Exploring an alternative metabolic pathway for production of adipic acid - a study on homocitrate synthase

Sammanfattning: Adipic acid is considered as the most valuable dicarboxylic acid with industrial use; however, its production is heavily petroleum-based. This non-natural metabolite can be produced de novo through different metabolic pathways, one of which is the alpha-ketoacid elongation pathway. This investigation revolved around the study of the enzyme that catalyses the first +1C elongation reaction of the pathway: homocitrate synthase. Homocitrate synthases expressed in Saccharomyces cerevisiae, Thermus thermophilus and Azotobacter vinelandii were chosen as candidates. These candidates were evaluated on features such as: identification of the catalytic pocket residues; substrate specificity and possibility to widen such specificity though rational mutagenesis; literature available on the topic; reproducibility of the enzyme expression conditions. The fitting candidate would be able to employ C5 and C6 ketoacid as substrates - either naturally or through mutagenesis -, be fairly simple to express, isolate and test for enzyme activity using different substrates. Of these candidates, homocitrate synthase from T. thermophilus was successfully expressed and tested for enzymatic activity against alpha-ketoglutarate. In silico protein models for homocitrate synthase from S. cerevisiae and A. vinelandii were produced, along with the identification of the residues in the catalytic pocket of the two enzymes. This investigation is concluded by the choice of homocitrate synthase expressed from A. vinelandii as the best fitting candidate (between the one examined) for the +1C elongation step of the alpha-keto acid pathway finalized to the production of adipic acid.