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Cubist Pharmaceuticals Inc., 65 Hayden Avenue, Lexington, MA 02421, USA
Correspondence
Marie-Françoise Coëffet-Le Gal
mlegal{at}cubist.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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dptD mutation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). One member of the family, daptomycin, which is produced by feeding decanoic acid to the fermentation (Huber et al., 1988), has been commercialized as Cubicin® for the treatment of skin and skin-structure infections caused by Gram-positive pathogens (Arbeit et al., 2004; Eisenstein, 2004; Kirkpatrick et al., 2003). The A21978C lipopeptides are synthesized by a large non-ribosomal peptide synthetase (NRPS) multienzyme composed of three subunits, DptA, DptBC and DptD, each containing two to six modules to direct the incorporation of the 13 amino acids (Fig. 1a). Each module has a complete set of domains responsible for amino acid condensation (C), adenylation (A) and thiolation (T) (Marahiel et al., 1997; Schwarzer et al., 2003), and three of the modules have epimerase (E) domains to convert L-amino acids to D-amino acids at positions 2, 8 and 11 (Miao et al., 2005). The daptomycin NRPS subunits are encoded by three very large genes, dptA (17.5 kb), dptBC (22.0 kb) and dptD (7.1 kb) (Miao et al., 2005) arranged in tandem (Fig. 1b). Two genes, dptE (1.8 kb) and dptF (270 bp), directly upstream of dptA, show sequence similarities to acyl-CoA ligase and acyl carrier protein genes, respectively, and are likely to be involved in the acylation of the N-terminal amino acid, Trp, which initiates lipopeptide biosynthesis.
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). The terminal NRPS gene, dptD, can be deleted and successfully complemented in trans by genes from related lipopeptide NRPS pathways that encode similar subunits, but with alternative modules to generate active new antibiotics (Miao et al., 2006b). This prompted us to evaluate whether dptA could also be complemented in trans. Extending the trans-complementation platform to include dptA would further increase the opportunities to generate novel lipopeptide antibiotics by module exchanges (Baltz et al., 2006). However, this approach is potentially more challenging as little is known about the transcriptional organization of the dpt pathway. The stop and start codons of the NRPS genes, dptA, dptBC and dptD, overlap and the intergenic regions between dptE, dptF and dptA are relatively small, less than 70 bp. It is possible that a promoter resides in the intergenic region upstream of dptE, or even further upstream, and that dptE, dptF and dptA, as well as dptBC and dptD, are transcribed on a single large mRNA. In Streptomyces coelicolor, it has been shown that there is a single promoter upstream of the three terminally overlapping NRPS genes encoding the calcium-dependent antibiotic CDA (Ryding et al., 2002). If this is also the case for the daptomycin NRPS, it may be necessary to compensate for potential polar effects on downstream genes when engineering an upstream gene such as dptA.
To investigate the possibility of using additional engineered NRPS subunits (dptA and dptD) to generate novel analogues of daptomycin, and to acquire a better understanding of the transcriptional organization of the NRPS genes, we have explored the transcription of the dpt gene cluster using RT-PCR, and the reconstitution of the daptomycin biosynthetic pathway by expressing the three dpt NRPS genes from different chromosomal loci. We also evaluated the effects on product yield of placing a strong constitutive promoter, ermEp* (Bibb et al., 1994), either in front of dptBC at the native locus, or in front of dptA or dptEFA expressing from the attB
C31 locus and demonstrated the utility of this system by producing hybrid lipopeptides by heterologous subunit exchanges with lptD (Miao et al., 2006a, b) and cdaPS3 (Hojati et al., 2002).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Streptomyces strains were routinely grown on trypticase soy agar or broth at 30 °C. AS-1 agar (Baltz, 1980), used for bioassays, and fermentation medium F10A and other procedures (Miao et al., 2005, 2006b) were described previously. Escherichia coli DH10B (Invitrogen) was used for general cloning and ML22 was used as a donor strain for conjugative transfer of plasmids to S. roseosporus. E. coli BW25113 was used in all the recombination experiments (Datsenko & Wanner, 2000). Staphylococcus aureus SA42 is susceptible to daptomycin and was used for microbiological assays. Antibiotics were added to the culture media at previously described concentrations (Kieser et al., 2000; Sambrook et al., 1989).
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dptA dptD deletion mutants.dptA mutants were constructed by homologous recombination in UA378 (dptD : : ermE), which was derived from UA117, an A21978C producer with a recessive mutation (rpsL7) conferring resistance to streptomycin (SmR) (Fig. 1c
, Table 1). Mutants were constructed using a markerless dptA deletion cassette. Fragments of DNA upstream and downstream of dptA were subcloned or amplified by PCR and directionally cloned into HindIII–XbaI sites in pRHB538 (Hosted & Baltz, 1997) to leave an artificial XhoI site between them: the 3.2 kb upstream fragment (dpt:48381–51590, the coordinates refer to NCBI accession no. AY787762, the dpt gene cluster) included a portion of dptN and all of dptE and dptF; the 1.5 kb downstream fragment (dpt:69010–70520) consisted primarily of the 5' end of the dptBC gene. This construction deleted most of dptA, leaving a small open reading frame encoding 25 amino acids. Another version of this cassette was constructed by inserting at the XhoI site, upstream of dptBC, a fragment containing ermEp* amplified by PCR from pHM11a (Motamedi et al., 1995) using primers m97F (5'-CTCGAGGCGAGTGTCCGTTCGAGTGG-3') and m98R (5'-CTCGAGCATATGGGTCCTCCTGTGGAC-3'). Each deletion plasmid was introduced into S. roseosporus UA378 by conjugation from E. coli (Bierman et al., 1992; Hosted & Baltz, 1997). The pRHB538 vector carries an apramycin resistance (AmR) marker and a dominant wild-type rpsL allele (SmS) that allows for the direct selection of gene replacements in SmR hosts carrying a mutant rpsL (Hosted & Baltz, 1997). Growth at 39 °C, a non-permissive temperature for plasmid replication (orits), in the presence of Am selects for exconjugants with the plasmid integrated into the chromosome by a single crossover (AmR). Subsequent selection on AS-1 agar plates containing Sm facilitates identification of recombinants that have eliminated vector sequences (SmS) after a second crossover exchanging the targeted gene with an in-frame deletion or a replacement cassette. Attempts to grow S. roseosporus at 39 °C during this experiment were not successful, but exconjugants with the desired in-frame deletions of dptA were identified by unique PCR products representing the new junctions that result from successful double crossovers. Positive candidate clones were streaked, and individual colonies were reconfirmed by PCR. A strain with the in-frame deletion of dptA was designated UA474, and one with the deletion of dptA and insertion of ermEp* upstream of dptBC was designated UA475 (Fig. 1d).
The dptA complementation plasmids were constructed by 
Red-mediated recombination (Datsenko & Wanner, 2000) from pCV1, a BAC clone with a 128 kb insert that contains the entire dpt gene cluster and flanking genes (Miao et al., 2005). The regions flanking the dptE, dptF and dptA group (‘dptEFA’) were replaced with marker gene cassettes: the region upstream of dptEFA (dpt:552–45576) was replaced by aadA1 (which confers resistance to spectinomycin, NCBI accession no. AP002527), and the region downstream of dptA (dpt:69106–127392) was replaced by aph(2'') (which confers resistance to gentamicin: Kao et al., 2000). The resulting plasmid, pLT01 (Fig. 2a), includes a very small fragment (dpt:1–551) from the 5' end of the original insert in pCV1 and a 23.2 kb fragment (dpt:45861–69105) that extends from inside dptP to within the 5' end of dptBC, at 49 nt downstream of the start codon for dptBC. The DNA region (3560 bp) upstream of the dptEFA group has been kept in the pLT01 construct as there was no evidence of an obvious promoter sequence immediately upstream of dptE.
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Red-mediated exchange with tetA (which confers resistance to tetracycline, NCBI accession no. J01830) (Fig. 2b, c). The pStreptoBAC V vector backbone derived from pCV1 (Miao et al., 2005) includes sequences for apramycin resistance [AmR; aac(3)IV], conjugative transfer (oriT) and plasmid integration (int/attP) (Bierman et al., 1992) in the bacterial chromosome (Table 1).
Construction of recombinant strains, bioassays and quantification of lipopeptides.
All complementation plasmids were introduced by conjugation from E. coli and integrated into the S. roseosporus chromosome. The pLT plasmids integrate at attBC31, while pRB04, the dptD complementation plasmid (Miao et al., 2006b), integrates at attBIS117 (Table 1). Integrations at these loci are neutral with respect to the production of A21978C (D. Alexander, personal communication). Plasmid pRB04 was introduced into the dptA dptD hosts and hygromycin-resistant (HmR) exconjugants (the phenotype conferred by pRB04), were then used as recipients for the pLT plasmids. Since the latter conferred AmR, HmRAmR exconjugants were patched on AS-1 agar, grown for 5 days at 30 °C and then screened for antimicrobial activity using a nutrient soft agar overlay containing CaCl2 (5 mM final) and Staph. aureus SA42. At least ten recombinants which produced zones of inhibition after 18 h at 37 °C were then fermented in F10A broth in shake flasks at 30 °C. After 5 days, aliquots of each culture were clarified by centrifugation and evaluated for A21978C lipopeptides, by HPLC and LC-MS (Miao et al., 2005, 2006b). Bioassays were also conducted by placing 50 µl clarified broth into 5 mm diameter wells cut in plates of AS-1 agar containing 5 mM CaCl2 and Staph. aureus, and inspecting for zones of inhibition after incubating the plates overnight at 37 °C. For each group of strains, three siblings were fermented and analysed, and one representative strain from each set was refermented in triplicate to obtain accurate yields. Comparisons of yields between the different strains were made from fermentations carried out concurrently.
Extraction of RNA and RT-PCR.
S. roseosporus UA343, an A21978C-producing strain, was grown in duplicate in 25 ml TSB in 125 ml baffled flasks at 30 °C and 250 r.p.m. After 4 days, the mycelium was harvested by rapid filtration through Miracloth (Calbiochem) and immediately frozen by immersion into liquid nitrogen. The frozen mycelium was ground under liquid nitrogen and total RNA was extracted using the RNeasy Midi kit (Qiagen). The sample was treated twice with DNase I from the DNA-free kit (Ambion). The integrity of total RNA was assessed by gel electrophoresis to visualize 16S and 23S rRNA and from this, to infer the quality of the mRNA. Test PCRs supplemented with DMSO (Expand Long Template kit, Roche) were performed with primers P219 and P220 (5'-GTATTCGACACACCCGACCG-3' and 5'-GAGGAGAGCTGTAGACCG-3'; V. Miao, unpublished) to amplify a 516 bp region of the rpsL gene (Hosted & Baltz, 1997) from S. roseosporus DNA (positive control) and DNase I-treated RNA using a 55 °C annealing temperature. The treated RNA was considered suitable for RT-PCR if there was no amplified rpsL fragment present in the PCR reaction.
RT-PCR was performed using 20–23 nt primers (Table 2) and the Superscript one-step RT-PCR kit with Platinum Taq (Invitrogen), supplemented with RNAguard RNase Inhibitor (Amersham Pharmacia) and DMSO on 500 ng RNA, in a total volume of 20 µl. The RT-PCR programme for all reactions was: 50 °C for 30 min, 94 °C for 2 min, followed by one cycle of 94 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min, followed by 29 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, and finally, 72 °C for 5 min. Two independent RNA preparations were used and RT-PCR was repeated in three experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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G=–26 kcal mol–1; 109 kJ mol–1) that may be involved in transcription termination. The predicted start codon of dptA is separated from the end of dptF by only 15 nt, while dptE and dptF are separated by 68 nt. Given the small intergenic regions between the genes, it is possible that a promoter resides in the 397 nt intergenic region upstream of dptE, or even further upstream, and leads to transcription of the core genes on a single transcript.
To determine if the core dpt genes, as well as dptG and dptH genes downstream of dptD, are transcribed from a large polycistronic mRNA, we designed primers to amplify mRNA across the boundaries of the genes (Table 2). RNA from 4-day-old cultures of S. roseosporus UA343 grown in TSB was tested and RT-PCR products of the sizes predicted for amplification across the junctions of adjacent genes, dptE–dptF, dptF–dptA, dptA–dptBC, dptBC–dptD, dptD–dptG, dptG–dptH were obtained (Fig. 3). This suggested that the contiguous genes from dptE to dptH may be transcribed on a polycistronic transcript, or on overlapping large transcripts.
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dptA dptD host strains] and expressed from the ermEp* promoter when plasmid pRB04 is inserted at the attBIS117 site; the recombinant produced 
90 % of the control A21978C factors (Miao et al., 2006b). In light of the possibility that many of the dpt genes may be transcribed as a polycistronic message, the deletions of the upstream gene, dptA, were designed to be in-frame. The dptA mutants were constructed by homologous recombination in UA378 using PCR to screen AmS colonies for double-crossover mutants. The frequency of in-frame deletion was 1–2 % of AmS colonies, demonstrating that homologous double-crossover recombination is efficient in S. roseosporus (Hosted & Baltz, 1997). The resulting dptA dptD strain, UA474, confirmed by PCR, should allow any promoters upstream of dptA to direct the transcription of dptBC and any other genes normally co-transcribed with dptBC (Fig. 1d). The same method was used to construct UA475, a deletion strain with an insertion of ermEp* upstream of dptBC (dptA ermEp* : : dptBC dptD), in addition to any native promoters (Fig. 1d). UA474 and UA475 were both confirmed for the loss of A21978C production in fermentation broths by microbiological and HPLC analyses.
Expression of complementation plasmids in S. roseosporus
Six combinations of dptA dptD deletions doubly complemented with dptEFA and dptD outside of the dpt gene cluster were constructed. First, pRB04, which contains the dptD gene fused to the ermEp* promoter, was integrated at the attBIS117 site in the chromosome of strains UA474 and UA475, resulting in strains MF6 and MF40, respectively (Table 1). The dptEFA-containing plasmids pLT01, pLT02 or pLT03 were then introduced at the attBC31 site in MF6 and MF40 to generate six genotypes that represent the daptomycin NRPS genes with different combinations of promoters expressed from three different sites in the chromosome (Table 1). All complementations appeared successful when assayed microbiologically (Fig. 4), except MF171. We studied at least three recombinants for each group of complementations, and found that all members of the same group had the same fermentation and production characteristics. Therefore, we present detailed data for one representative strain from each group.
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) to that described previously (elution between 11 and 12 min) (Debono et al., 1987). S. roseosporus MF54, which has native dpt promoter sequences for dptEFA and dptBC, produced about 200 mg l–1 A21978C factors, or 58 % of control (Table 3). S. roseosporus MF77 and MF85, which have ermEp* upstream of dptEFA or dptA, respectively (Fig. 2b, c), produced 273 and 267 mg l–1 A21978C factors, or 80 % and 79 % of control. Thus, the presence of ermEp* in the dptA complementation plasmids clearly caused improved production relative to dptA expressed from a native promoter(s) (Table 3).
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). However, when dptA and dptBC were expressed individually from ermEp* (MF110), the yields improved to 171 mg l–1, or 50 % of control. Thus, of the six combinations tested, the best expression was obtained when the two ectopically expressed genes, dptA and dptD, were expressed from ermEp*, and the dptBC gene, located in the original dpt locus, was expressed from its native promoter(s). There was no observable consequence of the positioning of ermEp* in front of dptEFA or dptA genes on the levels of production of A21978C factors.
Heterologous trans-complementation to produce hybrid lipopeptides
In a previous study (Miao et al., 2006b), the heterologous complementation of dptD by the lptD or the cdaPS3 genes, driven by ermEp* and inserted into the attBIS117 site, using plasmids pMF30 and pMF26, produced yields of hybrid molecules of 25 % and 50 %, respectively.
Having established robust production of A21978C factors in strains containing dptA and dptD inserted in ectopic positions of the chromosome under control of the ermEp* promoter, we investigated the heterologous expression of lptD and cdaPS3 in a strain that was deleted for dptD, but that has dptA expressing ectopically from ermEp*, and dptBC expressing from its native promoter. MF125, containing the cloned lptD gene from the A54145 gene cluster (Miao et al., 2006a) inserted into the attBIS117 site, produced 135 mg l–1 or 40 % of the control, of the hybrid lipopeptides containing a mixture of Ile and Val at position 13, as previously described in a dptD deletion mutant (Miao et al., 2006b) (Table 3). The HPLC profile was similar to the one obtained for the native and engineered A21978C factors, with retention times ranging between 11 and 12 min. MF193, containing the cloned cdaPS3 inserted in the attBIS117site, produced 235 mg l–1 or 69 % of the control, of the hybrid lipopeptide containing Trp in position 13 (Table 3). Those yields are superior to the yields observed previously in the dptD single complementation (Miao et al., 2006b), demonstrating a positive effect of the ermEp* driving the expression of dptA.
S. roseosporus MF202, which has dptA expressed from its native promoter(s), dptBC and lptD expressed from ermEp*, produced only 38 mg l–1 of hybrid lipopeptides, or 11 % of control (Table 3). When dptA and dptBC were expressed individually from ermEp* (MF141), the yields improved slightly to 64 mg l–1, or 19 % of control. Similarly, MF214, where dptA expressed from its native promoter(s), dptBC and cdaPS3 expressed from ermEp*, produced 33 mg l–1 of hybrid lipopeptide, or 10 % of control (Table 3). No exconjugants were obtained when dptBC and dptEFA or dptA were driven by ermEp*. Thus, as previously shown for dptA and dptD double homologous complementation, the best expression was obtained when dptA, and lptD or cdaPS3 were expressed from ermEp* and the dptBC gene was expressed from its native promoter at its native locus (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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dptD deletion mutation in S. roseosporus can be complemented in trans with a dptD gene under the control of ermEp* (Miao et al., 2006b). The dptD gene was also complemented with the heterologous genes lptD from the A54145 pathway (Miao et al., 2006a) and cdaPS3 from the S. coelicolor pathway (Hojati et al., 2002), both under control of ermEp*. In this study, we have further explored this NRPS expression system and reconstituted the daptomycin pathway by in trans complementation of two NRPS subunits from ectopic loci in the S. roseosporus chromosome. We demonstrated that it is possible to have robust production of A21978C when genes encoding DptBC, DptE and DptF proteins are expressed from the native locus, while those for subunits DptA and DptD are expressed from other chromosomal sites.
The organization of the daptomycin gene cluster was a consideration during design of the double trans-complementation, since deletion of dptA might disrupt transcription of the dptBC gene downstream. Large polycistronic transcripts have been found in some secondary metabolic pathways: a transcript greater than 16 kb, including six cephamycin C biosynthetic genes, is driven from the pcbAB promoter in Nocardia lactamdurans (Enguita et al., 1998) and a transcript of 35 kb, including seven erythromycin biosynthetic genes, is generated from the eryAI promoter in Saccharopolyspora erythraea (Reeves et al., 1999). The successful RT-PCR amplification across adjacent dpt genes from UA343 (dptE–dptF, dptF–dptA, dptA–dptBC, dptBC–dptD, dptD–dptG, dptG–dptH) allows the possibility of a single transcriptional unit. The forward primers were situated well upstream of the initiation codon of the downstream gene to exclude the possibility of encompassing untranslated leader sequences. In transcripts from other secondary metabolite biosynthetic pathways, there may be little or no leader (Reeves et al., 1999), although transcription starting 133–180 nt upstream of the translation start has been reported, and in one case 69–70 nt were inside the 3' end of the gene upstream (Enguita et al., 1998). The forward primer for the dptA–dptBC junction is over 600 nt upstream of the presumed initiation codon of dptBC, making capture of a product representing a leader sequence highly unlikely. Purine-rich sequences 5–7 nt upstream of the presumed translational starts of the terminally overlapping NRPS genes that may facilitate ribosome binding leave open the possibility of translational coupling of a large polycistronic transcript for the entire 48.8 kb region. This type of organization and possible translational coupling has been described in the tylosin biosynthetic gene cluster (Cundliffe et al., 2001).
Our model of a promoter upstream of dptE regulating transcription of a large polycistronic message, including the NRPS genes and other genes downstream, guided construction of the deletion host/complementation plasmid combinations. In the host UA474 and its derivative, MF6, the deletion of dptA leaves a small open reading frame to maintain the natural terminal overlap between dptA and dptBC. Restoration of A21978C production in MF6 after complementation, with the dptEFA genes outside the daptomycin gene cluster, showed that dptBC did not have a unique promoter within the deleted region of dptA, and further suggests that dptBC is regulated by a promoter upstream, either within dptF, dptE or further upstream of dptE: in an intact gene cluster, this promoter would also determine transcription of dptA in addition to dptE, dptF and dptBC. The functional expression of dptA from pLT01, which lacks an added promoter but contains dptE, dptF and three genes upstream (truncated dptP, dptM and dptN), is consistent with the presence of a native promoter upstream of dptA. While the possibilities of additional or secondary promoters are not excluded, the current observations showed that expression of the genes remaining at the native locus, dptE, dptF and dptBC, can be complemented by ectopic expression of dptEFA and dptD to restore antibiotic production. Similarly, in Bacillus subtilis (Guenzi et al., 1998), the physical dissociation of the thioesterase from the last amino acid of the surfactin synthetase did not affect the level of expression of the lipopetides.
We also investigated the impact of different promoters on lipopeptide production. The lipopeptide yield with dptA expressed in trans from a native promoter, dptBC from its native locus from a native promoter, and dptD in trans from ermEp* was about 58 % of control (Table 3). In previous work (Miao et al., 2006b), complementation of a single dptD mutation by dptD in trans from ermEp* yielded about 90 % of control. However, when both dptA and dptD were expressed ectopically from ermEp*, and dptBC from a native promoter at the native locus, the yield was improved to 80 % of control; equivalent results were obtained by positioning ermEp* in front of dptEFA or dptA (Table 3).
Given the excellent yields associated with ermEp* regulation of dptA and dptD, it was surprising that when the dptBC gene at the native locus is regulated by ermEp*, and dptA was expressed from a native promoter, the lipopeptide production was only 9 % of control. It is possible that the overproduction of the DptBC subunit from the native locus adversely affected the stoichiometry of the three subunits, or the expression of other genes.
The results in this study demonstrate that the dpt NRPS genes need not be expressed from the native dpt locus to obtain high yields of A21978C factors. The dptA and dptD genes can be conveniently expressed from the ermEp* promoter from ectopic positions in the chromosome. Using this configuration, we showed that dptD could be replaced by the heterologous lptD and cdaPS3 to produce daptomycin analogues with substitutions at position 13 at 40 % and 69 % of control yields, respectively. This compares favourably with the constructs containing dptA (and all other dpt genes except dptD) present in the native dpt locus, where recombinants with lptD and cdaPS3 produced 25 % and 50 % of control (Miao et al., 2006b). The apparent improvement in yields may be due to the presence of ermEp* driving the expression of dptA. This cloning system is now well suited to explore module exchanges in dptA, coupled with subunit exchanges for dptD to generate additional derivatives of daptomycin for evaluation. The demonstration of robust ectopic expression of two NRPS subunits in daptomycin biosynthesis, using ermEp* to drive transcription, also suggests that ectopic expression driven by strong constitutive promoters might also work in other multi-modular, multi-subunit NRPS biosynthetic pathways to engineer peptide or mixed peptide/polyketide derivatives not readily amenable to chemical synthesis or scale up.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 24 March 2006;
revised 22 June 2006;
accepted 23 June 2006.
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