Evolution of non-linear polyketide biosynthesis in the aureothin and neoaureothin pathways

CHRISTIAN HERTWECK
Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie
Hans-Knöll-Institut und Lehrstuhl für Biomolekulare Chemie
Beutenbergstraße 11a
D-07745 Jena
christian.hertweck@hki-jena.de
www.hki-jena.de

References
K. Ishida, G. Christiansen, W.Y. Yoshida, R. Kurmayer, M. Welker, N. Valls, J. Bonjoch, C. Hertweck, T. Borner, T. Hemscheidt & E. Dittmann Biosynthesis and Structure of Aeruginoside 126A and 126B, Cyanobacterial Peptide Glycosides Bearing a 2-Carboxy-6-Hydroxyoctahydroindole Moiety. Chem. Biol. 2007, 14, 565-576.
Aeruginosins represent a group of peptide metabolites isolated from various cyanobacterial genera and from marine sponges that potently inhibit different types of serine proteases. Members of this family are characterized by the presence of a 2-carboxy-6-Zydroxyoctahydroindole (Choi) moiety. We have identified and fully sequenced a NRPS gene cluster in the genome of the cyanobacterium Planktothrix agardhii CYA126/8. Insertional mutagenesis of a NRPS component led to the discovery and structural elucidation of two glycopeptides that were designated aeruginoside 126A and aeruginoside 126B. One variant of the aglycone contains a 1-amino-2-(N-amidino-Delta(3)-pyrrolinyl)ethyl moiety at the C terminus, the other bears an agmatine residue. In silico analyses of the aeruginoside biosynthetic genes aerA-aerI as well as additional mutagenesis and feeding studies allowed the prediction of enzymatic steps leading to the formation of aeruginosides and the unusual Choi moiety.

G. Lackner, A. Schenk, Z. Xu, Z. Yunt, K. Reinhardt, J. Piel & C. Hertweck
Biosynthesis of Pentangular Polyphenols: Deductions from the Benastatin and Griseorhodin Pathways J. Am. Chem. Soc. 2007, in press.

N. Traitcheva, H. Jenke-Kodama, J. He, E. Dittmann & C. Hertweck Non-Colinear Polyketide Biosynthesis in the Aureothin and Neoaureothin Pathways: An Evolutionary Perspective ChemBioChem 2007, 8, in press.

C. Hertweck, A. Luzhetskyy, Y. Rebets & A. Bechthold Type II Polyketide Synthases: Gaining a Deeper Insight into Enzymatic Teamwork Nat. Prod. Rep. 2007, 24, 162-190.
This review covers advances in understanding of the biosynthesis of polyketides produced by type II PKS systems at the genetic, biochemical and structural levels.

O. Kniemeyer O, Lessing F, Scheibner O, Hertweck C, Brakhage AA. Optimisation of a 2-D gel electrophoresis protocol for the human-pathogenic fungus Aspergillus fumigatus. Curr. Genet. 2006, 49, 178-189.
Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. One of the important questions concerning A. fumigatus is the identification of pathogenicity determinants. To obtain a comprehensive overview about the proteins produced at different physiological conditions that are related to the infectious process a proteomic approach has been applied. Here, 2-D gel electrophoresis for filamentous fungi was optimised concerning removal of interfering compounds, protein extraction and separation methods. A trichloroacetic acid-based precipitation method of proteins with their subsequent solubilisation by the use of a combination of CHAPS with a second sulfobetaine detergent gave the best results. The optimised protocol was evaluated by the analysis of the proteomes of A. fumigatus grown on two different carbon sources, i.e., glucose and ethanol. Carbon catabolite repression has not been studied in detail at the protein level in A. fumigatus yet. In addition, growth on ethanol leads to activation of the glyoxylate cycle which was shown to be essential for pathogenesis in bacteria and fungi. In A. fumigatus, differential patterns of enzymes of the gluconeogenesis, glyoxylate cycle and ethanol degradation pathway during growth on glucose and ethanol were observed.

R. Winkler, M.E.A. Richter, U. Knüpfer, D. Merten & C. Hertweck. Regio- and Chemoselective Enzymatic N-Oxygenation In Vivo, In Vitro, and in Flow Angew. Chem. 2006, 118, 8184-8186 (Angew. Chem. Int. Ed. 2006, 45, 8016-8018).

K. Ishida, K. Maksimenka, K. Fritzsche, K. Scherlach, G. Bringmann & C. Hertweck
the Boat-Shaped Polyketide Resistoflavin Results from Re-Facial Central Hydroxylation of the Discoid Metabolite Resistomycin. J. Am. Chem. Soc. 2006, 128, 14619-14624.
Resistoflavin is a rare boat-shaped pentacyclic polyketide metabolite of Streptomyces resistomycificus with marked antibacterial activity. By a series of experiments we have disclosed that the optically active molecule is derived from the discoid polyketide resistomycin by an unusual, enantioface-differentiating hydroxylation, which leads to the capped pentacyclic ring system. In vivo and in vitro experiments unequivocally demonstrate that this reaction is catalyzed by RemO, an FAD-dependent monooxygenase. In addition, we were able to establish the absolute configuration of 1 and thus the stereochemical course of this rare enzymatic reaction by extensive computational methods. Comparison of the experimental CD spectrum with those quantum chemically calculated for (R)-1 and (S)-1 revealed the R-configuration of 1. Consequently, the enzyme-catalyzed hydroxylation takes place from the Re-face of 2 with loss of aromaticity in favor of a chiral carbinol center. While other oxygenases involved in polyketide tailoring functionalize the periphery of polyphenols, RemO is unique in its ability to catalyze a central, nonperipheral hydroxylation of a fused ring system.

Hsiao NH, Söding J, Linke D, Lange C, Hertweck C, Wohlleben W, Takano E.
ScbA from Streptomyces coelicolor A3(2) has homology to fatty acid synthases and is able to synthesize gamma-butyrolactones. Microbiology. 2007, 153, 1394-404.
gamma-Butyrolactones play an important role in the regulation of antibiotic production and differentiation in Streptomyces. However the biosynthetic pathway for these small molecules has not yet been determined, and in vitro synthesis has not been reported. The function of the AfsA family of proteins, originally proposed to catalyse gamma-butyrolactone synthesis, has been in debate. To clarify the function of the AfsA family, and to understand the synthesis of the gamma-butyrolactones, we performed in silico analysis of this protein family. AfsA proteins consist of two divergent domains, each of which has similarity to the fatty acid synthesis enzymes FabA and FabZ. The two predicted active sites in ScbA, which is the AfsA orthologue found in Streptomyces coelicolor, were mutated, and gamma-butyrolactone biosynthesis was abolished in all four constructed mutants, strongly suggesting that ScbA has enzymic activity.

S. Bergmann, J. Schümann, K. Scherlach, C. Lange, A. A. Brakhage & C. Hertweck
Genomics-Driven Discovery of PKS-NRPS Hybrid Metabolites from Aspergillus nidulans
Nature Chem. Biol. 2007, 3, 213-217.
In the postgenomic era it has become increasingly apparent that the vast number of predicted biosynthesis genes of microorganisms is not reflected by the metabolic profile observed under standard fermentation conditions. In the absence of a particular (in most cases unknown) trigger these gene loci remain silent. Because these cryptic gene clusters may code for the biosynthesis of important virulence factors, toxins, or even drug candidates, new strategies for their activation are urgently needed to make use of this largely untapped reservoir of potentially bioactive compounds. The discovery of new microbial metabolites through genome mining has proven to be a very promising approach. Even so, the investigation of silent gene clusters is still a substantial challenge, particularly in fungi. Here we report a new strategy for the successful induction of a silent metabolic pathway in the important model organism Aspergillus nidulans, which led to the discovery of novel PKS-NRPS hybrid metabolites.

M. Müller, B. Kusebauch, G. Liang, C.M. Beaudry, D. Trauner & C. Hertweck. Photochemical Origin of SNF4435C/D and Formation of Orinocin by 'Polyene Splicing'
Angew. Chem. 2006, 118, 7999-8002 (Angew. Int. Ed. 2006, 45, 7835-7838).

M. Müller, J. He & C. Hertweck. Dissection of the Late Steps in Aureothin Biosynthesis.
ChemBioChem 2006, 7, 37-39.

R. Winkler & C. Hertweck. Sequential Enzymatic Oxidation of Aminoarenes to Nitroarenes via Hydroxylamine. Angew. Chem. Int. Ed. 2005, 44, 4083-4087.

J. He & C. Hertweck. Functional Analysis of the Aureothin Iterative Type I Polyketide Synthase. ChemBioChem 2005, 6, 908-912.
The modular-type polyketide synthase (PKS) that is involved in aureothin (aur) biosynthesis represents one of the first examples in which a single PKS module (AurA) is used in an iterative fashion. Here we report on the heterologous expression of an engineered AurAB fusion protein that unequivocally proves the iterative nature of AurA. In addition, point mutations reveal that aur PKS module 4 participates in polyketide biosynthesis despite its aberrant acyltransferase domain.

M. Ziehl, J. He, H.-M. Dahse & C. Hertweck. Mutasynthesis of Aureonitrile, an Aureothin Derivative with Significantly Improved Cytostatic Effect. Angew. Chem. Int. Ed. 2005, 44, 1202-1205.

J. He, M. Müller & C. Hertweck. Formation of the Aureothin Tetrahydrofuran Ring by a Bifunctional Cytochrome P450 Oxygenase. J. Am. Chem. Soc. 2004, 126, 16742-16743.

J. He & C. Hertweck. Biosynthetic Origin of the Rare Nitro Aryl Moiety of the Polyketide Antibiotic Aureothin: Discovery of an Unprecedented N-Oxygenase. J. Am. Chem. Soc. 2004, 126, 3694-3695.

J. He & C. Hertweck. Iteration as Programmed Event During Polyketide Assembly; Molecular Analysis of the Aureothin Biosynthesis Gene Cluster. Chem. Biol. 2003, 10, 1225-1232.
Analysis of the type I modular polyketide synthase (PKS) involved in the biosynthesis of the rare nitroaryl polyketide metabolite aureothin (aur) from Streptomyces thioluteus HKI-227 has revealed only four modules to catalyze the five polyketide chain extensions required. By heterologous expression of the aur PKS cluster, direct evidence was obtained that these modules were sufficient to support aureothin biosynthesis. It appears that one module catalyzes two successive cycles of chain extension, one of the first examples of a PKS in which such iteration or "stuttering" is required to produce the normal polyketide product. In addition, lack of a specified loading domain implicates a novel PKS priming mechanism involving the unique p-nitrobenzoate starter unit. The 27 kb aur gene cluster also encod
es a novel N-oxidase, which may represent the first member of a new family of such enzymes.

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