Uschi Sundermann

Enzyme-Directed Mutasynthesis: A Combined Experimental and Theoretical Approach to Substrate Recognition of a Polyketide Synthase

Acyltransferase domains control the extender unit recognition in Polyketide Synthases (PKS) and thereby the side-chain diversity of the resulting natural products. The enzyme engineering strategy presented here allows the alteration of the acyltransferase substrate profile to enable an engineered biosynthesis of natural product derivatives through the incorporation of a synthetic malonic acid thioester. Experimental sequence-function correlations combined with computational modeling revealed the origins of substrate recognition in these PKS domains and enabled a targeted mutagenesis. We show how a single point mutation was able to direct the incorporation of a malonic acid building block with a non-native functional group into erythromycin. This approach, introduced here as enzyme-directed mutasynthesis, opens a new field of possibilities beyond the state of the art for the combination of organic chemistry and biosynthesis toward natural product analogues.

Minimally Invasive Mutagenesis Gives Rise to a Biosynthetic Polyketide Library

Not in the public domain: Site-directed mutagenesis of megasynthases was the key to the generation of a library of polyketides in bacteria. Redox derivatizations are used to change the bioactivity profile of the compounds.

Substrate Flexibility of a Mutated Acyltransferase Domain and Implications for Polyketide Biosynthesis.

Polyketides are natural products frequently used for the treatment of various diseases, but their structural complexity hinders efficient derivatization. In this context, we recently introduced enzyme-directed mutasynthesis to incorporate non-native extender units into the biosynthesis of erythromycin. Modeling and mutagenesis studies led to the discovery of a variant of an acyltransferase domain in the erythromycin polyketide synthase capable of accepting a propargylated substrate. Here, we extend molecular rationalization of enzyme-substrate interactions through modeling, to investigate the incorporation of substrates with different degrees of saturation of the malonic acid side chain. This allowed the engineered biosynthesis of new erythromycin derivatives and the introduction of additional mutations into the AT domain for a further shift of the enzymes substrate scope. Our approach yields non-native polyketide structures with functional groups that will simplify future derivatization approaches, and provides a blueprint for the engineering of AT domains to achieve efficient polyketide synthase diversification.

Quantification of N-acetylcysteamine activated methylmalonate incorporation into polyketide biosynthesis

Summary Polyketides are biosynthesized through consecutive decarboxylative Claisen condensations between a carboxylic acid and differently substituted malonic acid thioesters, both tethered to the giant polyketide synthase enzymes. Individual malonic acid derivatives are typically required to be activated as coenzyme A-thioesters prior to their enzyme-catalyzed transfer onto the polyketide synthase. Control over the selection of malonic acid building blocks promises great potential for the experimental alteration of polyketide structure and bioactivity. One requirement for this endeavor is the supplementation of the bacterial polyketide fermentation system with tailored synthetic thioester-activated malonates. The membrane permeable N-acetylcysteamine has been proposed as a coenzyme A-mimic for this purpose. Here, the incorporation efficiency into different polyketides of N-acetylcysteamine activated methylmalonate is studied and quantified, showing a surprisingly high and transferable activity of these polyketide synthase substrate analogues in vivo.

Data in support of substrate flexibility of a mutated acyltransferase domain and implications for polyketide biosynthesis.

Enzyme-directed mutasynthesis is an emerging strategy for the targeted derivatization of natural products. Here, data on the synthesis of malonic acid derivatives for feeding studies in Saccharopolyspora erythraea , the mutagenesis of DEBS and bioanalytical data on the experimental investigation of studies on the biosynthetic pathway towards erythromycin are presented.

The Development of DNA Sequencing: From the Genome of a Bacteriophage to That of a Neanderthal

In 1977 Sanger et al. reported the first genome sequence ever determined: the roughly 5000 base pairs of a bacteriophage genome. In the same year Sanger published his didesoxy method for DNA sequencing, an experimental technique which in the following decades would revolutionize modern biochemistry and bring Sanger his second Nobel Prize in Chemistry. The aim of deciphering the human genome spurred a tremendous jump in the development of the Sanger sequencing technology (Table 1). The Human Genome Project (HGP), initiated in 1990, led to a factorylike upscaling of sequencing capacities in the participating institutes. Through optimization and automatization of each step of the sequencing process, the elucidation of complex genomes slowly came within reach. In the early 1990s, the improvements in Sanger technology enabled the sequencing of small bacterial genomes and already in 1996, the genome of Saccharomyces cerevisiae, baker s yeast, was described. In 2001, one decade after its project s commencement, the first draft of the human genome was published independently and in parallel by the Human Genome Consortium and Celera Genomics. 6] This initial draft was brought close to completion in 2004. The HGP certainly was a milestone in biochemistry, but the methods applied were time-consuming and expensive. A broader application, whether in personalized medicine or for the routine sequencing of microorganisms, still seemed too ambitious at that time. Despite the significant drop in sequencing costs during the HGP from approximately 10 US

Aufbau einer biosynthetischen Polyketid‐Bibliothek durch minimalinvasive Mutagenese

to 0.09 US

Natural Product Lego.

per nucleobase (see Table 1), the total costs of the human genome sequencing summed up to roughly three billion US

Reduzierte Polyketide. Beiträge zur Erforschung und Manipulation der Substratspezifität von Polyketidsynthasen

. It was in early 2010, only few years after its commencement, that the Neanderthal Genome Project, led by Svante P bo in Leipzig, was brought to completion. The approximately 3.2 billion base pairs of the Neanderthal genome were deciphered from 40000-year-old small fragments of ancient DNA. Clearly, the starting position for this project was significantly less favorable than for the HGP, owing to the poor condition of the old genetic material and its comparably limited availability. But an impressive jump in DNA-sequencing technology pushed forward a significantly faster and more economical genome analysis of our prehistoric relative. This jump in development is heralding a new era in biochemical research. The very first steps towards a truly broad application of genome sequencing were taken independently through “sequencing by synthesis” developed by 454 Life Sciences led by Jonathan Rothberg, and through “multiplex polony sequencing” developed by Shendure et al. Both groups used fluorescence detection, which enabled simultaneous sequencing of several hundred thousand DNA fragments from tiny amounts of template—a major improvement over the 96-well format used in the didesoxy method. This impressive parallelization was one reason that the first version of the genome sequencer of 454 Life Sciences was already operating at a sixth of the cost of the Sanger method. However, early in its development, sequencing by synthesis experienced initial difficulties. Of major Table 1: Comparison of cost and expenditure of time for different sequencing techniques. Only commercially available techniques of the first and second generation are considered. Mbp: 1 10 base pairs; Gbp: 1 10 base pairs.