Lignin upgrading to muconic acid by recombinant Pseudomonas putida KT2440: DO-stat fed-batch process development for strain evaluation

Lignin, the second most abundant biopolymer on Earth after cellulose, is underutilized in biochemical conversion processes to produce renewable fuels and chemicals, where it is typically used to generate heat and power. However, the aromatic molecules found in this complex polymer are an excellent carbon source for some organisms, which are able to metabolize heterogeneous slates of aromatic molecules to a few central intermediates and subsequently direct them into central metabolism. Coupling metabolic engineering of these natural pathways with fermentation engineering enables the efficient production of renewable chemical building blocks from aromatic species found in lignin. In the current work, we will present different bioreactor cultivation strategies using strains of Pseudomonas putida KT2440 engineered to produce muconic acid, which is a precursor to adipic acid. Diverse lignin aromatic models (e.g., benzoate, coumarate, and ferulate) have been utilized, and DO-stat fed-batch fermentations strategies have been developed to avoid toxicity effects by these aromatics and decrease catabolic repression by other carbon sources present in the cultivation media. Muconate titers up to 40 g/L and yields of 100% have been achieved to date. Downstream separations and catalysis of muconate resulting from these cultivations have been also performed, showing high yields of adipic acid, suggesting a viable path forward for this integrated technology to upgrade lignin to value-added compounds.