Engineering synthetic pathways for adipic acid biosynthesis

Sammanfattning: Utilization of petroleum in consumer product manufacturing has caused irreversible and detrimental environmental damages globally. Recent awareness of apparent impact of such pollutions on land, sea, and air urgently prompts development of technologies to produce consumer goods independently of petroleum and sustainably. Motivation of such development points towards use of renewable materials such as lignocellulosic biomass and to further raises a necessity to develop technologies to convert biomass into platform monomers. The thesis addresses the in vivo and in silico assessment of various enzymes involved in different metabolic pathways for microbial production of adipic acid from simple sugar molecules. The aim of the work presented in this thesis was to map out the comprehensive metabolic pathways leading to adipic acid biosynthesis as well as the corresponding enzymatic components. Reduction and deamination parts in one of the theoretical pathways based on L-lysine metabolism were assessed. Molecular docking studies accompanied with molecular dynamics studies as well as interaction energy studies shed light on to possible binding mechanisms for Old yellow enzyme class enzymes. Potential mechanism and feasibility of α,β-reduction of L-lysine pathway intermediate compounds were explained in terms of substrate interaction in the enzyme binding pockets. Deamination reaction of L-lysine was determined to be less likely to occur. D-β-lysine was chosen to be the better intermediate for α, β-elimination. Removal of β-amino group from β-lysine is deemed to be more feasible if an enzyme is additionally developed to accommodate the unnatural substrate. In terms of reversed β-oxidation pathway and implementation, Corynebacterium glutamicum was chosen as the host chassis for extending the synthetic pathway toward adipic acid. Stepwise construction of 5-step synthetic pathway demonstrated functionality of the condensation and the first reduction steps. Non-decarboxylative condensation of acetyl-CoA and succinyl-CoA was demonstrated in C. glutamicum in vivo for the first time. Reduction of 3-oxoadipyl-CoA was performed in C. glutamicum for the first time as well. Biosynthesis of 3-hydroxyadipate as well as secretion was detected in the cultivation broth using GC/MS methodology. Potential bottleneck hindering the pathway was identified to be the reduction step of 3-hydroxyadipyl-CoA. In order to alleviate the problem, expression optimization and choosing an alternative enzyme is suggested. Along the course of implementing the reversed β-oxidation pathway, a new pathway was observed and experimented on. A new strategy for reaching adipic acid from 3-oxoadipate via cis,cis-muconic acid was  pursued. Bio-conversion of benzoic acid to cis,cis-muconic acid was successful and molecular docking studies were carried out further improve knowledge regarding the currently refuted knowledge regarding enoate reductases for their ability to reduce cis,cis-muconic acid.