Rachel E. Lill

The discovery and development of marine compounds with pharmaceutical potential

An assessment of the current status of marine anticancer compounds is presented along with a case study on the aquaculture of Lissodendoryx n. sp. 1, a sponge that produces the antimitotic agents halichondrin B and isohomohalichondrin B. The use of polymer therapeutics to enhance the properties of marine natural products is considered.

Active-site residue, domain and module swaps in modular polyketide synthases

Sequence comparisons of multiple acyltransferase (AT) domains from modular polyketide synthases (PKSs) have highlighted a correlation between a short sequence motif and the nature of the extender unit selected. When this motif was specifically altered in the bimodular model PKS DEBS1-TE of Saccharopolyspora erythraea, the products included triketide lactones in which acetate extension units had been incorporated instead of propionate units at the predicted positions. We also describe a cassette system for convenient construction of hybrid modular PKSs based on the tylosin PKS in Streptomyces fradiae and demonstrate its use in domain and module swaps.

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.

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.

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.

Engineered biosynthesis of novel spinosyns bearing altered deoxyhexose substituents

Novel spinosyns have been prepared by biotransformation, using a genetically engineered strain of Saccharopolyspora erythraea, in which the beta-D-forosamine moiety in glycosidic linkage to the hydroxy group at C17 is replaced by alpha-L-mycarose.

Structure guided design of improved anti-proliferative rapalogs through biosynthetic medicinal chemistry

A combination of molecular modelling and rational biosynthetic engineering of the rapamycin polyketide synthase was used to generate rapalogs lacking O- and C-linked methyl groups at positions 16 and 17 respectively. These rapalogs displayed enhanced inhibition of cancer cell lines and were produced at titres close to those of the parent strain. By recapitulating these experiments in higher-producing rapamycin strains, combined with the ectopic expression of gene products acting late in the biosynthetic pathway in order to minimise the accumulation of intermediates, gram-quantities of novel rapalogs bearing multiple structural changes were produced.