erythraea after integration of the vector into the chromosome by homologous recombination, indicating the possibility that the S. coelicolor promoter/activator functions appropriately in S. erythraea. pCJR24-mediated recombination was also used to place the entire gene set for the erythromycin-producing polyketide synthase under the control of the actI promoter. The resulting strain produced copious quantities of erythromycins and precursor macrolides when compared with wild-type S. erythraea. The use of this system provides the means for rational strain improvement of antibiotic-producing actinomycetes. © 1998 Elsevier Science B.V. All rights reserved. Keywords: ActII–ORF4; Streptomyces coelicolor; Saccharopolyspora erythraea; actI promoter; homologous recombination; erythromycin

1. Introduction Numerous broad host-range cloning vectors are now available for use in actinomycetes (Hopwood et al., 1985, Lydiate et al., 1985, Hagege et al., 1993). Overexpression of native and heterologous genes has been achieved using such vectors either with strong constitutive promoters such as ermE* (Bibb et al., 1985) or with regulatable promoters which allow the control of expression of target genes. For example, the tipA gene of Streptomyces lividans is strongly induced by low levels of the antibiotic peptide thiostrepton (Murakami et al., 1989) and the tipA promoter (P ) has been used tipA * Corresponding author. Tel.: +44 1223 333656; Fax: +44 1223 333656; E-mail: [email protected] Abbreviations: bp, base pair(s); Ec, Escherichia coli; GC, gas chromatography; hcn, high copy number; kb, kilo base(s) or 1000 bp; ampr, bla-gene; lcn, low copy number; P , actI promoter; MCS, multiple actI cloning site; PCR, polymerase chain reaction; TE, thioesterase; TSB, tryptone soy broth; tsr, gene encoding thiostrepton resistance. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 32 7 - 8

for controlled gene expression in low copy number ( lcn) and high copy number (hcn) plasmids ( Takano et al., 1995 and references therein), and in integrative vectors (Smokvina et al., 1990). However, it is not always convenient to utilize thiostrepton to induce gene expression and the tipA gene encoding the protein needed for expression is reportedly not present in all Streptomyces ( Takano et al., 1995). In S. coelicolor, the biosynthetic gene cluster for the isochromanequinone polyketide actinorhodin contains a pathway-specific activator gene (actII–ORF4) ( Fernandez-Moreno et al., 1991) which is required for transcription from at least some of the act promoters (Parro et al., 1991). A similar activator gene (redD) is required in S. coelicolor for transcription of genes for biosynthesis of the red pigment undecylprodigiosin ( Takano et al., 1992). Members of this class of regulatory protein have been found in other Streptomyces, usually within gene clusters for aromatic polyketide biosynthesis (Chater and Bibb, 1997; Pe´rez-Llarena et al., 1997), but not yet in clusters for biosynthesis of

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complex polyketides such as the macrolides. The exact molecular basis for the action of activators of the ActII–Orf4 class remains obscure, although recent work provides evidence that DnrI binds directly to its target promoters ( Tang et al., 1996). In addition, an intriguing structural similarity between members of this family of activators and the OmpR- family of DNA-binding domains has led to the proposal that they act by binding to tandem repeats in the promoters of structural genes in the respective biosynthetic gene clusters ( Wietzorrek and Bibb, 1997). Introduction of additional copies of actII–ORF4 into S. coelicolor causes increased and premature production of actinorhodin (Gramajo et al., 1993). The presence of actII–ORF4 on an hcn plasmid in Streptomyces lividans also causes overproduction of actinorhodin from an endogenous gene cluster (D.A. Hopwood and H.M. Kieser, unpublished data quoted by Fernandez-Moreno et al., 1991). Similarly, extra copies of the redD gene cause overproduction of undecylprodigiosin in S. lividans (Narva and Feitelson, 1990; Malpartida et al., 1990). The DnrI activator reportedly can complement actII–ORF4 mutants (StutzmanEngwall et al., 1992) but redD cannot (Floriano and Bibb, 1996). A plasmid vector pRM5, based on the lcn plasmid SCP2* (Lydiate et al., 1985), has previously been developed using the activator gene actII–ORF4 for regulated expression in a strain of S. coelicolor (CH999) from which the act genes have largely been deleted (McDaniel et al., 1993). Derivatives of this plasmid have been used to express both native and heterologous polyketide synthase (PKS ) genes and gene sets (McDaniel et al., 1993, Kao et al., 1994, Bedford et al., 1995). However, in contrast to tipA, the use of the actII–ORF4/actI promoter combination has not been reported previously in host strains other than S. coelicolor and the very closely related Streptomyces lividans. When a cloned DNA fragment containing 32 kb of the act gene cluster was introduced into Streptomyces parvulus, it led to the production of an actinorhodin-like pigment, establishing that the cloned act DNA, together with factors from the heterologous host, together provide all the ingredients required for actinorhodin biosynthesis (Malpartida and Hopwood, 1984). It remains unclear, however, which act- or host-derived factor is responsible for activation of the gene cluster. Some individual act promoters are known to be active in other hosts, including Escherichia coli (Caballero et al., 1991). In contrast, the actI promoter was found to be inactive in E. coli, Bacillus subtilis and S. lividans (Parro et al., 1991). Even the mutually overlapping and divergent promoters for actI and actIII are apparently of different types and possibly subject to differential regulation (Parro et al., 1991). The aim of this study was to develop a high-level expression system for use in the erythromycin-producing

strain of S. erythraea. A key question to be addressed was whether the actinorhodin actI promoter, together only with its cognate activator gene actII–ORF4, would be sufficient, in S. erythraea as in S. coelicolor, to allow expression of downstream genes in response to nutritional signals during the transition to stationary phase. Here we describe the construction of new actII–ORF4/P -containing vectors and (although the actI involvement of actII–ORF4/P -has not yet been foractI mally proved) exemplify their utility in first, allowing heterologous expression of recombinant PKS genes, and secondly, in triggering significant over-expression of chromosomally located PKS genes.

2. Materials and methods 2.1. Bacterial strains, plasmids and growth conditions Escherichia coli DH10B (GibcoBRL, Gaithersburg, MD, USA) was grown in 2×TY medium as described by Sambrook et al. (1989). The vector pUC19 was obtained from Pharmacia and pMF1015 and pMV400 were kindly provided by F. Malpartida (Centro Nacional de Biotecnologia, Madrid, Spain). E. coli transformants were selected with 100 mg/ml ampicillin. Streptomyces coelicolor CH999 was obtained from D.A. Hopwood (John Innes Institute, Norwich, UK ) and was routinely maintained on R2YE agar and YEME for liquid cultures (Hopwood et al., 1985) at 30°C. The Saccharopolyspora erythraea NRRL-2338-red variant strain was obtained from J.M. Weber and was routinely maintained on R2T20 agar ( Yamamoto et al., 1986) and TSB (Difco, Detroit, MI, USA) for liquid cultures at 30°C. 2.2. Manipulation of recombinant DNA Standard methods were used for restriction enzyme analysis, PCR, ligations, transformation of E. coli and isolation of plasmid DNA from E. coli (Sambrook et al., 1989). Methods used for protoplast transformation of S. coelicolor protoplasts and isolation of plasmid DNA from S. coelicolor are described in Hopwood et al. (1985). Protoplast formation and transformation procedures for S. erythraea were adapted from Weber and Losick (1988) as described by Gaisser et al. (1997). Southern hybridizations were carried out with probes labelled with digoxigenin using the DIG DNA labelling kit (Boehringer Mannheim, Mannheim, Germany). Sequencing of DNA fragments generated by PCR amplification was performed by the method of Sanger et al. (1977), using automated DNA sequencing on doublestranded DNA templates with an applied Biosystems 373A sequencer.

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2.3. Analysis of production of triketides To test for the production of triketide lactones plasmid-containing S. coelicolor was grown in YEME supplemented with 5 mg/ml thiostrepton for 5 days at 30°C and cells removed by centrifugation. S. erythraea integrants were selected for resistance to thiostrepton (50 mg/ml in R2T20 agar). Production cultures were grown in sucrose–succinate medium (Caffrey et al., 1992) for 3 days at 30°C and cells removed by centrifugation. In each case the supernatant was adjusted to below pH 3 and extracted three times with an equal volume of ethyl acetate. The extracts were dried over MgSO , evaporated to dryness and dissolved in an 4 appropriate volume of methanol. Gas chromatography/mass spectrometry was carried out with chemical ionisation on a Finnigan/MAT GCQ instrument, as described in Weissman et al. (1997). 2.4. Analysis of production of macrolides Production of erythromycin by S. erythraea strains was assessed by plate bioassay using B. subtilis ATCC 6633 as the indicator organism (Gaisser et al., 1997), and by electrospray mass spectrometry on a BioQ (Micromass, Manchester, UK ) spectrometer (Gaisser et al., 1997).

3. Results and discussion

3.1. Overexpression of a DEBS1-TE gene in S. erythraea strain TER43 The DEBS1-TE gene encodes the loading module and first two extension modules of the erythromycin-producing polyketide synthase (6-deoxyerythronolide B synthase, DEBS ), fused at the C-terminus to the DEBS thioesterase ( TE ) domain (Corte´s et al., 1995). This gene is located on the chromosome in the S. erythraea strain TER43 (Corte´s et al., 1995), and TER43 produces a specific triketide lactone (1B) ( Fig. 1) when cultured in a defined liquid medium (Corte´s et al., 1995). The coding region of the DEBS1-TE gene has also been previously cloned into a derivative (pRM52, Oliynyk et al., 1996) of the very large SCP2*-derived lcn vector pRM5 (McDaniel et al., 1993) to produce pRMTE (Brown et al., 1995). When the gene was expressed from pRMTE in S. coelicolor CH999 under the control of the actII–ORF4 activator and actI promoter, a mixture of triketide 1B with the 6-nor-triketide 1A (the major product) was produced ( Fig. 1) (Brown et al., 1995). SCP2*-based plasmids are not stably maintained in S. erythraea (J. C., unpublished data). To test the ability of actII–ORF4/P -containing plasmids to function as actI

Fig. 1. Triketide lactones produced by expression from the actI promoter. 1B is produced by S. erythraea strain TER43 (Section 3.1); 1A and 1B are produced by S. erythraea strain TER43/pRMTE (Section 3.1) and both S. coelicolor CH999(pCJR30) and S. erythraea strain JC2/pCJR30 (Section 3.5); the 4-nor-triketide lactones 2A and 2B are produced by S. coelicolor(pRMTE) and the strain S. erythraea JC2/pCJR26 (Section 3.4).

integrative vectors in S. erythraea plasmid pRMTE was used to transform protoplasts of S. erythraea TER43 ( Yamamoto et al., 1986) and stable thiostrepton resistant colonies were isolated. One of these was selected and designated S. erythraea TER43/pRMTE. Extracts of S. erythraea TER43/pRMTE when analysed for production of lactones by electrospray mass spectrometry (operated in the positive ion mode) showed the presence of both 1A and 1B ( Fig. 1), in the ratio 1A:1B of 1:2. The combined yield of triketide lactones was 150 mg/l, compared with 10 mg/l obtained by fermentation of S. erythraea TER43 under identical conditions (Corte´s et al., 1995) and 20–30 mg/l obtained by expression from pRMTE in S. coelicolor CH999 in a defined medium (Brown et al., 1995). Southern analysis (data not shown) indicated that several copies (>3–5) of pRMTE were present in the chromosome of S. erythraea TER43/pRMTE. This variable number of additional copies could have arisen after initial integration, by duplication of the entire plasmid sequence. These data established that high-level expression from the actI promoter was possible in S. erythraea, but they did not allow a distinction to be drawn between an effect of multiple gene copies of DEBS1-TE and actII–ORF4, and an enhanced rate of transcription from P , stimulated by ActII–ORF4, or even to actI transcription by readthrough from adjacent vector sequences. 3.2. Construction of pCJR24 pCJR24 ( Fig. 2) is a small (4.5 kb compared with 20 kb for pRM5) expression vector designed for integ-

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Fig. 2. Construction of the expression vector pCJR24 was as follows: A 970-bp DNA fragment (containing the actII–ORF4 activator gene) was amplified using PCR from pMF1015 (Parro et al., 1991) using primers which introduced a flanking SpeI restriction site upstream of the gene and an AflII site downstream. This fragment was introduced into AatII-digested and end-repaired pUC19 to yield pCJR18. A 215-bp DNA fragment (containing the bidirectional promoter pair P /P ) was amplified from pMV400 (Fernandez-Moreno et al., 1991) using synthetic primers actIII actI which introduced an AflII site suitably positioned to serve as the start codon for genes to be expressed from P and an Nde I site suitably actIII positioned for expression of genes from P . This PCR product was digested with AflII and NdeI and cloned into pCJR18 previously digested actI with AflII and NdeI to generate pCJR19. A 1.1-kb HindIII–SphI fragment containing the tsr gene was amplified from pIJ922 (Lydiate et al., 1985) introducing the flanking sites in the primers. This fragment was digested with HindIII and SphI and introduced into similarly digested pCJR19 to give pCJR24.

ration into an actinomycete host via inserted DNA sequences that are homologous to a region of the host chromosome. Like pRM5, it includes a ColEI replicon to allow it to replicate in E. coli, the ampicillin resistance gene to allow selection in E. coli, the thiostrepton resistance gene to allow selection in most actinomycetes, the actII–ORF4 gene, and the promoter P . Included actI within the amplified sequences are a 20-bp AT-rich inverted repeat upstream of the actII–ORF4 gene, and the putative ribosome binding site for actII–ORF4, and also the putative ribosome binding site for the thiostrepton resistance gene and a potential stem–loop structure,

which may act as a signal for transcription termination, downstream of the thiostrepton resistance gene. 3.3. Construction of S. erythraea strain JC2 For use with pCJR24-derived plasmids, an S. erythraea host cell was genetically engineered to remove almost all of the eryA region, which encodes the erythromycin-producing polyketide synthase, except for the region of eryAIII encoding the chain-terminating TE. S. erythraea JC2 was constructed by homologous recombination starting from S. erythraea NRRL2338 (red

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variant) ( Hessler et al., 1997). The plasmid used for the deletion of the eryA region (pDEL702) was constructed as follows. The 1.4-kbp SmaI segment containing the start codon of eryAI was cloned into pUC18 to give p612SL, the segment was excised as a BamHI–SacI fragment using the MCS of pUC18, and subcloned into plasmid pDEL, a derivative of plasmid pT7-18 (Roberts et al., 1993). pDEL contains the SacI–KpnI fragment of eryAIII which encodes the C-terminus of DEBS3 from which a BglII–SacI fragment had been excised. Plasmid pDEL was digested with BamHI, treated with alkaline phosphatase, and ligated to plasmid pIJ702 ( Katz et al., 1983) which had been linearized with BglII. The resulting plasmid pDEL702 was used to transform S. erythraea NRRL2338 (red variant) protoplasts following the procedure described by Yamamoto et al. (1986). Thiostrepton resistant colonies were isolated and subcultured four times in non-selective liquid medium (TSB) followed by preparation and regeneration of protoplasts. Thiostrepton sensitive colonies were isolated, chromosomal DNA was prepared and analysed by Southern hybridization. One such clone was designated S. erythraea JC2. Supernatants from S. erythraea JC2 cultures were analysed for erythromycin production by electrospray mass spectrometry (Gaisser et al., 1997) and no production of erythromycin A or erythronolide B was detected. S. erythraea JC2 produced no erythromycin A, as judged by bioassay using an erythromycinsensitive strain of B. subtilis, unless fed with erythronolide B (Gaisser et al., 1997).

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3.5. Construction and application of pCJR29 pCJR29 contains the same features as pCJR24 but resembles pRM5 in containing the SCP2* origin of replication (Bibb and Hopwood, 1981). The construction of pCJR29 is illustrated in Fig. 4. In hosts that can maintain plasmids with the SCP2* origin, pCJR29 should exist as an autonomously replicating plasmid from which heterologous genes may be expressed. pRM5 and its derivatives, from the results presented here, ought also to function in many streptomycete hosts other than S. coelicolor and S. lividans. To exemplify the use of pCJR29, the coding region for DEBS1-TE (Corte´s et al., 1995) was cloned as an NdeI–XbaI fragment from pNTEP2 (Oliynyk et al., 1996) into pCJR29 to generate pCJR30. This expression plasmid was used to transform either S. coelicolor CH999 (McDaniel et al., 1993) as previously described for pRMTE (Brown et al., 1995) or S. erythraea JC2 as described above for pCJR26. In S. coelicolor CH999 pCJR30 is maintained as a self-replicating plasmid, whereas in S. erythraea JC2 integration occurs by homologous recombination into the thioesterase-encoding domain of eryAIII. Southern analysis showed multiple copies of pCJR30 (>3–5) in thiostrepton-resistant colonies. Both S. coelicolor CH999/pCJR30 and S. erythraea JC2/pCJR30 produced triketide lactones 1A and 1B ( Fig. 1) in the ratio 1A:1B of 1:2 (data not shown). However, the smaller size of the pCJR29 vector greatly facilitates the cloning steps and the ease of transformation of S. erythraea strains.

3.4. Construction of pCJR26 and high-level production of novel triketide lactones in S. erythraea JC2 A pRM52-based plasmid (pRM-AT2) was previously used (Oliynyk et al., 1996) to demonstrate the production of novel 4-nor-triketide lactones 2A and 2B (Fig. 1) in S. coelicolor, through expression of a hybrid DEBS1-TE gene in which the AT domain of module 1 had been replaced by the acetate-specific AT derived from module 2 of the rapamycin-producing PKS (Schwecke et al., 1995; Haydock et al., 1995; Aparicio et al., 1996). The coding region for the mutant DEBS1-TE was cloned from pD1-AT2 (Oliynyk et al., 1996) as an NdeI–XbaI fragment into pCJR24 to generate pCJR26 ( Fig. 3) and pCJR26 was used to transform S. erythraea JC2 protoplasts. The presence of multiple (3–6) copies of the DEBS1-TE gene was confirmed by Southern blot analysis (data not shown) of colonies resistant to thiostrepton. The strain S. erythraea JC2/pCJR26 when grown in a defined medium (Caffrey et al., 1992) produced the lactones 2A and 2B (Fig. 1) at the same levels as S. coelicolor (pRM-AT2) (Oliynyk et al., 1996), as determined by GCMS analysis and comparison with authentic synthetic samples, in the ratio 2A:2B of 1:3.

3.6. Overproduction of erythromycins and their macrolide precursors by S. erythraea NRRL2338/pRM52 and S. erythraea JC103 To illustrate the use of pCJR24-derived plasmids in the overexpression of chromosomally located genes in actinomycetes, two S. erythraea strains were constructed, which overexpress the eryA PKS gene set. S. erythraea NRRL2338/pRMTE was constructed by transformation of wild type S. erythraea NRRL2338 (red variant) protoplasts with pRMTE, which bears the gene for DEBS1-TE under actII–ORF4/P -mediated control, actI followed by selection for stable thiostrepton resistant colonies. Southern analysis (data not shown) of one such colony confirmed that the plasmid had integrated specifically into the chromosomal copy of the eryAI gene. Growth of S. erythraea NRRL2338/pRMTE in a defined medium under conditions where the wild type S. erythraea NRRL2338 produces 10–15 mg/l total erythromycins led to the production of a complex mixture of erythromycins in greatly increased amounts (100–150 mg/l total ) together with triketide lactones 1A and 1B and considerable amounts of erythronolide B

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Fig. 3. Construction of pCJR26, designed to express the nor-triketide lactones 2A and 2B on integration into S. erythraea. Transformation into S. erythraea JC2 protoplasts was carried out following the procedure of Yamamoto et al. (1986) and thiostrepton-resistant colonies were isolated. The integrity of the strain was confirmed by Southern hybridization. The strain S. erythraea JC2/pCJR26 was incubated in a defined medium (Caffrey et al., 1992) at 30°C for 4–5 days and cultures were extracted three times with ethyl acetate. Analysis of the products using a Finnegan GC-MS showed the presence of triketide lactones 2A and 2B (Fig. 1).

and 6-deoxyerythronolide B precursors, as shown by HPLC-MS analysis (data not shown). The product mixture contained a significant proportion of specific polyketide products in which the starter unit was derived from acetate rather than propionate, indicating that polyketide synthesis under these conditions outstrips the supply of propionate precursor within the cells. To obtain overexpression of erythromycin production without the formation of triketide lactone side-products, S. erythraea strain JC103 was then constructed, as follows. First, the 1.6-kbp DNA segment encoding part of the loading domain of the erythromycin PKS was amplified by PCR employing the CloneAmp procedure (Rashtchian, 1992) using as template the DNA of plasmid pNTEP2. The PCR product (1.6 kbp) was digested with uracil DNA glycosylase for 30 min at 37°C in the presence of 25 ng of pAMP18 vector DNA (Gibco BRL), the mixture was cooled on ice and used to transform E. coli TG1recO. The desired plasmid pARLD was identified by its restriction map. Secondly, a 1.6-kbp fragment of plasmid pARLD was excised using PacI and NheI, purified by gel electrophoresis, and ligated to plasmid pCJR24 which had been cut with PacI and XbaI to obtain plasmid pNHE. Approximately 5 mg pNHE was used to transform S. erythraea NRRL2338 (red variant) protoplasts and stable thiostrepton resistant colonies were selected. One of these colonies was selected and subjected to Southern analysis (data not shown) which confirmed that the plasmid had integrated specifically into the chromosomal copy of the eryAI gene in the area that encodes the N-terminal loading domain. Growth of this strain, S. erythraea JC103, under the same conditions as used for NRRL2338/pRMTE, also led to an increase in macrolide production compared with wild type, but without concomitant formation of 1A and 1B. Yields of erythro-

mycins from cultures treated identically were 20 mg/l from S. erythraea NRRL2338 (red variant) and 50 mg/l from S. erythraea JC103. These data taken together demonstrate that pRM52, pCJR24 and pCJR29 can be used for the expression of polyketide synthase genes in actinomycete hosts other than S. coelicolor and S. lividans, which removes a significant limitation on the analysis and use of these genes. Analysis of the proteins present in cell-free extracts from S. erythraea strain TER43 (pRMTE ), which overproduces triketide lactones 1A and 1B ( Fig. 1), showed pronounced increases in the level of the DEBS1-TE protein (data not shown). The mechanism by which the expression occurs has not yet been formally proved to involve either the cloned actI promoter or the adjacent actII–ORF4 gene. This would require the mapping of the promoter start site, and elimination of the possibility of transcriptional readthrough from vector sequences. If the actI promoter does indeed function in S. erythraea, previous evidence suggests that it would require activation, since the actI promoter in contrast to the actIII and other act promoters, is not active even in S. lividans (Parro et al., 1991). If the cloned actII–ORF4 gene is essential for the observed expression, then it is likely that the presence of multiple copies of the plasmid in the chromosome, supplying multiple copies of the activator gene, plays a role in the observed overexpression. It is also possible that the host strain supplies additional essential factors (e.g. that interact with or modify ActII–ORF4), which would limit the use of these expression vectors to specific hosts. However, the ability to mediate the activation of a modular PKS gene cluster in its native host is likely to find important future application in rational strain improvement of antibiotic-producing actinomycetes.

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Fig. 4. Construction of pCJR29 was as follows: The SCP2* origin was excised from pIJ922 (Lydiate et al., 1985) on a BamHI–SstI fragment and cloned into pUC19 to generate a bifunctional plasmid pCJR16. The tsr gene was amplified from pIJ922 (Lydiate et al., 1985) by PCR as a HindIII–XhoI fragment and introduced into pCJR16 to generate pCJR25. The 2-kb HindIII–SphI fragment from pCJR25, including the tsr gene and a portion of the SCP2* origin, was then introduced into pCJR19 to give pCJR28. The plasmid pCJR29 was then constructed by introducing the remainder of the origin as an SphI fragment from pCJR25. Introduction of the coding region of DEBS1-TE as an NdeI–XbaI fragment from pNTEP2 gave the expression vector pCJR30.

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4. Conclusions (1) Plasmid vectors containing the actI promoter from the biosynthetic gene cluster for the aromatic polyketide actinorhodin and the gene for the cognate activator protein ActII–ORF4 have been used for high-level expression of recombinant genes in S. erythraea. (2) A new, small integrative plasmid pCJR24 has been constructed which can be used conveniently to deliver recombinant genes into the S. erythraea chromosome for stable expression. (3) Plasmid pCJR29 has been constructed which has the same features as pCJR24 but contains, in addition, the SCP2* origin of replication, so that gene expression can be studied in the broad range of actinomycetes that stably maintain plasmids with this origin. (4) Strains S. erythraea NRRL2338/pRMTE and JC103, which each contain only one full set of erythromycin PKS genes, nevertheless significantly overproduced erythromycin-related metabolites when pRM5-derived and pCJR24-derived plasmids respectively were used to transform these strains. The increased levels of specific polyketide products obtained are therefore not due to the presence of additional copies of the structural genes. Formal proof is still required for the respective roles in this expression system of the actI promoter and of the actII–ORF4 gene.

Acknowledgement We wish to thank F. Malpartida for providing us with cloned act DNA used in the construction of pCJR24. J. Lester and K. Pennock carried out the sequencing analysis and G. Kearney, G. Bo¨hm and M. Brown performed analysis of macrolides and small lactones. This work was supported by a grant from the UK BBSRC Biotechnology Directorate, and from the European Union BIOTECH programme PL9322067 and Human Capital and Mobility programme, network project CT94-0570. We are grateful to Bristol Myers Squibb Co. for the kind gift of thiostrepton.

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