Engineering enzymes for the synthesis of 6-aminohex-2-enoic acid: first steps to green production of adipic acid

Research Project, 2017 – 2020

The proposed project fits contribute to the shift from oil-based economy to bio-based economy. Industrial biotechnology processes have been developed in this direction; however it is now time to build on those to advance towards the biosynthesis of a wider range of chemicals. The proposed project aims at engineering known enzymes with certain desired activity in order to modify their substrate specificity and catalyzing the biosynthesis of 6-aminohex-2-enoic acid (6-AHEA) from lysine (Lys). This achievement would pave the way to bio-based production of adipic acid that is widely used for nylon production. The enzyme activity necessary to directly convert Lys to 6-AHEA is an ammonia-lyase cleaving the carbon-nitrogen bond at the α-carbon of Lys. Lysine ammonia lyases are not known, while ammonia lyases acting on histidine or 3-methylaspartate are among the known and characterized ammonia lyases that we aim at engineering in order to convert their specificity from their natural substrate to Lys. The conversion of Lys into 6-AHEA could be alternatively achieved via three enzymatic steps involving the oxidation, dehydrogenation and
dehydration of Lys. Of these three steps only the first one is catalyzed by a known oxidase (EC 1.4.3.14) and our aim is to engineer phosphoglycerate dehydrogenase and fumarate dehydratase to change their substrate specificity towards 6-amino-2-oxohexanoic acid and 6-amino-2-hydroxyhexanoic acid, respectively. The proposed project combines state-of-the-art methodologies of computational structural biology that will assist in the generation and selection of mutant variants potentially able to catalyze the desired reactions. In particular, the computational approach will be combined with the generation and characterization of the suggested variants, with saturation mutagenesis and DNA shuffling, which will increase the possibilities to introduce further positive modifications not predicted by the computational approach.