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

Kirsty J. McLean, Marcus Hans, Ben Meijrink, Wibo B. Van Scheppingen, Aad Vollebregt, Kang Lan Tee, Jan Metske Van Der Laan, David Leys, Andrew W. Munro, Marco A. Van Den Berg

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Abstract

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 P450Prava 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 P450Prava 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.

Original languageEnglish
Pages (from-to)2847-2852
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number9
Early online date17 Feb 2015
DOIs
Publication statusPublished - 3 Mar 2015
Externally publishedYes

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    McLean, K. J., Hans, M., Meijrink, B., Van Scheppingen, W. B., Vollebregt, A., Tee, K. L., Van Der Laan, J. M., Leys, D., Munro, A. W., & Van Den Berg, M. A. (2015). Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum. Proceedings of the National Academy of Sciences of the United States of America, 112(9), 2847-2852. https://doi.org/10.1073/pnas.1419028112