title: Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.
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 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.
authors.affiliation: Manchester Institute of Biotechnology, Faculty of Life Sciences, The University of Manchester, Manchester M1 7DN, United Kingdom; and.
authors.forename: Kirsty J
authors.identifier: http://orcid.org/0000-0002-7193-5044
authors.initials: KJ
authors.lastname: McLean
authors.affiliation: DSM Biotechnology Center, 2613 AX, Delft, The Netherlands.
authors.forename: Marcus
authors.initials: M
authors.lastname: Hans
authors.affiliation: DSM Biotechnology Center, 2613 AX, Delft, The Netherlands.
authors.forename: Ben
authors.initials: B
authors.lastname: Meijrink
authors.affiliation: DSM Biotechnology Center, 2613 AX, Delft, The Netherlands.
authors.forename: Wibo B
authors.initials: WB
authors.lastname: van Scheppingen
authors.affiliation: DSM Biotechnology Center, 2613 AX, Delft, The Netherlands.
authors.forename: Aad
authors.initials: A
authors.lastname: Vollebregt
authors.affiliation: Manchester Institute of Biotechnology, Faculty of Life Sciences, The University of Manchester, Manchester M1 7DN, United Kingdom; and.
authors.forename: Kang Lan
authors.initials: KL
authors.lastname: Tee
authors.affiliation: DSM Biotechnology Center, 2613 AX, Delft, The Netherlands.
authors.forename: Jan-Metske
authors.initials: JM
authors.lastname: van der Laan
authors.affiliation: Manchester Institute of Biotechnology, Faculty of Life Sciences, The University of Manchester, Manchester M1 7DN, United Kingdom; and.
authors.forename: David
authors.initials: D
authors.lastname: Leys
authors.affiliation: Manchester Institute of Biotechnology, Faculty of Life Sciences, The University of Manchester, Manchester M1 7DN, United Kingdom; and andrew.munro@manchester.ac.uk marco.berg-van-den@dsm.com.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
authors.affiliation: DSM Biotechnology Center, 2613 AX, Delft, The Netherlands andrew.munro@manchester.ac.uk marco.berg-van-den@dsm.com.
authors.forename: Marco A
authors.initials: MA
authors.lastname: van den Berg
mesh_terms: D001483:Base Sequence; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010408:Penicillium chrysogenum / Q000201:enzymology / Q000235:genetics; D017035:Pravastatin / Q000096:biosynthesis*; D013237:Stereoisomerism
chemical_list: D005656:Fungal Proteins; D003577:Cytochrome P-450 Enzyme System; D017035:Pravastatin
keywords: P450 engineering; cholesterol; fermentation; pravastatin; statin