Molekularbiologische und biochemische Untersuchungen zur Biosynthese der Atmungsketteninhibitoren Myxothiazol und Melithiazol

Im Rahmen der vorgelegten Dissertation wurden die Biosynthese-Gencluster der Atm ungsketten-Inhibitoren Myxothiazol und Melithiazol, die von verschiedenen Myxoba kterien produziert werden, genetisch und biochemisch analysiert. Das Myxothiazol -Biosynthesegencluster wurde aus Stigmatella aurantiaca DW4/3-1 kloniert und seq uenziert. Die Sequenzanalyse zeigt, dass Myxothiazol durch eine ungewöhnliche Ko mbination mehrerer Polyketidsynthasen und nichtribosomaler Peptidsynthetasen bio synthetisiert wird. Diese enthalten zahlreiche neuartige Domänentypen, die sich gut mit der chemischen Struktur des Myxothiazols korrelieren lassen. Um deren Fu nktion während der Biosynthese zu untersuchen, wurde ein unter Erhalt des Lesera hmens ablaufendes Mutageneseverfahren etabliert. Über den Nachweis veränderter M yxothiazol-Derivate in der Mutante sollte die für die betreffende Proteindomäne postulierte Funktion bestätigt werden. Die Anwendung dieses Verfahrens auf zahlr eiche Gene führte mit einer Ausnahme zu Nullproduzenten. Offensichtlich handelt es sich bei der Myxothiazolsynthetase um ein hochspezialisiertes System, dass ni cht geeignet ist, veränderte Intermediate zu prozessieren. Der zweite Teil der Arbeit beschreibt die Identifizierung und Charakterisierung des Melithiazol-Biosynthesegenclusters aus Melittangium lichenicola Me l46. Meli thiazol ähnelt dem Myxothiazol strukturell, besitzt jedoch interessante Untersch iede. Basierend auf dem Sequenzvergleich mit dem Myxothiazol-Biosynthesegenclust er konnten Hypothesen zur Evolution der beiden Biosynthesen aufgestellt werden. Um die Bildung des Methylesters im Melithiazol zu untersuchen, wurden zwei Prote ine der Melithiazolsynthetase heterolog im Myxothiazolproduzenten exprimiert. Au f diese Weise konnte gezeigt werden, dass die beiden Proteine auch das Säureamid Myxothiazol A in sein Methylesteranalogon das Myxothiazol Z überführen.The biosynthetic gene clusters of the electron transport inhibitors Myxothiazol and Melithiazol, which are produced by different strains of myxobacteria, were g enetically and biochemically analysed. The Myxothiazol biosynthetic gene cluster was cloned from Stigmatella aurantiaca DW4/3-1. Sequence analysis reveals that Myxothiazol is formed by a unique combi nation of several polyketide synthases and nonribosomal peptide sythetases. Thes e contain several unusual domain types, which correlate with the chemical struct ure. In order to investigate their function during biosynthesis a genetic system for markerless mutagenesis in S. aurantiaca DW4/3-1 was established. Detection of the predicted Myxothiazol derivate in the mutant should prove the postulated function of the altered gene. A series of mutants in the Myxothiazol megasynthet ase were constructed, which do not produce novel Myxothiazol derivates. It is as sumed that the Myxothiazol biosynthetic system does not process changed intermed iates. The second part of the thesis describes identification and characterization of t he Melithiazol biosynthetic gene cluster from Melittangium lichenicola Me l46. M elithiazol is similar to Myxothiazol but shows some remarkable differences. Base d on sequence comparisons suggestions for the evolution of the two biosynthetic pathways were made. In order to investigate the formation of the methylester-structure in Melithiazo l two proteins of the Melithiazolsynthetase were heterologously expressed in the Myxothiazol producer. It could be shown that these proteins could also convert the amide Myxothiazol A into the methyl-ester Myxothiazol Z

A unique mechanism for methyl ester formation via an amide intermediate found in myxobacteria.

Secondary metabolism involves a broad diversity of biochemical reactions that result in a wide variety of biologically active compounds. Terminal amide formation during the biosynthesis of the myxobacterial electron-transport inhibitor, myxothiazol, was analyzed by heterologous expression of the unique nonribosomal-peptide synthetase, MtaG, and incubation with a synthesized substrate mimic. These experiments provide evidence that the terminal amide is formed from a carrier protein-bound myxothiazol acid that is thioesterified to MtaF. This intermediate is transformed to an amide by extension with glycine and subsequent oxidative cleavage by MtaG. The final steps of melithiazol assembly involve a highly similar protein-bound intermediate (attached to MelF, a homologue of MtaF), which is transformed to an amide by MelG (homologue of MtaG). In this study, we also show that the amide moiety of myxothiazol A can be hydrolyzed in vivo to the formerly unknown free myxothiazol acid by heterologous expression of melJ in the myxothiazol producer Stigmatella aurantiaca DW4/3-1. The methyltransferase MelK can finally methylate the acid to give rise to the methyl ester, which is produced as the final product in the melithiazol A biosynthetic pathway. These experiments clarify the role of MelJ and MelK during melithiazol assembly

NFDI4Chem - Towards a National Research Data Infrastructure for Chemistry in Germany

The vision of NFDI4Chem is the digitalisation of all key steps in chemical research to support scientists in their efforts to collect, store, process, analyse, disclose and re-use research data. Measures to promote Open Science and Research Data Management (RDM) in agreement with the FAIR data principles are fundamental aims of NFDI4Chem to serve the chemistry community with a holistic concept for access to research data. To this end, the overarching objective is the development and maintenance of a national research data infrastructure for the research domain of chemistry in Germany, and to enable innovative and easy to use services and novel scientific approaches based on re-use of research data. NFDI4Chem intends to represent all disciplines of chemistry in academia. We aim to collaborate closely with thematically related consortia. In the initial phase, NFDI4Chem focuses on data related to molecules and reactions including data for their experimental and theoretical characterisation.This overarching goal is achieved by working towards a number of key objectives:Key Objective 1: Establish a virtual environment of federated repositories for storing, disclosing, searching and re-using research data across distributed data sources. Connect existing data repositories and, based on a requirements analysis, establish domain-specific research data repositories for the national research community, and link them to international repositories.Key Objective 2: Initiate international community processes to establish minimum information (MI) standards for data and machine-readable metadata as well as open data standards in key areas of chemistry. Identify and recommend open data standards in key areas of chemistry, in order to support the FAIR principles for research data. Finally, develop standards, if there is a lack.Key Objective 3: Foster cultural and digital change towards Smart Laboratory Environments by promoting the use of digital tools in all stages of research and promote subsequent Research Data Management (RDM) at all levels of academia, beginning in undergraduate studies curricula.Key Objective 4: Engage with the chemistry community in Germany through a wide range of measures to create awareness for and foster the adoption of FAIR data management. Initiate processes to integrate RDM and data science into curricula. Offer a wide range of training opportunities for researchers.Key Objective 5: Explore synergies with other consortia and promote cross-cutting development within the NFDI.Key Objective 6: Provide a legally reliable framework of policies and guidelines for FAIR and open RDM