MedicalResearch.com Interview with:
Professor Andrew W. Munro FRSC FSB
Professor of Molecular Enzymology
Manchester Institute of Biotechnology
Faculty of Life Sciences University of Manchester
MedicalResearch: What is the background for this study? What are the main findings?
Dr. Munro: Statins are blockbuster drugs that inhibit the key enzyme in cholesterol synthesis: 3-beta-hydroxymethylglutaryl CoA reductase (HMG-CoA reductase), which catalyzes the rate-limiting step in the biosynthesis of cholesterol. As a consequence, statin drugs reduce levels of low-density lipoprotein (LDL-) cholesterol, are effective against hypercholesterolemia and reduce the risk of atherosclerosis and heart attack. One of the major statin drugs is pravastatin, which is derived from a fungal natural product called compactin. The process of conversion of compactin into pravastatin involves the use of an oxygen-inserting enzyme called a cytochrome P450 (or P450), which catalyzes the hydroxylation of compactin to form pravastatin. In order to produce a more cost-efficient and streamlined route to pravastatin production, our teams from the University of Manchester (UK) and DSM (Delft, The Netherlands) developed a single-step process for pravastatin production. This process involved harnessing the productive efficiency of an industrial strain of the beta-lactam (penicillin-type) antibiotic producing fungus Penicillium chrysogenum. The beta-lactam antibiotic genes were deleted from this organism, and replaced by those encoding for compactin biosynthesis (transferred from a different Penicillium species). This led to high level production of compactin, but also to substantial formation of a partially degraded (deacylated) form. To get around this problem and in order to further improve compactin production, the enzyme responsible for the deacylation (an esterase) was identified and the gene encoding this activity was deleted from the production strain. The final stages of development of the novel, one-step pravastatin production process involved the identification of a suitable P450 enzyme that could catalyze the required hydroxylation of compactin. A bacterial P450 was identified that catalyzed hydroxylation at the correct position on the compactin molecule. However, the stereoselectivity of the reaction was in favour of the incorrect isomer – forming predominantly epi-pravastatin over the desired pravastatin. This was addressed by mutagenesis of the P450 – ultimately leading to a variant (named P450Prava) that hydroxylated compactin with the required stereoselectivity to make pravastatin in large amounts. Determination of the structure of P450Prava in both the substrate-free and compactin-bound forms revealed the conformational changes that underpinned the conversion of the P450 enzyme to a pravastatin synthase. The expression of P450Prava in a compactin-producing strain of P. chrysogenum enabled pravastatin production at over 6 g/L in a fed-batch fermentation process, facilitating an efficient, single-step route to high yield generation of pravastatin.
MedicalResearch: What should clinicians and patients take away from your report?
Dr. Munro: Pravastatin is a leading statin drug that is widely used to lower cholesterol levels and in the prevention of cardiovascular disease. The implementation of an improved route to the large scale synthesis of pravastatin should enable a more cost-effective process for its industrial production. The report also shows that industrially important fungal strains developed for the production of antibiotics can be effectively modified and repurposed to produce different types of important medicines. In addition, this work highlights how protein engineering can be used to generate enzymes with desirable catalytic properties, in this case facilitating efficient synthesis of the biologically active pravastatin drug at the expense of its inactive isomer epi-pravastatin.
MedicalResearch: What recommendations do you have for future research as a result of this study?
Dr. Munro: Many important medicines are developed from natural products derived from microorganisms. The work in this study demonstrates how the redesign of an industrially high-producing fungal strain can enable its exploitation for synthesis of different types of molecules in large yield – in this case an effective conversion of a beta-lactam (penicillin-type) antibiotic producer into a strain that produces the cholesterol-lowering drug pravastatin in large amounts. Future research in this area could involve further repurposing of such strains to allow more efficient production of different medicines and other industrially important molecules. This research study is a successful illustration of the emerging field of synthetic biology – in which different pieces of catalytic machinery have been adapted and assembled into a single fungal production unit, improving our ability to make a valuable statin drug.
Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum
McLean KJ, Hans M, Meijrink B, van Scheppingen WB, Vollebregt A, Tee KL, van der Laan JM, Leys D, Munro AW, van den Berg MA.
Published online before print February 17, 2015, doi: 10.1073/pnas.1419028112 PNAS March 3, 2015 vol. 112 no. 9 2847-2852
MedicalResearch.com Interview with: Professor Andrew W. Munro FRSC FSB (2015). Single Step Process May Speed Production of Statins