UC Berkeley

Fluorination is an important tool for the discovery and production of synthetic pharmaceuticals, as it allows fine-tuning their pharmacokinetic properties and metabolic stability. However, unlike chemists, Nature rarely utilizes fluorine to enhance its molecules, and the few known naturally occurring organofluorines generally lack structural complexity. Despite advances in chemical fluorination methods, the regiospecific fluorination of complex natural products remains a challenge. Thus, we have taken a metabolic engineering approach to develop a novel platform for fluorine incorporation into polyketide natural products. Polyketide natural products are synthesized from one starter unit and multiple acyl-CoA extender units. We designed a modular system in which an inactivating mutation was introduced to the acyltransferase (AT) domain of interest to reduce the native activity of the module, while an alternative fluorine-selective AT enzyme was provided in trans to incorporate fluorine as a fluoromalonyl-CoA extender unit. A malonate transporter and a malonyl-CoA synthetase were introduced to support the intracellular accumulation of fluoromalonate and its enzymatic activation into fluoromalonyl-CoA extender unit, respectively. By engineering a single module construct of 6-deoxyerythronolide B (6dEB) synthase (DEBS), fluorinated triketide lactone was produced as the major polyketide product at mg/L levels in an Escherichia coli host. Extending these engineering concepts to a complete DEBS pathway allowed us to produce novel fluorine-containing 6dEB analogs in E. coli production host in a regiospecific manner. This platform can allow the precise incorporation of fluorine into many positions in the backbones of polyketides. These results open the door to producing a new class of drug-like molecules with potentially diverse and improved properties.