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

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.Homocitrate synthase, a ubiquitous but unique enzyme Plastic is a ubiquitous material, much needed in our everyday life. However, petroleum-based production of plastic is the cause for waste littering and greenhouse gasses emissions during manufacturing. Instead, biobased plastics are composed of building blocks obtained from renewable carbon sources. Some of these building blocks can also be non-natural metabolites, such as adipic acid. Adipic acid is industrialized mostly in the form of nylon 6,6, a polymer employed in the automotive, textile, electric, and many other fields. There are different pathways that can be used to produce de novo adipic acid in microorganisms, however some are more efficient than others. The pathway involving carbon chain elongation of alpha-ketoacids in the citric acid cycle, for example, can be greatly improved. For this reason, in-depth studies concerning each reaction and catalyst are needed to improve the efficiency of the pathway. This investigation revolved around the study of the enzyme that catalyzes 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 ketoacid (alpha-ketoglutarate) and C6 ketoacid (alpha-ketoadipate) - either naturally or through mutagenesis -, be fairly easy to express and test for enzyme activity using different substrates. The practical work revolved around expression of these homocitrate synthases, however only homocitrate synthase from Thermus thermophilus was successfully expressed and tested for enzymatic activity against alpha-ketoglutarate. Protein modelling was also performed. Since homocitrate synthases in Saccharomyces cerevisiae and Azotobacter vinelandii have yet to be crystallized, in silico protein models for these two candidates were produced during this investigation. These models allowed to conduct a search into the catalytic pocket residues of the various homocitrate synthase candidates, which were investigated through alignment of protein sequences and structures. Most of the residues belonging in the active site of homocitrate synthase in Saccharomyces cerevisiae and Azotobacter vinelandii were identified and localized. These notions could of use in order to determine rational mutagenesis of the active site of other homocitrate synthases, attempting to widen substrate specificity. Moreover, the lid motif – a “gatekeeping” structure that allows the access to the enzyme´s catalytic pocket – was identified in the homocitrate synthase in Saccharomyces cerevisiae, and visualized. 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. Master’s Degree Project in Molecular Biology 60 credits 2019 Department of Biology, Lund University Advisor: Rajni Hatti-Kaul Division of Biotechnology, Department of Chemistr