Synthetic pathway engineering in Corynebacterium glutamicum for production of adipic acid
Poster (konferens), 2020
We are interested in production of adipic acid from renewable sources using a gram-positive bacterium, Corynebacterium glutamicum ATCC 13032, as a target for metabolic engineering. We aim to construct a synthetic pathway that would allow biosynthesis of adipic acid and to implement it into the host chassis for utilizing simple sugar, glucose, as the starting raw material. The chosen pathway stems off from intermediates from the central metabolism and requires 5 synthetic biochemical steps before reaching adipic acid. The pathway is equipped with a promiscuous enzyme to allow leakage at each metabolic step and byproduct formation in order to monitor the efficiency of each step of the pathway. In order to ensure translation, each gene introduced was codon-optimized to C. glutamicum. Introducing the synthetic genes one by one into C. glutamicum as well as flask-level cultivation of engineered strains in a semi-defined medium allowed detection of each byproduct of the pathway leading up to adipic acid by GC/MS based methods. Further, the codon-optimized version of the 5 synthetic genes were constructed in 2 separate operons each with an inducible promoter. Finally, we analyzed the translational efficiency of the genes and modified the constructs for a better operon expression. We implement additional sample preparation methods for isolating and concentrating adipic acid content for better analysis. The results shown here will be used to further develop
and complete biosynthesis of adipic acid from a C. glutamicum chassis.