Acyltransferase LovD as a Simvastatin Synthase: Mutational Deconvolution, Design, Substrate Scope and Immobilization

Résumé

In this doctoral thesis, a systematic study of the acyltransferase enzyme LovD from the fungus Aspergillus terreus, which was previously modified by directed evolution for the improved production of the anti-cholesterol drug simvastatin from monacolinic acid J, is performed. First, an analysis of the features affecting the catalytic activity of the enzyme throughout the different rounds of directed evolution (competition between thioesterase, acyltransferase and hydrolase activities, inhibition by substrate and thermal stability), is described, and new variants with fewer mutations and comparable catalytic activities are designed. Second, a rational design of native LovD is performed using computational methods for sequence diversification and molecular dynamics. Then, the value of vinyl and para-nitrophenyl esters as efficient alternatives to thioesters as acyl donor agents is described. Finally, the immobilization of a designed LovD variant on different solid supports with different characteristics is accomplished, and the properties of the resulting biocatalysts are analyzed in detail using biophysical techniques. The conditions for the efficient synthesis of simvastatin using continuous flow reactors with potential interest for industrial production, were optimized.