Hamish McArthur

Engineering specificity of starter unit selection by the erythromycin‐producing polyketide synthase

Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ by KSQ, a ketosynthase‐like decarboxylase domain. Consistent with this, the acyltransferase (AT) domains of all KSQ‐containing loading modules are shown here to contain a key arginine residue at their active site. Site‐specific replacement of this arginine residue in the oleandomycin (ole) loading AT domain effectively abolished AT activity, consistent with its importance for catalysis. Substitution of the ole PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery) loading module gave polyketide products almost wholly either acetate derived or propionate derived, respectively, instead of the mixture found normally. An authentic extension module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately when engineered in the place of the ole loading AT domain, and gave rise to substantial amounts of C13‐methylerythromycins, as predicted. The role of direct acylation of the ketosynthase domain of ex‐tension module 1 in chain initiation was confirmed by demonstrating that a mutant of the triketide synthase DEBS1‐TE, in which the 4′‐phosphopante‐theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro‐ducts in detectable amounts.

Engineering of complex polyketide biosynthesis — insights from sequencing of the monensin biosynthetic gene cluster

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 360–367.

Peptide antibiotics : discovery, modes of action, and applications

Introduction to the Peptide Antibiotics Harry W. Taber Sources of Antimicrobial Peptides Chemistry and Applications of Synthetic Antimicrobial Peptides David Andreu and Luis Rivas Lanthionine-Containing Bacterial Peptides Ulrike Pag and Hans-Georg Sahl Unmodified Peptide-Bacteriocins (Class II) Produced by Lactic Acid Bacteria Ingolf F. Nes, Helge Holo, Gunnar Fimland, Havard Hildeng Hauge, and Jon Nissen-Meyer Insect Cationic Antimicrobial Peptides Charles Hetru, Jules A. Hoffmann, and Robert E. W. Hancock Mammalian Antimicrobial Peptides Charles L. Bevins and Gill Diamond Potential Applications of Peptides Exploitation of Lantibiotic Peptides for Food and Medical Uses Maire P. Ryan, Colin Hill, and R. Paul Ross Amphibian Antimicrobial Peptides Michael A. Zasloff Index

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.

Recent developments in the area of macrolide antibiotics

There have been exciting developments in the area of macrolide antibiotics recently. This activity largely stems from the discovery and development of a new class of agents called ketolides. They are more potent than erythromycin A and active against macrolide-resistant pathogens. At the same time, our knowledge of the mechanism of action of macrolides has been refined and resistance mechanisms have been elucidated. By employing new chemical methods and combinatorial biosynthesis, qualitatively different kinds of derivatives are being synthesised. Alternative non-infective uses for macrolide templates are also being explored. These areas are reviewed primarily in relation to 14-membered macrolides.