Exploring lovastatin pathway enzymes for biocatalyst application.

摘要

Statins are extremely important drugs for treating cardiovascular diseases. Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes the rate-limiting step in cholesterol biosynthesis. The importance of statins to atherosclerosis has been compared to that of penicillin to infectious diseases. Statins can reduce death induced by coronary heart disease and heart attack rates by 20 to 50 percent, depending on the height of the initial blood cholesterol level and other risk factors for heart diseases. There are six statins approved by the FDA, three of which (lovastatin, simvastatin and pravastatin) are natural product derived. Lovastatin is produced by Aspergillus terreus as a secondary metabolite and is the first FDA approved statin. Simvastatin is a semi-synthesized derivative of lovastatin and contains a dimethyl group at C-2' of the side chain at C8. The subtle structural modification renders simvastatin more potent in the reduction of total and low-density lipoprotein cholesterol (LDL-C), with decreased hepatotoxicity and weakened side effects. Simvastatin was Merck's best selling drug it recorded &simMy research focused on the characterization of lovastatin biosynthetic pathway enzymes, mainly LovD and LovF. Through a thorough investigation of the enzymatic mechanism of LovD and LovF, I wished to gain deeper insight into polyketide biosynthesis including the polyketide programming rules as well as product releasing mechanism. Through characterization of LovD, we demonstrated that the protein-protein interaction between LovD and LovF was not necessary for the LovD catalyzed biosynthesis of lovastatin and its analogs. However, the protein- protein interaction facilitates highly efficient release and transfer of the diketide product to an accepting acyl substrate. In addition, LovD displayed broad substrate specificity toward the side chain as well as the acyl carrier.The second and also the main research goal was the application of lovastatin biosynthetic pathway enzymes for the biosynthesis of simvastatin. Lovastatin is an important and successful cholesterol lowering drug. Its pathway enzymes, especially LovD, are good candidates for engineering of lovastatin analog production. Using the broad substrate specificity property of LovD, I turned LovD into a very powerful biocatalyst for simvastatin biosynthesis. With the optimization of substrate, metabolic engineering of the host, protein engineering of LovD, and process engineering of the whole cell biocatalytic reactions, the LovD mediated simvastatin biosynthesis significantly decreased the cost of simvastatin manufacturing and outperformed the current organic semisynthetic methods.