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.pravastatin | P450 engineering | fermentation | statin | cholesterol

No abstract

The regio-and stereoselective oxidative transformation of unactivated C-H to C-OH is a major challenge for traditional organic synthesis but with enzymes these reactions often proceed in a one-step synthesis of complex molecules a large number of which have industrial added value as pharmaceuticals, flavors and fragrances and fine chemicals. In this chapter a range of enzymes that perform this are described.The most studied of the enzymes that carry out this transformation is the cytochrome P450 groups of enzymes that have been extensively studied for hydroxylation of organic substrates in very regio-and stereospecific manner. This is a very large and diverse superfamily of hemoproteins found in all domains of life. In mammals these proteins oxidize steroids, fatty acids and xenobiotics and are important for the detoxification and clearance of various compounds from the body, hormone synthesis and breakdown, cholesterol synthesis and vitamin D metabolism. Linked to their role of xenobiotic breakdown they can also convert pharmaceutically active substances into more active metabolites that are synthetically challenging to produce and hence as biocatalysts can be used to produce more effective syntheses of these pharmaceutical targets in both small scale preparations as analytical standards and as synthetic methods. The most common reaction catalyzed by P450 is a monooxygenase reaction, e.g. insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water: RH þ O 2 þ 2H þ þ 2e À ! ROH þ H 2 O , stereo-, and chemoselective hydrocarbon oxyfunctionalization reactions are important in industrial organic synthesis but difficult to achieve by chemical means 1 . As an alternative to the present chemical and oxygenase-based biocatalytic oxyfunctionalization processes, molybdenum (Mo)-containing dehydrogenases have a high potential in technical applications, since they use water as the oxygen donor and produce rather than 5.5 Regioselective Aromatic Hydroxylation of Quinaldine 153