Polyketide Modification Research Articles

Chemoenzymatic synthesis of fluorinated polyketides.

Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-selective incorporation of fluorine in complex molecules have improved in recent years, approaches for the biosynthetic incorporation of fluorine in natural compounds are still rare. Here, we report a strategy to introduce fluorine into complex polyketides during biosynthesis. We exchanged the native acyltransferase domain of a polyketide synthase, which acts as the gatekeeper for the selection of extender units, with an evolutionarily related but substrate tolerant domain from metazoan type I fatty acid synthase. The resulting polyketide-synthase/fatty-acid-synthase hybrid can utilize fluoromalonyl coenzyme A and fluoromethylmalonyl coenzyme A for polyketide chain extension, introducing fluorine or fluoro-methyl units in polyketide scaffolds. We demonstrate the feasibility of our approach in the chemoenzymatic synthesis of fluorinated 12- and 14-membered macrolactones and fluorinated derivatives of the macrolide antibiotics YC-17 and methymycin.

The Substrate‐Protein and Protein‐Protein Interactions of Fatty Acid and Polyketide Mega‐Synthases

Polyketide derived natural products are a large and diverse class of secondary metabolites used in the pharmaceutical and agricultural industry. These include antibiotics, immunosuppressant's, chemotherapeutics and insecticides. Examples of valuable polyketide compounds include tetracycline (antibiotic), lovastatin (cholesterol‐lowering) and rapamycin (immunosuppressant). The multi‐domain enzyme complex that is responsible for the biosynthesis of polyketides is known as the polyketide synthase (PKS). Similar to the PKS, the fatty acid synthase (FAS) is a multi–domain enzyme complex that utilizes acyl‐CoA's as building blocks to generate linear poly‐beta keto intermediates that can be subsequently processed via reduction, dehydration or other acyl‐chain modifying reactions. Both FAS and PKS utilize the acyl carrier protein (ACP) that functions as the transporter protein that is covalently tethered to the nascent polyketide intermediate. Polyketide and fatty acid production require three basic biosynthetic steps: (1) Polyketide elongation, (2) polyketide modification and (3) polyketide release from the ACP. How the ACP interacts with enzyme domains from all of these steps is not well understood. Unlike FAS, PKS utilize a much more diverse polyketide starter units, modification reactions that give rise to the rich chemical diversity of polyketide natural products. One of these important modifications includes the regio‐specific cyclization and aromatization of linear poly‐b‐keto intermediates. How the different PKS promote different cyclization patterns is not well understood. Lastly, how polyketide ACP‐tethered intermediates are released from the PKS machinery needs further investigation. An elucidation of the mechanism by which these bioactive polyketides are cyclized and/or released while being tethered to the ACP would be highly significant in the prediction, identification and engineered biosynthesis of new polyketides. Herein we present the structural enzymology of the effect of protein‐substrate and protein‐protein interactions on the product outcomes for these mega‐synthases.

The Determinants of Activity and Specificity in Actinorhodin Type II Polyketide Ketoreductase

In the actinorhodin type II polyketide synthase, the first polyketide modification is a regiospecific C9-carbonyl reduction, catalyzed by the ketoreductase (actKR). Our previous studies identified the actKR 94-PGG-96 motif as a determinant of stereospecificity. The molecular basis for reduction regiospecificity is, however, not well understood. In this study, we examined the activities of 20 actKR mutants through a combination of kinetic studies, PKS reconstitution, and structural analyses. Residues have been identified that are necessary for substrate interaction, and these observations have suggested a structural model for this reaction. Polyketides dock at the KR surface and are steered into the enzyme pocket where C7-C12 cyclization is mediated by the KR before C9-ketoreduction can occur. These molecular features can potentially serve as engineering targets for the biosynthesis of novel, reduced polyketides.

Expanding the scope of aromatic polyketides by combinatorial biosynthesis.

Combinatorial biosynthesis is a technology for mixing genes responsible for the biosynthesis of secondary metabolites, in order to generate products for compound libraries serendipitously or to cause desired modifications to natural products. Both of these approaches are extremely useful in drug discovery. Streptomyces and related species are abundant in bioactive secondary metabolites and were therefore the first microbes to be used for combinatorial biosynthesis. Polyketides are the most abundant medicinal agents among natural products. Structural diversity and a wide scope of bioactivities are typical of the group. However, the common feature of polyketides is a biosynthetic process from simple carboxylic acid residues. In molecular genetics, polyketides are sub-classified as types I and II, called modular and aromatic polyketides respectively. The best-known bioactivities of aromatic polyketides are their antibacterial and antitumor effects. Genetic analysis of aromatic polyketides has resulted in almost 30 cloned and identified biosynthetic gene clusters. Several biosynthetic enzymes are flexible enough to allow their use in combinatorial biosynthesis to create high diversity compound libraries. This review describes the state of the art of combinatorial biosynthesis, giving anthracyclines as examples. Contiguous DNA sequences for antibiotics, cloned from four different anthracycline producers, provide tools for rapid lead optimization or other structural modification processes, and not only for anthracyclines. Two gene cassettes enabling fast and flexible structural modification of polyketides are introduced in this paper.

Genetic Analysis of Biosynthesis of Polyketide Anthelmintic Macrolide Avermectin in Streptomyces avermitilis.

Within the 82 kb biosynthetic gene cluster for polyketide anthelmintic macrolide avermectin, the central 65 kb segment was found to be required for aglycon biosynthesis. Analysis of a 82 kb segment DNA from avermectin producer, Streptomyces avermitilis, revealed that it contains four large open reading frames (ORFs) encoding giant multifunctional polypeptides of the avermectin type-I polyketide synthase (AVES 1, AVES 2, AVES 3 and AVES 4). These clustered polyketide synthase genes responsible for avermectin biosynthesis together encode 12 homologous sets of enzyme activities (modules), each catalyzing a specific round of polyketide chain elongation and modification of β-carbon. The clustered genes encoding polyketide synthase are organized as two sets of six modular repeats, aveA1-aveA2 and aveA3-aveA4, which are convergently transcribed. The total of 55 constituent active sites were found in these four polyketide synthases but among two domains would not be functional in the process of the polyketide-chain elongation. The biosynthetic gene cluster for avermectin contains 14 additional open reading frames, some of which encode polypeptides governing other key steps in avermectin biosynthesis. Between the two sets of polyketide synthase genes lie two genes involved in postpolyketide modification. On the right of the large polyketide synthase genes is a set of genes involved in biosynthesis of methylated deoxysugar, L-oleandrose, and its transglycosylation to polyketide-derived aglycons. This cluster includes nine genes but one is not functional in the biosynthesis of avermectin. On the left side of polyketide synthase genes, Two open reading frames encoding methyltransferase and non-polyketide synthase ketoreductase involved in post- polyketide modification are located and an adjacent gene encodes a regulatory function which may be involved in activation of the transcription of avermectin biosynthetic genes.