Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum

The cholesterol-lowering blockbuster drug pravastatin can be produced by stereoselective hydroxylation of the natural product compactin. We report here the metabolic reprogramming of the antibiotics producer Penicillium chrysogenum toward an industrial pravastatin production process. Following the successful introduction of the compactin pathway into the β-lactam–negative P. chrysogenum DS50662, a new cytochrome P450 (P450 or CYP) from Amycolatopsis orientalis (CYP105AS1) was isolated to catalyze the final compactin hydroxylation step. Structural and biochemical characterization of the WT CYP105AS1 reveals that this CYP is an efficient compactin hydroxylase, but that predominant compactin binding modes lead mainly to the ineffective epimer 6- epi -pravastatin. To avoid costly fractionation of the epimer, the enzyme was evolved to invert stereoselectivity, producing the pharmacologically active pravastatin form. Crystal structures of the optimized mutant P450 Pᵣₐᵥₐ bound to compactin demonstrate how the selected combination of mutations enhance compactin binding and enable positioning of the substrate for stereo-specific oxidation. Expression of P450 Pᵣₐᵥₐ fused to a redox partner in compactin-producing P. chrysogenum yielded more than 6 g/L pravastatin at a pilot production scale, providing an effective new route to industrial scale production of an important drug. Significance Statins are successful widely used drugs that decrease the risk of coronary heart disease and strokes by lowering cholesterol levels. They selectively inhibit the key regulatory enzyme of the cholesterol synthesis pathway, thus lowering levels of plasma LDL (bad) cholesterol. Pravastatin is one of the leading and most effective statins, derived from the natural product compactin. However, pravastatin production involves a costly dual-step fermentation and biotransformation process. Here we present a single-step fermentative method for production of the active drug pravastatin. Reprogramming of the antibiotics-producing fungus Penicillium chrysogenum , with discovery and engineering of an enzyme involved in the hydroxylation of compactin, enables high level fermentation of the correct form of pravastatin to facilitate efficient industrial-scale statin drug production.