SummaryThe analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in monensin biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway-specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently-proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI , monBII , monCI and monCII . In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI , encoding a flavin-linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in monensin biosynthesis.

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.

Polyether ionophores, such as monensin A, are known to be biosynthesised, like many other antibiotic polyketides, on giant modular polyketide synthases (PKSs), but the intermediates and enzymes involved in the subsequent steps of oxidative cyclisation remain undefined. In particular there has been no agreement on the mechanism and timing of the final polyketide chain release. We now report evidence that MonCII from the monensin biosynthetic gene cluster in Streptomyces cinnamonensis, which was previously thought to be an epoxide hydrolase, is a novel thioesterase that belongs to the alpha/beta-hydrolase structural family and might catalyse this step. Purified recombinant MonCII was found to hydrolyse several thioester substrates, including an N-acetylcysteamine thioester derivative of monensin A. Further, incubation with a hallmark inhibitor of such enzymes, phenylmethanesulfonyl fluoride, led to inhibition of the thioesterase activity and to the accumulation of an acylated form of MonCII. These findings require a reassessment of the role of other enzymes implicated in the late stages of polyether ionophore biosynthesis.

Stereochemistry resolved: The introduction of hybrid polyketide synthases in Streptomyces cinnamonensis has shown that the E forms of a tetraketide and pentaketide are intermediates in the biosynthesis of the antibiotic ionophore monensin A (see structure). Furthermore, the trans double bond present in these intermediates has been identified as one of the targets for the epoxidase that initiates oxidative cyclization.

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