The structures of complex polyketide natural products, such as erythromycin, are programmed by multifunctional polyketide synthases (PKSs) that contain modular arrangements of functional domains. The colinearity between the activities of modular PKS domains and structure of the polyketide product portends the generation of novel organic compounds—“unnatural” natural products—by genetic manipulation. We have engineered the erythromycin polyketide synthase genes to effect combinatorial alterations of catalytic activities in the biosynthetic pathway, generating a library of >50 macrolides that would be impractical to produce by chemical methods. The library includes examples of analogs with one, two, and three altered carbon centers of the polyketide products. The manipulation of multiple biosynthetic steps in a PKS is an important milestone toward the goal of producing large libraries of unnatural natural products for biological and pharmaceutical applications.

To identify the minimum set of polykedde synthase (PKS) components to accuratey control the course of this on by Itself. In the presence ofa downstream enzyme, the flux through one branch of the cyclization pathway increa relative to the other. We propose that these altatie spicitie may be due to the ability of d ream enzyme to asate with the minimal PKS and to selectively inhibit a particular branch of the cyclization pathway.Polyketides are a large family of structurally diverse natural products with a broad range ofbiological activities, including antibiotic and pharmacological properties. Polyketide synthases (PKSs) are structurally and mechanistically related to fatty acid synthases (1-4). Both classes are multifunctional enzymes that catalyze repeated decarboxylative condensations between acyl thioesters (usually acetyl, propionyl, malonyl, or methylmalonyl). The main difference between PKSs and fatty acid synthases is that, following each condensation, PKSs introduce enormous structural variability into the product by omitting all, part, or none of the typical fatty acid synthase reductive cycle comprising a ketoreduction, dehydration, and enoylreduction on the P-keto group of the growing polyketide chain.Within the PKSs, recent molecular genetics and biochemical studies have revealed two different mechanisms for the control of polyketide specificity. In one, exemplified by the PKS for the macrolide antibiotic erythromycin, the synthase provides separate sets of active sites for each condensation and reduction cycle, and product structure is dictated by the number and arrangement of these active sites (5, 6). In the second class, represented by the actinomycete PKSs for aromatic polyketides, this relationship is not apparent because each synthase contains a single set of iteratively used active sites for all condensation and reduction cycles.Studies on PKS gene clusters, including those based on sequence analysis (7-9), functional complementation in vivo

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Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzyme complexes that are organized into modules, where each module carries the domains needed to catalyze the condensation of an extender unit onto a growing polyketide chain. Each module also dictates the stereochemistry of the chiral centers introduced into the backbone during the chain elongation process. Here we used domain mutagenesis to investigate the role of the acyl transferase (AT) domains of individual modules in the choice and stereochemical fate of extender units. Our results indicate that the AT domains of DEBS do not influence epimerization of the (2S)-methylmalonyl-CoA extender units. Hence, stereochemical control of the methyl-branched centers generated by DEBS most likely resides in the ketosynthase (KS) domains of the individual modules. In contrast, several recent studies have demonstrated that extender unit specificity can be altered by AT domain substitution. In some of these examples, the resulting polyketide was produced at considerably lower titers than the corresponding natural product. We analyzed one such attenuated mutant of DEBS, in which the methylmalonyl transferase domain of module 2 was replaced with a malonyl transferase domain. As reported earlier, the resulting PKS produced only small quantities of the expected desmethyl analogue of 6-deoxyerythronolide B. However, when the same hybrid module was placed as the terminal module in a truncated 2-module PKS, it produced nearly normal quantities of the expected desmethyl triketide lactone. These results illustrate the limits to modularity of these multifunctional enzymes. To dissect the role of specific amino acids in controlling AT substrate specificity, we exchanged several segments of amino acids between selected malonyl and methylmalonyl transferases, and found that a short (23-35 amino acid) C-terminal segment present in all AT domains is the principal determinant of their substrate specificity. Interestingly, its length and amino acid sequence vary considerably among the known AT domains. We therefore suggest that the choice of extender units by the PKS modules is influenced by a "hypervariable region", which could be manipulated via combinatorial mutagenesis to generate novel AT domains possessing relaxed or altered substrate specificity.