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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 9; pp. 2847 - 2852
• Main Authors: McLean, Kirsty J., Hans, Marcus, Meijrink, Ben, van Scheppingen, Wibo B., Vollebregt, Aad, Tee, Kang Lan, van der Laan, Jan-Metske, Leys, David, Munro, Andrew W., van den Berg, Marco A.
• Format: Journal Article
• Language: English
• Published: National Academy of Sciences 03-03-2015
• ISSN: 0027-8424; 1091-6490

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 producerPenicillium chrysogenumtoward an industrial pravastatin production process. Following the successful introduction of the compactin pathway into the β-lactam–negativeP. chrysogenumDS50662, a new cytochrome P450 (P450 or CYP) fromAmycolatopsis 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 P450Pravabound to compactin demonstrate how the selected combination of mutations enhance compactin binding and enable positioning of the substrate for stereo-specific oxidation. Expression of P450Pravafused to a redox partner in compactin-producingP. chrysogenumyielded more than 6 g/L pravastatin at a pilot production scale, providing an effective new route to industrial scale production of an important drug.