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

Research output: Contribution to journalArticle

  • External authors:
  • Kirsty J Mclean
  • Marcus Hans
  • Ben Meijrink
  • Wibo B van Scheppingen
  • Aad Vollebregt
  • Kang Lan Tee
  • Jan-Metske van der Laan
  • Marco van den Berg

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.

Bibliographical metadata

Original languageEnglish
Pages (from-to)2847-2852
Number of pages5
JournalProceedings of the National Academy of Sciences
Volume112
Issue number9
DOIs
StatePublished - 3 Mar 2015