Quantification of N-acetylcysteamine activated methylmalonate incorporation into polyketide biosynthesis

Quantification of N-acetylcysteamine activated methylmalonate incorporation into polyketide biosynthesis Stephan Klopries1, Uschi Sundermann1,2 and Frank Schulz*1,2 Full Research Paper Address: 1Fakultät für Chemie, Technische Universität Dortmund, Otto-Hahn-Str. 6, 44221 Dortmund, Germany and 2Abteilung für Chemische Biologie, Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany Email: Frank Schulz* - * Corresponding author Open Access Beilstein J. Org. Chem. 2013, 9, 664–674. doi:10.3762/bjoc.9.75 Received: 10 October 2012 Accepted: 11 March 2013 Published: 05 April 2013 Associate Editor: A. Kirschning © 2013 Klopries et al; licensee Beilstein-Institut. License and terms: see end of document. Keywords: biosynthesis; coenzyme A; malonic acid; polyketide; polyketide synthase Abstract 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. Introduction Polyketides are ubiquitous natural products and find widespread application in current medicine and agriculture. Polyketide synthases (PKS), giant multienzyme complexes, play a pivotal role in their biosynthesis. PKS generate molecular complexity and diversity through a number of stepwise condensations in analogy to fatty acid synthases but with optional and varying degrees of reduction in each step (Figure 1) [1-3]. Ad- ditional diversity is introduced by the incorporation of different carboxylic acid starter units and a range of different extender units, usually coenzyme A-activated malonic acid derivatives, with varying substituents at C-2 [4,5]. Current experiments to generate biosynthetic polyketide diversity focus on different aspects of the biosynthetic reaction 664 Beilstein J. Org. Chem. 2013, 9, 664–674. Figure 1: The most intensively studied PKS, deoxyerythronolide B synthase (DEBS), which catalyzes the key steps in the biosynthesis of the antibiotic erythromycin. DEBS catalyzes the extension of a propionate starter unit with six equivalents of methylmalonyl-coenzyme A (MM-CoA). After six rounds of decarboxylative Claisen condensations and varying degrees of reduction of the initially formed β-keto thioesters, the polyketide core of erythromycin is released from the enzyme via a terminal esterase [6-8]. Abbreviations: AT: acyltransferase, ACP: acyl carrier protein, KS: ketosynthase, KR: ketoreductase, DH: dehydratase, ER: enoylreductase, TE: thioesterase. cascade. Mainly by genetic replacement or deletion of various fragments of PKS, alterations in chain length [9-11], redox pattern [12-14], stereochemistry [15-17], and starter unit diversity [18-20] were in several instances introduced, giving rise to a significant number of polyketide derivatives to date. However, the accessible extender unit diversity is currently restricted to a small number of different malonate units and changes in the polyketide side chain pattern are highly sought after [4,17,2126]. Different strategies can be pursued to introduce non-native extender units into the PKS machinery. They rely on the replacement of an acyltransferase domain of a given PKS module with another domain possessing different substrate specificity by using various different strategies. Subsequently, thioester activated non-native malonate derivatives can be synthesized by additional heterologously expressed biosynthetic pathways leading to the respective derivative of malonylCoA [17] or malonyl-ACP [27], which are supplied in vivo to the mutated PKS. Another option is the exogenous supply of the malonate derivative [28], typically activated as N-acetylcysteamine thioesters (malonyl-SNAC) [29-40]. Despite its apparent simplicity, the latter option is not well characterized for in vivo applications. Especially the bioavailability and the coupled acceptance efficiency to the corresponding CoA-esters remain to be clarified. This has recently become highly relevant, as the first example for an acyltransferase domain with artificially broadened substrate specificity has been constructed and introduced in the biosynthetic pathway towards erythromycin in place of the native AT6 domain [39]. This has led to the formation of 2-propargylerythromycin through the incorporation of 2-propargylmalonate into the biosynthetic pathway, activated as SNAC-thioester and supplied to the bacterial fermentation. In this step, the exogenously supplied synthetic building block competes with the endogenous methylmalonyl-CoA (MM-CoA) for acceptance by the same enzyme domain; hence, the result of this experiment was the formation of the wild-type erythromycin product as a mixture with the new propargylated derivative. It is now of interest to judge the efficiency of SNAC- versus CoA-activation. This can show to which extent the activation influences the choice of the extender units in the case that a promiscuous acyltransferase domain can catalyze the incorporation of a natural and a non-natural building block. 665 Beilstein J. Org. Chem. 2013, 9, 664–674. We here report the characterization of deuterated methylmalonyl-SNAC ester incorporation into polyketides in vivo and therefore in direct competition to endogenous MM-CoA, to determine the relative incorporation efficiency and the concentrations required to saturate polyketide synthases with the artificial extender unit donor. This determines the impact of SNACversus CoA-activation and gives rise to rapid optimization of feeding experiments using exogenously supplied malonatederivatives. These experiments point the way towards the incorporation of non-native malonate derivatives into biosynthetic pathways en route to new polyketide derivatives. Results and Discussion Synthesis of D3-labeled malonyl-SNACesters The incorporation of artificially activated exogenous methylmalonate is most straightforwardly monitored by LC/ESI–MS. This requires stable-isotope-labeled material, preferably a D 3 -label. For adding exogenous malonates to bacterial fermentations, millimolar concentrations of the SNAC-activated (...truncated)