Engineering ammonia-lyases for lysine transformation: first steps to green production of adipic acid

Poster (konferens), 2017

Adipic acid is one of the most important dicarboxylic acid for commercial purposes, mainly used as building block for nylon polymers. The current chemical production process has serious consequences for the environment. Therefore, the implementation of a by a bio-based process using renewable feedstocks would be highly beneficial for the society and the environment. 
The construction of a microbial metabolic pathway to produce adipic acid using L-lysine as precursor is a potential alternative. The first step of the proposed pathway converts L-lysine into 6-aminohex-2-enoic acid (6-AHEA) via removal of the α-amino group. Chemical methods for this deamination reaction are known; however, no enzymes able to carry out this reaction on L-lysine have been disclosed yet. The main goal of the current research is the generation a novel enzyme activity for the conversion of L-lysine to 6-AHEA. 
The enzymatic activity necessary to catalyze the required deamination is defined as ammonia lyase and results in the removal of the α-amino group. Histidine ammonia-lyase (HAL) and 3-methylaspartate-ammonia-lyase (MAL), enzymes acting on histidine and 3-methylaspartate, respectively, were selected to be engineered to catalyze the deamination of lysine. 
HAL from Pseudomonas putida and MAL from Clostridium tetanomorphum and Carboxydothermus hydrogenoformans were expressed in E.coli and purified. The capability of the enzymes to deaminate lysine was tested. No deamination activity was observed, while the inhibitory effect of L-lysine on HAL activity was shown.
Computational structural biology methodologies were applied on MAL and combined with protein engineering techniques in order to design mutant enzyme variants potentially active on L-lysine. In-silico saturation mutagenesis tools were used to model all the possible mutations in the active site or its surroundings that are expected to increase affinity for the L-lysine substrate. Following the results obtained, the residues C361, M389 and L384 were mutagenized. The mutant variants were produced and purified, and the activity on L-lysine tested by monitoring the production of 6-AHEA and the release of ammonia.