Sabine Gaisser

Analysis of seven genes from the eryAI –eryK region of the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

Abstract The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA, and lying between eryA and the gene eryK, which is known to encode the C-12 hydroxylase, has been sequenced and shown to contain seven additional open reading frames (ORFs 13–19). On the basis of sequence similarities, roles are proposed for several of these ORFs in the biosynthesis of the deoxysugar mycarose and the deoxyaminosugar desosamine. A chromosomal mutant carrying a deletion in ORF15 has been constructed and shown to accumulate 3-O-mycarosyl-erythronolide B, as expected for an eryC mutant. Similarly, a chromosomal mutant carrying a deletion in ORF16 has been constructed and shown to accumulate erythronolide B, as expected for an eryB mutant.

A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea

The biological activity of polyketide antibiotics is often strongly dependent on the presence and type of deoxysugar residues attached to the aglycone core. A system is described here, based on the erythromycin‐producing strain of Saccharopolyspora erythraea, for detection of hybrid glycoside formation, and this system has been used to demonstrate that an amino sugar characteristic of 14‐membered macrolides (d‐desosamine) can be efficiently attached to a 16‐membered aglycone substrate. First, the S. erythraea mutant strain DM was created by deletion of both eryBV and eryCIII genes encoding the respective ery glycosyltransferase genes. The glycosyltransferase OleG2 from Streptomyces antibioticus, which transfers l‐oleandrose, has recently been shown to transfer rhamnose to the oxygen at C‐3 of erythronolide B and 6‐deoxyerythronolide B. In full accordance with this finding, when oleG2 was expressed in S. erythraea DM, 3‐O‐rhamnosyl‐erythronolide B and 3‐O‐rhamnosyl‐6‐deoxyerythronolide B were produced. Having thus validated the expression system, endogenous aglycone production was prevented by deletion of the polyketide synthase (eryA) genes from S. erythraea DM, creating the triple mutant SGT2. To examine the ability of the mycaminosyltransferase TylM2 from Streptomyces fradiae to utilise a different amino sugar, tylM2 was integrated into S. erythraea SGT2, and the resulting strain was fed with the 16‐membered aglycone tylactone, the normal TylM2 substrate. A new hybrid glycoside was isolated in good yield and characterized as 5‐O‐desosaminyl‐tylactone, indicating that TylM2 may be a useful glycosyltransferase for combinatorial biosynthesis. 5‐O‐glucosyl‐tylactone was also obtained, showing that endogenous activated sugars and glycosyltransferases compete for aglycone in these cells.

Analysis of eryBI, eryBIII and eryBVII from the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

Abstract The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic β-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A␣mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis.

Optimizing natural products by biosynthetic engineering: Discovery of nonquinone Hsp90 inhibitors

A biosynthetic medicinal chemistry approach was applied to the optimization of the natural product Hsp90 inhibitor macbecin. By genetic engineering, mutants have been created to produce novel macbecin analogues including a nonquinone compound (5) that has significantly improved binding affinity to Hsp90 (Kd 3 nM vs 240 nM for macbecin) and reduced toxicity (MTD > or = 250 mg/kg). Structural flexibility may contribute to the preorganization of 5 to exist in solution in the Hsp90-bound conformation.

Rapamycin biosynthesis: elucidation of gene product function

The function of gene products involved in the biosynthesis of the clinically important polyketide rapamycin were elucidated by biotransformation and gene complementation.

Molecular Characterization of Macbecin as an Hsp90 Inhibitor

Macbecin compares favorably to geldanamycin as an Hsp90 inhibitor, being more soluble, stable, more potently inhibiting ATPase activity (IC50 = 2 microM) and binding with higher affinity (Kd = 0.24 microM). Structural studies reveal significant differences in their Hsp90 binding characteristics, and macbecin-induced tumor cell growth inhibition is accompanied by characteristic degradation of Hsp90 client proteins. Macbecin significantly reduced tumor growth rates (minimum T/C: 32%) in a DU145 murine xenograft. Macbecin thus represents an attractive lead for further optimization.

Parallel pathways for oxidation of 14‐membered polyketide macrolactones in Saccharopolyspora erythraea

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.

A locus coding for putative non-ribosomal peptide/polyketide synthase functions is mutated in a swarming-defective Proteus mirabilis strain

Abstract We describe a large bacterial locus that, unusually, encodes components typically required for both the non-ribosomal synthesis of peptides and also polyketide/fatty acid synthase function. Two tandem ABC transporter genes in this putative nrp (non-ribosomal peptide/polyketide) operon suggest that the principal product may be secreted. Immediately distal to the nrp operon is a gene, irpP, encoding a small peptide similar to the Bacillus ComX pheromone that in its mature, extracellular form increases expression of unlinked non-ribosomal peptide synthesis genes. Transcription of both the nrp operon and irpP was up-regulated in iron-limiting culture conditions, consistent with the presence of a putative Fur repressor-binding site 5′ of irpP. The locus was isolated from Proteus mirabilis as the site of a TnphoA insertion causing impaired swarm cell differentiation and an aberrant swarming pattern. The mutation was in one of the transporter genes, but a comparable swarming defect resulted from interposon disruption of the putative nrp synthetase gene.

New erythromycin derivatives from Saccharopolyspora erythraea using sugar O-methyltransferases from the spinosyn biosynthetic gene cluster.

Using a previously developed expression system based on the erythromycin‐producing strain of Saccharopolyspora erythraea, O‐methyltransferases from the spinosyn biosynthetic gene cluster of Saccharopolyspora spinosa have been shown to modify a rhamnosyl sugar attached to a 14‐membered polyketide macrolactone. The spnI, spnK and spnH methyltransferase genes were expressed individually in the S. erythraea mutant SGT2, which is blocked both in endogenous macrolide biosynthesis and in ery glycosyltransferases eryBV and eryCIII. Exogenous 3‐O‐rhamnosyl‐erythronolide B was efficiently converted into 3‐O‐(2′‐O‐methylrhamnosyl)‐erythronolide B by the S. erythraea SGT2 (spnI) strain only. When 3‐O‐(2′‐O‐methylrhamnosyl)‐erythronolide B was, in turn, fed to a culture of S. erythraea SGT2 (spnK), 3‐O‐(2′,3′‐bis‐O‐methylrhamnosyl)‐erythronolide B was identified in the culture supernatant, whereas S. erythraea SGT2 (spnH) was without effect. These results confirm the identity of the 2′‐ and 3′‐O‐methyltransferases, and the specific sequence in which they act, and they demonstrate that these methyltransferases may be used to methylate rhamnose units in other polyketide natural products with the same specificity as in the spinosyn pathway. In contrast, 3‐O‐(2′,3′‐bis‐O‐methylrhamnosyl)‐erythronolide B was found not to be a substrate for the 4′‐O‐methyltransferase SpnH. Although rhamnosylerythromycins did not serve directly as substrates for the spinosyn methyltransferases, methylrhamnosyl‐erythromycins were obtained by subsequent conversion of the corresponding methylrhamnosyl‐erythronolide precursors using the S. erythraea strain SGT2 housing EryCIII, the desosaminyltransferase of the erythromycin pathway. 3‐O‐(2′‐O‐methylrhamnosyl)‐erythromycin D was tested and found to be significantly active against a strain of erythromycin‐sensitive Bacillus subtilis.

Direct production of ivermectin-like drugs after domain exchange in the avermectin polyketide synthase of Streptomyces avermitilis ATCC31272

Ivermectin, a mixture of 22,23-dihydroavermectin B1a9 with minor amounts of 22,23-dihydroavermectin B1b 10, is one of the most successful veterinary antiparasitic drugs ever produced. In humans, ivermectin has been used for the treatment of African river blindness (onchocerciasis) resulting in an encouraging decrease in the prevalence of skin and eye diseases linked to this infection. The components of ivermectin are currently synthesized by chemical hydrogenation of a specific double bond at C22-C23 in the polyketide macrolides avermectins B1a 5 and B1b 6, broad-spectrum antiparasitic agents isolated from the soil bacterium Streptomyces avermitilis. We describe here the production of such compounds (22,23-dihydroavermectins B1a 9 and A1a 11) by direct fermentation of a recombinant strain of S. avermitilis containing an appropriately-engineered polyketide synthase (PKS). This suggests the feasibility of a direct biological route to this valuable drug.