(27ay) Engineering Corynebacterium Glutamicum for Biomanufacturing of ?-Ketoadipate from Lignin-Derived Aromatics

A significant challenge in biomanufacturing remains the valorization of aromatic molecules, which can be readily attained from the lignin fraction of lignocellulosic biomass. In particular, lignin-derived aromatics can be bio-transformed into various petrochemical alternatives; however, they are typically toxic to conventionally used microbes at industrially relevant concentrations. To overcome this, we utilize Corynebacterium glutamicum, a soil microbe naturally capable of growing on diverse aromatic molecules, many of which are catabolized through the β-ketoadipate pathway. One intermediate of key interest is β-ketoadipate, which can be used as an alternative to adipic acid for the synthesis of bioplastics. To explore the ability of C. glutamicum ATCC 13032 to produce β-ketoadipate from lignin-derived aromatics, it was hypothesized that the deletion of the genes pcaI and pcaJ (encoding for subunits of β-ketoadipate:succinyl-coenzyme A transferase) would lead to the accumulation of β-ketoadipate. We used the sacB counterselection method to develop the strain and then evaluated it’s ability to produce β-ketoadipate from various model aromatic molecules. Shake-flask experiments conducted with 20 g/L glucose and 6 g/L of different individual aromatic substrates produced 1.73 g/L (from ferulate), 1.68 g/L (from vanillin), and 1.77 g/L (from p-coumarate) of β-ketoadipate. In all cases, since accumulation of intermediates was observed, it was hypothesized that additional glucose supplementation might enable the full conversion of aromatics to β-ketoadipate. To assess this, initial glucose was doubled to 40 g/L, which resulted in 2.16 g/L β-ketoadipate from p-coumarate; representing a 24% increase in yield, from 0.29 g/g to 0.36 g/g. Meanwhile, although the engineered strain displayed significantly decreased fitness when grown using model aromatics as the sole substrate, minimal growth was still observed; suggesting that additional strain engineering may be required to further improve β-ketoadipate production. Overall, this study is the first to report the biotransformation of lignin-derived aromatics to β-ketoadipate in C. glutamicum. Future work entails utilizing genome scale modeling, adaptive laboratory evolution (ALE), and reverse engineering of mutants to identify additional genome engineering strategies for enhancing β-ketoadipate production.