Filling the Gaps in the Kirromycin Biosynthesis: Deciphering the Role of Genes Involved in Ethylmalonyl-CoA Supply and Tailoring Reactions

Kirromycin is the main product of the soil-dwelling Streptomyces collinus Tü 365. The elucidation of the biosynthetic pathway revealed that the antibiotic is synthesised via a unique combination of trans-/cis-AT type I polyketide synthases and non-ribosomal peptide synthetases (PKS I/NRPS). This was the first example of an assembly line integrating the three biosynthetic principles in one pathway. However, information about other enzymes involved in kirromycin biosynthesis remained scarce.\ua0In this study, genes encoding tailoring enzymes KirM, KirHVI, KirOI, and KirOII, and the putative crotonyl-CoA reductase/carboxylase KirN were deleted, complemented, and the emerged products analysed by HPLC-HRMS and MS/MS. Derivatives were identified in mutants ΔkirM, ΔkirHVI, ΔkirOI, and ΔkirOII. The products of ΔkirOI, ΔkirOII, and kirHVI were subjected to 2D-NMR for structure elucidation. Our results enabled functional assignment of those enzymes, demonstrating their involvement in kirromycin tailoring. In the ΔkirN mutant, the production of kirromycin was significantly decreased. The obtained data enabled us to clarify the putative roles of the studied enzymes, ultimately allowing us to fill many of the missing gaps in the biosynthesis of the complex antibiotic. Furthermore, this collection of mutants can serve as a toolbox for generation of new kirromycins

Lysoquinone-TH1, a New Polyphenolic Tridecaketide Produced by Expressing the Lysolipin Minimal PKS II in Streptomyces albus

The structural repertoire of bioactive naphthacene quinones is expanded by engineering Streptomyces albus to express the lysolipin minimal polyketide synthase II (PKS II) genes from Streptomyces tendae Tü 4042 (llpD-F) with the corresponding cyclase genes llpCI-CIII. Fermentation of the recombinant strain revealed the two new polyaromatic tridecaketides lysoquinone-TH1 (7, identified) and TH2 (8, postulated structure) as engineered congeners of the dodecaketide lysolipin (1). The chemical structure of 7, a benzo[a]naphthacene-8,13-dione, was elucidated by NMR and HR-MS and confirmed by feeding experiments with [1,2-13C2]-labeled acetate. Lysoquinone-TH1 (7) is a pentangular polyphenol and one example of such rare extended polyaromatic systems of the benz[a]napthacene quinone type produced by the expression of a minimal PKS II in combination with cyclases in an artificial system. While the natural product lysolipin (1) has antimicrobial activity in nM-range, lysoquinone-TH1 (7) showed only minor potency as inhibitor of Gram-positive microorganisms. The bioactivity profiling of lysoquinone-TH1 (7) revealed inhibitory activity towards phosphodiesterase 4 (PDE4), an important target for the treatment in human health like asthma or chronic obstructive pulmonary disease (COPD). These results underline the availability of pentangular polyphenolic structural skeletons from biosynthetic engineering in the search of new chemical entities in drug discover

Analogs, Biosynthesis and chemical Analytics of the microbial Polyketides Iromycin and Lysolipin

Polyketides represent a large substance class with a broad range of activity. Biosynthetically, these natural products are formed by polyketide synthases (PKS) which are classified as three different types (PKS I, II and III). Differences within the polyketides are based on the setup of the biosynthetic enzyme complexes and the structural classes of the biosynthesized polyketides. The novel structural class of iromycins represent PKS I metabolites. Using the example of Streptomyces bottropensis Dra 17, experiments of precursor directed biosynthesis examined the loading of synthetic new starter units on the iromycin PKS to generate novel derivatives. Preferably, various SNAC esters were synthesized and fed together with L-isoleucine to the growing cultures of strain Dra 17. With these methods it was possible to produce four new derivatives that have been isolated and characterized by NMR spectroscopy. Another aim of this Ph.D. was to isolate a new metabolite from the biosynthetic gene cluster of lysolipin (a PKS II metabolite) in mg-scale and elucidate its structure by NMR spectroscopy. This substance attracted attention by a red natural color. In this work in addition to a reproducible production in liquid culture also a chromatographic sequence with the corresponding HPLC analytics was developed. The resulting compound is not known in the literature and was named Lysoquinone-TH1. For investigating the biogenesis of this new Lysolipin analog, feeding experiments with 13C acetate were performed. Valuable insights in the lysolipin biosynthesis were obtained. Within the studies to the biological profiling of the pure lysoquinone-TH1, this novel compound especially attracted attention in a biological activity assay.Polyketide stellen eine große Substanzklasse mit einem breiten Wirkungsspektrum dar. Biosynthetisch werden diese Naturstoffe durch Polyketidsynthasen (PKS) aufgebaut. Es kann zwischen verschiedenen Typen (PKS I, II und III) differenziert werden. Der Unterschied liegt in dem Aufbau des Biosyntheseapparates und den daraus aufgebauten Polyketiden. Die neu eröffnete Substanzfamilie der Iromycine stellt PKS I-Metabolite dar. Am Beispiel des Streptomyces bottropensis Dra 17 wurden im Rahmen der Vorläufer-;dirigierten Biosynthese versucht, neue Starteinheiten auf die für die Iromycin-Bildung verantwortliche PKS zu laden, um neue Derivate zu erzeugen. Dazu wurden verschiedene SNAC-Ester synthetisiert und diese zusammen mit L-Isoleucin im Rahmen der Vorläufer-dirigierten Biosynthese zugefüttert. Mit dieser Methode gelang es, vier neue Derivate zu produzieren, die isoliert und mittels NMR-Spektroskopie charakterisiert wurden. Ein weiteres Ziel dieser Arbeit war es, ein neues Stoffwechselprodukt auf Grundlage des Biosynthese¬genclusters des Lysolipins (ein PKS II-Metabolit) in mg-Mengen zu isolieren und in seiner Struktur mittels NMR-Spektroskopie aufzuklären. Diese Substanz fiel durch rote Eigenfarbe auf. Im Rahmen dieser Arbeit konnte neben einer reproduzierbaren Produktion aus Flüssigkulturen auch eine Chromatographiesequenz mit der entsprechenden HPLC-Analytik erarbeitet werden. Die erhaltene Verbindung ist bisher nicht bekannt und wurde als Lysochinon-TH1 bezeichnet. Zur Untersuchung der Biogenese des neuen Lysolipin-Analogons wurden Fütterungsexperimente mit 13C-Acetat durchgeführt. So dass wertvolle Einsichten zur Aufklärung der Biosynthese des Lysolipins gewonnen wurden. Im Rahmen des biologischen Profiling des Reinstoffs Lysochinon-TH1 fiel die neuartige Struktur vor allem in biologischen Aktivitätsassays auf