post-PKS Modifications Research Articles

Insights into Radicicol Biosynthesis via Heterologous Synthesis of Intermediates and Analogs

Resorcylic acid lactones are fungal polyketides that display diverse biological activities, with the potent Hsp90 inhibitor radicicol being an important representative member. Two fungal iterative polyketide synthases (IPKSs), Rdc5, the highly reducing IPKS, and Rdc1, the nonreducing IPKS, are required for the biosynthesis of radicicol in Pochonia chlamydosporia. In this study, the complete reconstitution of Rdc5 and Rdc1 activities both in vitro and in Saccharomyces cerevisiae uncovered the earliest resorcylic acid lactone intermediate of the radicicol biosynthetic pathway, (R)-monocillin II. The enzymatic synthesis of (R)-monocillin II confirmed the exquisite timing of the Rdc5 enoyl reductase domain. Using precursor-directed biosynthesis, the chemical modularity of the dual IPKS system was determined. Rdc1 readily accepted an N-acetylcysteamine thioester mimic of the reduced pentaketide product of Rdc5 to synthesize (R)-monocillin II with four additional iterations of polyketide elongation, indicating the C2' ketone group found in (R)-monocillin II is incorporated via the functions of Rdc1 instead of Rdc5. The involvement of the thioesterase domain in Rdc1 in macrolactonization was confirmed through both site-directed mutagenesis and domain deletion. The Rdc1 thioesterase domain was also shown to be tolerant of the opposite stereochemistry of the terminal hydroxyl nucleophile, demonstrated in the precursor-directed synthesis of the enantiomeric (S)-monocillin II. Finally, reconstitution of the halogenase Rdc2 was demonstrated both in vivo and in vitro in the synthesis of pochonin D and a new halogenated analog 6-chloro, 7',8'-dehydrozearalenol.

Recent advancements in the biosynthetic mechanisms for polyketide-derived mycotoxins.

Polyketides (PKs) are a large group of natural products produced by microorganisms and plants. They are biopolymers of acetate and other short carboxylates and are biosynthesized by multifunctional enzymes called polyketide synthases (PKSs). This review discusses the biosynthesis of four toxic PK, aflatoxins, fumonisins, ochratoxins (OTs), and zearalenone. These metabolites are structurally diverse and differ in their mechanisms of toxicity. However, they are all of concern in food safety and agriculture because of their toxic properties and their frequent accumulation in crops used for food and feed. The focus is on the recent advancements in the understanding of the molecular mechanisms for the biosynthesis of these mycotoxins. Several of the mycotoxin PKSs have been genetically and biochemically studied while other PKSs remain to be investigated. Multiple post-PKS modifications are often required for the maturation of the mycotoxins. Many of these modification steps for aflatoxins and fumonisins are well established while the post-PKS modifications for zearalenone and OTs remain to be biochemically characterized. More efforts are needed to completely illustrate the biosynthetic mechanisms for this important group of PKs.

Genetic engineering of macrolide biosynthesis: past advances, current state, and future prospects

Polyketides comprise one of the major families of natural products. They are found in a wide variety of bacteria, fungi, and plants and include a large number of medically important compounds. Polyketides are biosynthesized by polyketide synthases (PKSs). One of the major groups of polyketides are the macrolides, the activities of which are derived from the presence of a macrolactone ring to which one or more 6-deoxysugars are attached. The core macrocyclic ring is biosynthesized from acyl-CoA precursors by PKS. Genetic manipulation of PKS-encoding genes can result in predictable changes in the structure of the macrolactone component, many of which are not easily achieved through standard chemical derivatization or total synthesis. Furthermore, many of the changes, including post-PKS modifications such as glycosylation and oxidation, can be combined for further structural diversification. This review highlights the current state of novel macrolide production with a focus on the genetic engineering of PKS and post-PKS tailoring genes. Such engineering of the metabolic pathways for macrolide biosynthesis provides attractive alternatives for the production of diverse non-natural compounds. Other issues of importance, including the engineering of precursor pathways and heterologous expression of macrolide biosynthetic genes, are also considered.

Insights into the Biosynthesis of the Benzoquinone Ansamycins Geldanamycin and Herbimycin, Obtained by Gene Sequencing and Disruption

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.