Engineering synthetic pathways for adipic acid biosynthesis

Doctoral thesis, 2021

Utilization of petroleum in consumer product manufacturing is causing irreversible environmental damage. Its impact on land, sea, and air calls for the development of more sustainable technologies based on the use of renewable materials such as lignocellulosic biomass and its conversion into platform chemicals. Engineering microorganisms to produce chemicals is an important undertaking to address such issues and bio-based production of adipic acid especially has gained recent attention. In the present thesis I assess the in vivo and in silico action of enzymes involved in microbial production of adipic acid from simple sugar molecules. The aim of this work was to comprehensively map out the metabolic pathways leading to adipic acid biosynthesis and to investigate the enzymatic components of the L-lysine pathway, the reverse β-oxidation pathway, and cis,cis-muconic acid reduction.
Investigation of theoretical and in silico aspects in the deamination step in the L-lysine pathway revealed deamination of L-lysine was determined to be chemically difficult to occur. Removal of the β-amino group from β-D-lysine was deemed more feasible than the α-amino group from L-lysine, and an alternative route via β-D-lysine deamination was suggested. Homology modeling and molecular docking studies shed light on the substrate binding mechanisms of enzymes responsible for the reduction of the intermediates in the L-lysine pathway. Potential mechanism and feasibility of α,β-reduction were explained in terms of substrate interaction in the enzyme-binding pockets. Corynebacterium glutamicum was chosen as the host chassis for achieving adipic acid synthesis via reverse β-oxidation. Stepwise construction of a five-step synthetic pathway demonstrated functionality of each step in C. glutamicum. Biosynthesized and secreted 3-hydroxyadipate was detected in the cultivation broth using GC/MS. Weak trans-2-hexenedioic acid and adipic acid signals was observed using LC/MS after concentrating the cultivation broth. Dehydration of 3-hydroxyadipyl-CoA was identified as a potential bottleneck hindering this pathway. While implementing the reverse β-oxidation pathway, a new pathway involving cis,cis-muconic acid and 3-oxoadipic acid was observed and experimented on. The modified strategy for bio-conversion of benzoic acid to cis,cis-muconic acid was successful and molecular docking studies were carried out to better understand how oxidoreductases might reduce cis,cis-muconic acid.
Taking multiple approaches to generate adipic acid revealed different challenges in each pathway. One approach led to biosynthesis of adipic acid. Further investigation will allow multiple options for bio-based adipic acid production for better sustainability.