From lignin to bio-nylon: optimization of muconic acid production by engineered Pseudomonas putida for separations and chemical catalysis to adipic acid

Lignin, the second most abundant biopolymer on Earth after cellulose, is underutilized in modern biorefineries despite its unique aromatic nature. The technical barrier associated with lignin is mainly due to its intrinsic recalcitrance and heterogeneous structure. In the current work, we will present how some native bacteria, such as Pseudomonas putida KT2440, are able to simultaneously deconstruct lignin (through the release of ligninolytic enzymes), uptake their principal constituents (aromatics), and utilize these molecules as carbon and energy sources. We have also recently engineered P. putida KT2440 to produce muconic acid, which can be further catalytically be upgraded to adipic acid, which is a primary precursor for nylon-6,6 production. We have combined metabolic engineering and fermentation engineering strategies to enable efficient production of muconate from aromatics found in lignin such as benzoate, p-coumarate, and ferulate. Muconate titers up to 35 g/L and yields over 95% have been achieved to date. Downstream separations and catalysis also lead to high muconic acid purity (99.8%), high conversion yields to adipic acid (100%), as well as excellent thermal properties of our bio-nylon polymer. These results provide a step towards an integrated approach to utilize enriched-lignin substrates by using a single biological catalyst and a viable path forward for a biochemical integrated technology to upgrade lignin into renewable chemical building blocks and value-added compounds.