RESUMO
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 enzyme's 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.
Assuntos
Aciltransferases/metabolismo , Policetídeos/metabolismo , Aciltransferases/genética , Antibacterianos/química , Antibacterianos/metabolismo , Eritromicina/análogos & derivados , Eritromicina/biossíntese , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Policetídeo Sintases/química , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Mutação Puntual , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Engenharia de Proteínas , Aciltransferases/química , Aciltransferases/genética , Aciltransferases/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Eritromicina/metabolismo , Malonatos/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Policetídeo Sintases/química , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Especificidade por SubstratoRESUMO
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.