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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Supplementary material
  8. Acknowledgements
  9. References
  10. Supporting Information

The glycosyltransferases OleG1 and OleG2 and the cytochrome P450 oxidase OleP from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus have been expressed, either separately or from artificial gene cassettes, in strains of Saccharopolyspora erythraea blocked in erythromycin biosynthesis, to investigate their potential for the production of diverse novel macrolides from erythronolide precursors. OleP was found to oxidize 6-deoxyerythronolide B, but not erythronolide B. However, OleP did oxidize derivatives of erythronolide B in which a neutral sugar is attached at C-3. The oxidized products 3-O-mycarosyl-8a-hydroxyerythronolide B, 3-O-mycarosyl-8,8a-epoxyerythronolide B, 6-deoxy-8-hydroxyerythronolide B and the olefin 6-deoxy-8,8a-dehydroerythronolide B were all isolated and their structures determined. When oleP and the mycarosyltransferase eryBV were co-expressed in a gene cassette, 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B was directly obtained. When oleG2 was co-expressed in a gene cassette together with oleP, 6-deoxyerythronolide B was converted into a mixture of 3-O-rhamnosyl-6-deoxy-8,8a-dehydroerythronolide B and 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B, confirming previous reports that OleG2 can transfer rhamnose, and confirming that oxidation by OleP and attachment of the neutral sugar to the aglycone can occur in either order. Similarly, four different 3-O-mycarosylerythronolides were found to be substrates for the desosaminyltransferase OleG1. These results provide additional insight into the nature of the intermediates in OleP-mediated oxidation, and suggest that oleandomycin biosynthesis might follow parallel pathways in which epoxidation either precedes or follows attachment of the neutral sugar.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Supplementary material
  8. Acknowledgements
  9. References
  10. Supporting Information

The biosynthetic pathways to the macrolide antibiotics produced by actinomycete bacteria generally involve the assembly of an aglycone structure, followed by specific modifications which may include hydroxylation or other oxidative steps, methylation and glycosylation. As these modifications are often critical for activity (Liu and Thorson, 1994; Kaneko et al., 2000), there has been increasing interest in understanding the mechanism and specificity of these enzymes, and in harnessing this knowledge to engineer the biosynthesis of diverse novel hybrid macrolides with potentially improved activities. In the case of the 14-membered macrolide erythromycin A (Fig. 1) in Saccharopolyspora erythraea these late-stage modifications consist of hydroxylation at C-6 by the EryF cytochrome P450 (Haydock et al., 1991; Weber et al., 1991) to form erythronolide B; the attachment of mycarose via the hydroxyl group at C-3 catalysed by the mycarosyltransferase EryBV (Gaisser et al., 1997; Summers et al., 1997; Salah-Bey et al., 1998) followed by the attachment of desosamine via the hydroxyl group at C-5, catalysed by the desosaminyltransferase EryCIII (Summers et al., 1997; Salah-Bey et al., 1998) to produce erythromycin D, the first intermediate with antibiotic activity. Erythromycin D is subsequently converted to erythromycin A by hydroxylation at C-12 (Stassi et al., 1993) by EryK and by O-methylation on the mycarosyl group by the methyltransferase EryG (Paulus et al., 1990; Haydock et al., 1991), this order being preferred (Lambalot et al., 1995).

image

Figure 1. Structures of erythromycin A and oleandomycin.

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Similarly, the gene products of oleG2, oleY, oleP and oleG1 have been implicated (Rodriguez et al., 1995; Olano et al., 1998) in the late-stage modifications that lead to the 14-membered macrolide oleandomycin (Fig. 1) in Streptomyces antibioticus. The glycosyltransferases OleG2 and OleG1 have been shown to govern the attachment of the neutral sugar at C-3 and of desosamine at C-5 respectively (Doumith et al., 1999). More recently, OleG2 has been identified as transferring L-olivose, after which the L-olivose residue attached to the oleandomycin aglycon is converted into L-oleandrose by the action of the OleY methyltransferase (Rodriguez et al., 2001). As with erythromycin, transfer of the deoxyaminosugar to C-5 appears to follow attachment of the neutral sugar at C-3 (Rodriguez et al., 2001). However, there remain significant uncertainties about the sequence of events, not least about the timing of the oxidative steps which lead to the introduction of an epoxy group at C8-C8a (Fig. 1) and which require OleP (Rodriguez et al., 1995; Shah et al., 2000). The pioneering work of Spagnoli and colleagues (Spagnoli et al., 1983) showed that erythronolide B was bioconverted by a mutant of S. antibioticus blocked in endogenous aglycone biosynthesis into novel hybrid macrolides, including the same deoxysugars as oleandomycin, and with either the epoxy group at C8-C8a, or a hydroxyl group at C-8, or no oxidative modification at all. They speculated that oxidation happened after attachment of both sugars. However, it has since been shown that chemically synthesized aglycones, already oxidized at C8-C8a, are smoothly converted into oleandomycin by S. antibioticus (Tatsuta et al., 1990). Recently, the heterologous co-expression of OleP and of 6-deoxyerythronolide B synthase in Streptomyces lividans was shown to result in the production of at least six novel minor compounds derived by oxidation from 6-deoxyerythronolide B (Shah et al., 2000), which is consistent with the findings of Tatsuta and colleagues. One of these compounds was analysed by nuclear magnetic resonance spectroscopy (NMR) and identified as 8,8a-dihydroxy-6-deoxyerythronolide B (Shah et al., 2000), and these authors speculated that epoxidation precedes glycosylation. Rodriguez and colleagues (Rodriguez et al., 2001) have likewise suggested that the introduction of the C-8-epoxy group by OleP is the first modification event (Rodriguez et al., 2001).

An alternative view is that there may be more than one pathway for the late-stage modifications that lead to the production of oleandomycins. As anticipated by the work of Spagnoli and colleagues (Spagnoli et al., 1983), and the work on EryG and EryK discussed above, at least some modification enzymes appear to have a relaxed substrate specificity that would permit such parallel pathways to operate. This flexibility would also support their use in generating additional diversity in polyketide macrolide structures. Thus, the PikC cytochrome P450 of Streptomyces venezuelae is known to hydroxylate both a 12- and a 14-membered macrolide substrate (Xue et al., 1998). Even more strikingly, the glycosyltransferase OleG2, when expressed in S. erythraea, is capable of attaching L-rhamnose at position C-3 of the heterologous aglycones erythronolide B and 6-deoxyerythronolide B (Doumith et al., 1999; Gaisser et al., 2000).

In the present work, we have created hybrid macrolides by expressing oleG2, oleP and oleG1 in mutant strains of S. erythraea in which they can act on erythronolide templates. In these experiments, the relaxed specificity of OleG2 was used to introduce rhamnose as the neutral sugar (Doumith et al., 1999; Gaisser et al., 2000). A convenient method was used to construct gene cassettes of oleG2, oleP, oleG1 and eryBV in different combinations. Novel compounds were identified and isolated from the culture supernatant and their structures determined. In the course of this work, evidence was obtained that oxidation could either precede or follow the attachment of a neutral sugar, which suggests that such parallel pathways may operate in the biosynthesis of oleandomycin.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Supplementary material
  8. Acknowledgements
  9. References
  10. Supporting Information

The expression plasmids pSGOleP and pSGOleG1 were isolated as described in Experimental procedures. The isolation of plasmid pSG142 (the expression vector for eryBV) and pSGOleG2 (the pSG142-derived expression vector for oleG2) has been described previously (Gaisser et al., 2000).

Analysis of the substrate specificity of OleP using the S. erythraea mutant SGT2(ΔeryBVΔeryCIIIΔeryA) as strain background for feeding assays

Saccharopolyspora erythraea SGT2 (Gaisser et al., 2000) was transformed with the plasmid construct pSGOleP. The transformants were verified by isolating chromosomal DNA followed by polymerase chain reaction (PCR) analysis. Feeding experiments were carried out using each of 6-deoxyerythronolide B (1) (Fig. 2), erythronolide B, 3-O-mycarosylerythronolide B (7), 3-O-rhamnosylerythronolide B (Gaisser et al., 2000) and erythromycin A (Fig. 1). The compounds were added to cultures of the strains SGT2 and SGT2pSGOleP using methods described previously (Gaisser et al., 2000), incubated at 30°C for 3–5 days and analysed using electrospray mass spectrometry. Novel peaks were visible in supernatants of SGT2pSGOleP fed with 6-deoxyerythronolide B (m/z 434 [M]NH4+), 3-O-mycarosylerythronolide B (m/z 578 [M]NH4+ and m/z 580 [M]NH4+) and 3-O-rhamnosylerythronolide B (m/z 580 [M]NH4+ and m/z 582 [M]NH4+) (data not shown). Novel compounds were not detected in supernatants from cultures supplemented with erythronolide B or erythromycin A. MS/MS analysis of the novel compounds indicated the presence of small amounts of 8,8a-epoxy- or 8,8a-dihydroxy-derivatives of deoxyerythronolide B, 3-O-mycarosylerythronolide B, and 3-O-rhamnosylerythronolide B respectively. To confirm the structures of the compounds derived from 3-O-mycarosylerythronolide B, strain SGT2pSGOleP was grown and feeding experiments were carried out as described in Experimental procedures. The novel compounds were isolated and their structures were identified as 3-O-mycarosyl-8a-hydroxyerythronolide B (8) and 3-O-mycarosyl-8,8a-epoxyerythronolide B (9) (Fig. 2) respectively. The NMR assignments of these compounds are given in Supplementary material.

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Figure 2. Bioconversion of erythronolide substrates by cytochrome P450 OleP and desosaminyltransferase OleG1 in S. erythraea. The structures of compounds 2, 3, 4 (Shah et al., 2000), 6, 8. 9, 11 and 12 were all confirmed by NMR analysis. Substrates for the desosaminyltransferase OleG1, which attaches desosamine to the hydroxyl at C-5 of the macrolactone ring, are shown boxed.

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Analysis of the substrate specificity of OleP using the S. erythraea mutant SGT1 as strain background for feeding assays

To prevent the conversion of 6-deoxyerythronolide B to erythronolide B during feeding assays, a deletion of the eryF gene was introduced into the genome of the S. erythraea mutant strain DM (Gaisser et al., 2000) to create SGT1 (ΔeryBV, ΔeryCIII, ΔeryF) using standard microbiological techniques, and plasmids pSGOleP, pSGOleG2 and pSGOleG1 were used for its transformation as described in Experimental procedures. The transformants were verified by isolating chromosomal DNA followed by PCR analysis. Cultures of SGT1 and SGT1pSGOleP, and SGT1pSGOleG2 were grown, and the supernatants were analysed using electrospray mass spectrometry as described previously (Gaisser et al., 2000). The supernatants of SGT1 still contained 6-deoxyerythronolide B only. Three compounds isolated from a 4 l culture supernatant of SGT1pSGOleP (Fig. 3) were analysed by liquid chromotography-mass spectrometry (LCMS) and characterized by NMR spectroscopy and the product with m/z 401 [M-H2O]H+ was identified as the 8,8a-dihydroxy derivative of 6-deoxyerythronolide B (4) re-cently reported by others (Shah et al., 2000). The structure of the compound with m/z of 385 [M]H+ was revealed as 8,8a-dehydro-6-deoxyerythronolide B (2) and a further (minor) compound of the culture supernatant was identified as 6-deoxy-8-hydroxyerythronolide B (3). In the culture supernatant of SGT1pSGOleG2, a major new peak representing 3-O-rhamnosyl-6-deoxyerythronolide B (10) was detected (Fig. 3). The NMR assignments of the novel compounds are presented in Supplementary material.

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Figure 3. Electrospray mass spectrometry analyses of the culture supernatants of SGT1, SGT1pSGOleG2 and SGT1pSGOleP (A) and SGQ1, and SGT1pSGOleP (B) after feeding with 3-O-rhamnosyl-6-deoxyerythronolide B.

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Analysis of the substrate specificity of OleP using the S. erythraea mutant SGQ1 as strain background for feeding assays

The S. erythraea mutant SGQ1 (SGT2ΔeryF), which is deficient in endogenous macrolactone formation, was isolated using standard microbiological techniques and used for transformation with pSGOleG2, pSGOleP and pSGOleG1. Feeding experiments were carried out by adding sterile-filtered culture supernatants of SGT1 (containing 6-deoxyerythronolide B) to cultures of SGQ1, SGQ1pSGOleG2, SGQ1pSGOleP and SGQ1pSGOleG1 respectively, as described previously (Gaisser et al., 2000). Analysis of the culture supernatants confirmed that OleG2, and to a smaller extent OleP, accept 6-deoxyerythronolide B (1) as a substrate. To investigate whether OleP accepts 3-O-rhamnosyl-6-deoxyerythronolide B (10) as a substrate, culture supernatants of SGT1pSGOleG2 were sterile-filtered and added to cultures of SGQ1, SGQ1pSGOleG2, and SGQ1pSGOleP as described above. The analysis of these culture supernatants clearly showed that 3-O-rhamnosyl-6-deoxyerythronolide B is indeed a substrate for OleP (Fig. 3). The results of the MS/MS analysis indicated the presence of 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxy-erythronolide B (12) in the culture supernatants of SGQ1pSGOleP. To assess whether OleP accepts 3-O-mycarosyl-6-deoxyerythronolide B (5) (Fig. 2) as a substrate, culture supernatants of SGT1pSG142 (expressing eryBV) were sterile-filtered and added to cultures of SGQ1 and SGQ1pSGOleP. The analysis of the supernatant suggested the presence of 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B (6) in the culture supernatants of SGQ1pSGOleP.

Analysis of the substrate specificity of OleP using gene cassettes

To isolate 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B (12) and 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B (6) in sufficient amounts for NMR analysis, the gene cassettes pSGcasoleG2oleP and pSGcaseryBVoleP were isolated as described in Experimental procedures and used to transform S. erythraea SGT1. The culture supernatants of SGT1pSGcasoleG2oleP were analysed as described earlier and three major peaks were detected representing 3-O-rhamnosyl-6-deoxyerythronolide B (10) (m/z 550 [M]NH4+), 3-O-rhamnosyl-6-deoxy-8,8a-dehydroerythronolide B (11) (m/z 548 [M]NH4+) and 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B (12) (m/z 564 [M-H2O]NH4+). In the culture supernatants of SGT1pSGcaseryBVoleP, a major peak representing 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B (6) (m/z 567 [M-H2O]Na+) was detected. The structure of the novel compounds were confirmed by NMR analysis and the data are given in Supplementary material.

6-Deoxy-8,8a-dehydroerythronolide B as an intermediate of the C-8-epoxidation reaction

To establish whether 6-deoxy-8,8a-dehydroerythronolide B (2) represents a chemically competent intermediate in the epoxidation of 6-deoxyerythronolide B (1), a culture of SGT1pSGOleP was grown and samples were taken for analysis every 24 h. The amount of ‘2’ was found to reach a maximum after 3–4 days and then decrease (data not shown). Compound ‘2’ was added to cultures of SGQ1 and SGQ1pSGOleP, and LCMS analysis of the culture supernatants was carried out as described elsewhere (Gaisser et al., 2000). The results showed that in the presence of OleP, 6-deoxy-8,8a-dehydroerythronolide B (2) was converted to 6-deoxy-8,8a-dihydroxyerythronolide B (4) and a further, unknown compound. Preliminary MS data indicated that this compound could be 6-deoxy-8,8a-epoxyerythronolide B, but the amount was too low for NMR analysis (data not shown). A similar feeding assay was also carried out using 6-deoxy-8,8a-dihydroxyerythronolide B (4) and 6-deoxy-8-hydroxyerythronolide B (3) as substrates but no further conversion of these compounds was detected in the presence of OleP. An overview of the range of substrates on which OleP was found to act is illustrated in Fig. 2 and summarized in Table 1.

Table 1. Substrate range of OleG1 and OleP.
SubstrateOleG1OleP
Erythronolide BND
6-Deoxyerythronolide B+
3-O-Mycarosylerythronolide B++
3-O-Mycarosyl-6-deoxyerythronolide B++
3-O-Rhamnosylerythronolide B+
3-O-Rhamnosyl-6-deoxyerythronolide B+
3-O-Mycarosyl-8,8a-epoxyerythronolide B+ND
3-O-Mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B+ND
6-Deoxy-8,8a-dehydroerythronolide BND+
6-Deoxy-8,8a-dihydroxyerythronolide BND
6-Deoxy-8-hydroxyerythronolide BND
Erythromycin AND

Analysis of the substrate specificity of OleG1

To determine the substrate specificity of OleG1 the S. erythraea strains, SGT1 and SGT1pSGOleG1 were grown and analysed as described elsewhere (Gaisser et al., 2000). No differences were detected indicating that 6-deoxyerythronolide B (1) is not a substrate for OleG1 (data not shown). This result was confirmed by feeding sterile-filtered supernatant of SGT1 to SGQ1pSGOleG1 followed by the analysis of the culture supernatant (data not shown). Similar results were obtained when sterile-filtered culture supernatants of SGT1pSGOleG2 were added to SGQ1 and SGQ1pSGOleG1, suggesting that 3-O-rhamnosyl-6-deoxyerythronolide B (10) is not accepted as a substrate by OleG1 (data not shown). Cultures of SGQ1pSGOleG1 were grown and fed with 3-O-mycarosylerythronolide B (7), 3-O-mycarosyl-6-deoxyery-thronolide B (5), 3-O-rhamnosylerythronolide B, 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B (12), 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B (6) and 3-O-mycarosyl-8,8a-epoxyerythronolide B (9) as described previously (Gaisser et al., 2000).

Analysis of the culture supernatants using LCMS (Gaisser et al., 2000) showed that 3-O-rhamnosylerythronolide B and 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxy-erythronolide B were not accepted as substrates by OleG1 (data not shown). The analysis of the supernatants also revealed that 3-O-mycarosylerythronolide B is a substrate for OleG1, indicated by small peaks with m/z 734 and m/z 718, representing erythromycin A and erythromycin B respectively (data not shown). This result confirmed earlier findings that oleG1 can to some extent complement an eryCIII mutation in S. erythraea (Doumith et al., 1999). The analysis of the culture supernatant containing 3-O-mycarosyl-6-deoxyerythronolide B (5) also revealed novel peaks, with the major compound (m/z of 718) identified as 6-deoxyerythromycin A, because its MS/MS fragmentation pattern exactly matched that of an authentic sample. 3-O-mycarosyl-8,8a-epoxyerythronolide B (9) was also tested as a substrate for OleG1 (Gaisser et al., 2000). The presence of a small peak with m/z 718 [M]H+ and 740 [M]Na+ in the LCMS chromatogram indicates that this compound is also a substrate for OleG1. The MS/MS analysis suggests that the desosaminylated derivative of 3-O-mycarosyl-8,8a-epoxyerythronolide B (9) is not accepted as a substrate by EryG and EryK, indicated by the loss of 144 atomic mass units representing mycarose rather than cladinose, and a fragment with m/z 574 [M-144]H+ indicating that the C-12 hydroxylation has not occurred. 3-O-MYcarosyl-6-deoxy-8,8a-dihydroxyerythronolide B (6) was also further processed in the presence of OleG1 as indicated by a small peak of m/z 718 [M]H+. The substrate range tested for OleG1 is summarized in Table 1.

Analysis of the substrate specificity of OleG1 using gene cassettes

Results of the feeding assays were confirmed when the gene cassettes pSGcasoleG2oleG1olePhis and pSGcaseryBVolePoleG1his were used to transform S. erythraea SGT1 as described in Experimental procedures. A major peak of m/z 569 was detected in the LCMS spectrum of the culture supernatants of SGT1pSGcas-oleG2oleG1olePhis representing 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B (12) but as expected from the results of the feeding assays the desosaminylated derivative of this compound could not be detected. The analysis of the culture supernatants of SGT1pSGcasBVolePoleG1his indicated the presence of a mixture of products with two major peaks representing desosaminylated compounds with m/z of 702 and 718 respectively. To investigate these compounds in more detail, 4 l of cultures of SGT1pSGcaseryBVolePoleG1his were grown and the supernatant was analysed using LCMS (Gaisser et al., 2000). The analysis of the major compound (m/z 718) showed that it contained mycarose and desosamine as attached sugars (in other words, that it is a derivative of 6-deoxyerythromycin C), and its MS/MS fragmentation pattern was consistent with oxidation at C8-C8a having occurred to form either the C8-C8a epoxy- or the C8-C8a-dihydroxy group (these are indistinguishable in the analysis).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Supplementary material
  8. Acknowledgements
  9. References
  10. Supporting Information

The recent characterization of numerous macrolide biosynthetic pathways in actinomycete bacteria has triggered considerable interest in the specificity of the late-stage modifications undergone by these polyketide secondary metabolites (for reviews, see Hallis and Liu, 1999; Trefzer et al., 1999; Kaneko et al., 2000). In the present work, we have directly evaluated the substrate specificity of the cytochrome P450 enzyme OleP and of the desosaminyltransferase OleG1, first through feeding studies and then through the creation of S. erythraea bioconversion strains in which combinations of the genes oleG2, oleP, oleG1 and eryBV are expressed in artificial gene cassettes.

OleP has been shown to operate on a range of 14-membered macrolide substrates, as summarized in Fig. 2 and Table 1. No evidence was found that any derivative of erythromycin, with sugars attached via the hydroxy groups at both C-3 and C-5, could serve as a substrate for OleP (data not shown). However, in agreement with previous findings (Shah et al., 2000), 6-deoxyerythronolide B can be used directly as a substrate. The isolation for the first time of the olefinic compound (2), together with the demonstration that it can be further oxidized by OleP-containing S. erythraea strains, fully supports the earlier evidence of the intermediacy of such an olefin in OleP-mediated oxidation (Tatsuta et al., 1990). The final oxidation products are a compound which has been tentatively identified as the 8,8a-epoxymacrolactone, and the 8,8a-dihydroxy compound (4), which is likely to be derived from the 8,8a-epoxymacrolactone by adventitious hydrolysis (Shah et al., 2000). In contrast, the 8-hydroxy compound (3) and the 8,8a-dihydroxy compound (4) were not further modified on being re-incubated with OleP-containing S. erythraea strains, implying perhaps that if analogous compounds are seen in oleandomycin biosynthesis they are likely to be shunt metabolites.

The monoglycosides 3-O-rhamnosyl-6-deoxyerythronolide B (10) and 3-O-mycarosyl-6-deoxyerythronolide B (5) are also oxidized by OleP (Fig. 2 and Table 1). Further evidence for the intervention of olefinic intermediates in OleP-mediated oxidation was provided by the isolation of 3-O-rhamnosyl-6-deoxy-8,8a-dehydroerythronolide B (11) (Fig. 2 and Table 1). It was initially surprising that erythronolide B was not a substrate for oxidation by OleP, as Spagnoli and colleagues (Spagnoli et al., 1983) had shown that erythronolide B could readily be converted into oleandomycin-like macrolides. An explanation for this apparent paradox is offered by our finding (Fig. 2 and Table 1) that attachment of a neutral sugar (L-rhamnose or L-mycarose) at C-3 of erythronolide B converts it back into a substrate for OleP, despite the presence of the C-6 hydroxyl group. The isolation of 3-O-mycarosyl-8,8a-epoxyerythronolide B (9) in feeding experiments with this monoglycoside provides further support for the idea (Rodriguez et al., 1995) that OleP is solely responsible for the introduction of the 8,8a-epoxy group, although this question will not be completely resolved until oxidation experiments are carried out with purified OleP enzyme. As Tatsuta and colleagues (Tatsuta et al., 1990) had already shown that oleandolide is efficiently incorporated into oleandomycin, the oxidation of monoglycosylated erythronolides represents an alternative or parallel pathway to the final macrolide structure. We therefore examined whether the oleandomycin desosaminyltransferase OleG1 could recognize such oxidized intermediates as substrates.

First, we identified by experiment (data not shown) the optimal start codon for the expression of active OleG1 glycosyltransferase in S. erythraea, which differed from the published start codon (EMBL database accession no. AJ002638). The alignment of the N-terminal regions of several glycosyltransferases involved in macrolide biosynthesis underlines the unusual character of the OleG1 N-terminal region (data not shown). Comparison of the structures of the erythronolides accepted by OleG1 as substrates (Fig. 2) shows that the activity of this glycosyltransferase is less sensitive to the oxidation level at C8 than to the nature of the substituent at C-6, and (particularly) the identity of the sugar residue attached to C-3-OH of the aglycone. Only 3-O-mycarosyl- and not 3-O-rhamnosyl-substituted macrolactones were accepted as substrates. In contrast, the erythromycin desosaminyltransferase EryCIII readily accepts 3-O-rhamnosylerythronolide B, 3-O-(2′-O-methylrhamnosyl)erythronolide B and 3-O-(2′,3′-bis-O-methylrhamnosyl)erythronolide B as alternative substrates (Gaisser et al., 2001).

The data presented in this paper do not allow definite conclusions to be drawn about which of the parallel pathways for the late modifications in oleandomycin biosynthesis is actually preferred. The isolation of particular intermediates provides only a qualitative picture of events. However, initial transfer of the neutral sugar to the aglycone, catalysed by either OleG2 and EryBV, appeared in these experiments to compete efficiently with C-8 modification involving OleP. If the preferred pathway for oleandomycin biosynthesis starts with the transfer of the neutral sugar residue to C-3-OH of the aglycone by OleG2 [which is then converted to L- oleandrose by OleY, as proposed by Rodriguez and colleagues (Rodriguez et al., 2001) ] followed by the introduction of the 8,8a-epoxy- group by OleP, then the natural substrate for OleG1 should be 3-O-oleandrosyl-8,8a-epoxy-6-deoxyoleandolide B. As the genes for activated L-olivose and L-oleandrose synthesis are now available on plasmids (Aguirrezabalaga et al., 2000), the approach used here could be readily extended to prepare this substrate in S. erythraea, so that the idea could be tested.

OleG1, in particular, does not seem to be very flexible in its substrate specificity. However, the methods described here can be used to identify other modification enzymes with more relaxed specificities. The rapid assembly of expression gene cassettes for these enzymes, as demonstrated here, should greatly speed up this process by allowing many permutations and combinations to be tried in parallel.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Supplementary material
  8. Acknowledgements
  9. References
  10. Supporting Information

Bacterial strains, plasmids and growth conditions

The strains and vectors used in this work are shown in Table 2. Other bacterial strains and growth conditions were as described (Gaisser et al., 2000). Expression vectors in Saccharopolyspora erythraea were derived from plasmid pSG142 (Gaisser et al., 2000). Plasmid 3gh2 and plasmid eryF702 were the kind gifts of Drs C. Olano and J. Cortés respectively. 3-O-Mycarosylerythronolide B was kindly provided by B. Wilkinson (Glaxo Wellcome). Plasmid-containing S. erythraea were selected with 25 μg ml−1 of thiostrepton. To investigate the production of antibiotics, S. erythraea strains were grown in sucrose-succinate medium (Caffrey et al., 1992) as described (Gaisser et al., 1997) and the cells were harvested by centrifugation. For the preparation of 3-O-mycarosyl-8a-hydroxyerythronolide B (8) and 3-O-mycarosyl-8,8a-epoxyerythronolide B (9), 2.5 l of culture supernatant of SGT2pSGOleP was fed with 60 mg of 3-O-mycarosylerythronolide B (7). The culture was incubated at 30°C for 4 days and the supernatant was used to isolate the compounds using the methods described in this section. Then, 1.7 mg of 3-O-mycarosyl-8,8a-epoxyerythronolide B (9) and 1.9 mg of 6-deoxy-8-hydroxyerythronolide B (3) were each dissolved in 1 ml of methanol, and 200 μl was used for feeding assays. Next, 4.1 mg of 6-deoxy-8,8a-dehydroerythronolide B (2), 4.6 mg of 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B (12) and 9.8 mg of 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B (6) were each dissolved in 1 ml of methanol, and 100, 50 and 25 μl of these stock solutions were used for feeding assays.

Table 2. Strains of Saccharopolyspora erythraea and plasmids used in this work.
Strains/plasmidsCharacteristicsSource
Strains
NRRL 2338Wt-red variant Hessler et al. (1997)
SGT2ΔeryBVΔeryCIIeryA Gaisser et al. (2000)
SGT1ΔeryBVΔeryCIIIΔeryFThis study
SGQ1ΔeryBVΔeryCIIIΔeryFΔeryAThis study
Plasmids
pSG142Derivative of pSG2414 containing a 1.7 kb NcoI fragment from the ermE distal flank of the ery cluster Gaisser et al. (2000)
pSGOleG2Derivative of pSG142 where eryBV is replaced with oleG2 using NdeI and BglII restriction sites. Gaisser et al. (2000)
pSGOlePDerivative of pSG142 where eryBV is replaced with oleP using NdeI and BglII restriction sites.This study
pSGOleG1Derivative of pSG142 where eryBV is replaced with oleG1 using NdeI and BglII restriction sites.This study
pSGcasoleG2olePDerived from pSG142 to express oleG2 and olePThis study
pSGcaseryBVolePDerived from pSG142 to express eryBV and olePThis study
pSGcasoleG2oleG1olePhisDerived from pSG142 to express oleG2, oleP and oleG1This study
pSGcaseryBVolePoleG1hisDerived from pSG142 to express eryBV, oleP and oleG1This study

DNA manipulation and sequencing

DNA manipulations, polymerase chain reaction (PCR) and electroporation procedures were carried out as described by Sambrook and colleagues (Sambrook et al., 1989). Protoplast formation and transformation procedures of S. erythraea were as described (Gaisser et al., 1997). DNA sequencing was performed by the method of Sanger and colleagues (Sanger et al., 1977), using automated DNA sequencing on double-stranded DNA templates with an Applied Biosystems 373A sequencer. Sequence data were analysed using the Staden Programs (Staden, 1984) and the Genetics Computer Group (GCG, version 10) software package (Devereux et al., 1984).

Extraction and mass spectrometry

Extractions and mass spectrometry were carried out as described previously (Gaisser et al., 2000).

Isolation of the S. erythraea mutants SGT1 and SGQ1

Plasmid eryF702 (J. Cortés) was used to introduce an approximately 1 kb deletion of eryF (Weber et al., 1991) into the genome of the S. erythraea mutant strains DM and SGT2 (Gaisser et al., 2000). Transformations, selection of thiostrepton-resistant colonies, subculturing and the selection of thiostrepton-sensitive mutants were carried out as described previously (Gaisser et al., 1997). The S. erythraea mutants SGT1 and SGQ1 were isolated and the introduction of an approximately 1 kb deletion into the chromosome of these strains was verified by PCR. The presence of 6-deoxyerythronolide B in the culture supernatant of SGT1 was confirmed using methods described in this section.

Construction of expression plasmid for oleP

For expression of oleP, the primers OleP1 5′-CTCC AGCAAAGGACACACCCATATGACCGATACGCACA-3′ and OleP2 5′-CGGCAGATCTGCCGGCCGTCACCAGGAGAC GATCTGG-3′ were used to amplify oleP, using plasmid 3gh2 as a template. The PCR fragment was isolated, treated with T4 polynucleotide kinase and cloned into SmaI-cut pUC18. After transformation into Escherichia coli DH10B, the construct was isolated and the sequence of oleP was verified. After digestion with NdeI–BglII, a 1.3 kb fragment was isolated, ligated with the vector fragment of NdeI–BglIIdigested pSG142 and used to transform E. coli DH10B. Plasmid pSGOleP was isolated.

Construction of expression plasmid for oleG1

To establish which of the various possible start codons in the published sequence (accession no. AJ002638) is used for the expression of oleG1, various constructs were tested by measuring the complementation of the eryCIII mutation in S. erythraea SGT2 after feeding with 3-O-mycarosylerythronolide B using the techniques described above. Small amounts of erythromycin A that indicate the complementation of the eryCIII deletion by oleG1 were only observed when vector pSGOleG1 was used. Expression vectors using the previously published start codon or one of the following in-frame ATG codons did not complement the eryCIII mutation of SGT2 after feeding with 3-O-mycarosylerythronolide B (data not shown). This result suggests that the start codon used for the expression of oleG1 is the ATG overlapping the oleP1 stop codon. Plasmid pSGOleG1 was isolated using the primers 7390 5′-CCGCCATATGAGCATCGCGTCGAACGGC GCGCGCTCGGC-3′ and Ole2 5′-TCAGATCTCCGCCTTCC CGCCATCGCGCCGGTGGCAT-3′ to amplify oleG1. The cloning procedure was as described for the construction of the expression plasmid for oleP.

Construction of gene cassettes

The expression of gene cassettes in the S. erythraea mutant strains SGT1 and SGQ1 under the control of the actII-Orf4 regulator was used to generate novel compounds. The strategy to obtain gene cassettes with different combinations of the genes oleG2, eryBV, oleG1 and oleP is based upon a proposal by Drs A. Ranganathan and T. Schwecke (personal communication) to exploit the possibility of controlled scission by the restriction enzyme XbaI (see below). This method was adapted to build up cassettes vectorially and sequentially, gene by gene. First, a PCR fragment bearing oleG2 was isolated, into which a HindIII and a NdeI restriction site were introduced at the 5′-end of the fragment and a XbaI, a BglII and a EcoRI site were introduced at the 3′-end of the PCR fragment (Fig. 4). This PCR fragment was cloned into SmaI-cut pUC18 for sequencing analysis. The oleG2 gene was then recloned into pUC19 using the restriction enzymes HindIII and EcoRI, and plasmid pUCcasOleG2 was isolated. For gene cassettes containing eryBV, a similar strategy was used to isolate the plasmid pUC18eryBV. The genes oleP and oleGI were each amplified using PCR techniques, introducing XbaI sites at the 5′-ends, which were sensitive to the Dam methyltransferase of E. coli, and XbaI sites at the 3′-ends, which, in contrast, were resistant to the Dam methyltransferase. To retain the Shine–Dalgarno sequence 5′ of the respective gene, the pSG142-derived constructs were used as a template to amplify oleP and oleG1 (with or without a C-terminal histidine tag). The PCR fragments were cloned into SmaI-cut pUC18 using standard molecular biological techniques, and the DNA sequences of these clones were confirmed by sequence analysis. After transforming the constructs into a dam strain background, the DNA was isolated and digested using XbaI. The fragments were isolated and ligated into the XbaI-cut vector pUCcasOleG2 or pUC18eryBV. The orientation of the insert was confirmed using suitable restriction digests. The protection of the XbaI site at the 5′-end of the gene by selective methylation can thus be used to create gene cassettes of different length and gene order in an iterative fashion (Fig. 4). After building the gene cassettes in the pUC18/pUC19 derived constructs, each plasmid was digested using the restriction enzymes NdeI–BglII and the DNA fragment encoding the gene cassette was isolated and cloned into the NdeI–BglII-digested vector DNA of pSG142 (Gaisser et al., 2000) followed by transformation of the S. erythraea mutant strains.

image

Figure 4. . Overview of the strategy for production of gene cassettes.

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Supplementary material

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Supplementary material
  8. Acknowledgements
  9. References
  10. Supporting Information

The following material is available from

The NMR assignments of the following novel compounds:

1H NMR data for 3-O-mycarosyl-8a-hydroxyerythronolide B

13C NMR data for 3-O-mycarosyl-8a-hydroxyerythronolide B

1H NMR data for 3-O-mycarosyl-8,8a-epoxyerythronolide B

13C NMR data for 3-O-mycarosyl-8,8a-epoxyerythronolide B

1H NMR data for 6-deoxy-8,8a-dehydroerythronolide B

13C NMR data for 6-deoxy-8,8a-dehydroerythronolide B

1H NMR data for 6-deoxy-8-hydroxyerythronolide B (CDCl3)

13C NMR data for 6-deoxy-8-hydroxyerythronolide B (CDCl3)

1H NMR data for 3-O-rhamnosyl-6-deoxy-8,8a-dehydroerythronolide B

13C NMR data for 3-O-rhamnosyl-6-deoxy-8,8a-dehydroerythronolide B

1H NMR data for 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B

13C NMR data for 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B

1H NMR data for 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B

13C NMR data for 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Supplementary material
  8. Acknowledgements
  9. References
  10. Supporting Information

We thank the staff of the Wellcome Trust/BBSRC DNA Sequencing Facility, Department of Biochemistry. We thank P. Gates and R. Sheridan for technical assistance. We also thank D. A. Hopwood, J. M. Weber and B. Wilkinson for kind gifts of materials. The work was supported by a project grant to P.F.L. and J.S. from the European Community (GENOVA, QLRT-1999–00095).

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  4. Results
  5. Discussion
  6. Experimental procedures
  7. Supplementary material
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Supplementary material
  8. Acknowledgements
  9. References
  10. Supporting Information

The NMR assignments of the following novel compounds: 1H NMR data for 3-O-mycarosyl-8a-hydroxyerythronolide B 13C NMR data for 3-O-mycarosyl-8a-hydroxyerythronolide B 1H NMR data for 3-O-mycarosyl-8,8a-epoxyerythronolide B 13C NMR data for 3-O-mycarosyl-8,8a-epoxyerythronolide B 1H NMR data for 6-deoxy-8,8a-dehydroerythronolide B 13C NMR data for 6-deoxy-8,8a-dehydroerythronolide B 1H NMR data for 6-deoxy-8-hydroxyerythronolide B (CDCl3) 13C NMR data for 6-deoxy-8-hydroxyerythronolide B (CDCl3) 1H NMR data for 3-O-rhamnosyl-6-deoxy-8,8a-dehydroerythronolide B 13C NMR data for 3-O-rhamnosyl-6-deoxy-8,8a-dehydroerythronolide B 1H NMR data for 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B 13C NMR data for 3-O-rhamnosyl-6-deoxy-8,8a-dihydroxyerythronolide B 1H NMR data for 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B 13C NMR data for 3-O-mycarosyl-6-deoxy-8,8a-dihydroxyerythronolide B

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