Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity

Abstract

The conversion of biomass-derived sugars and aromatic molecules to cis,cis-muconic acid (referred to hereafter as muconic acid or muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces muconate production. A recent study showed that activity of the PCA decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of muconate from the aromatic lignin monomer p-coumarate by 50% and resulted in a titer of >15 g/L. In strains engineered to produce muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92±0.48 g/L muconate. This study demonstrates that overcoming the PCA decarboxylase bottleneck can increase muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions.

Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae

“…In E. coli, it was demonstrated that AroY-B stimulates the activity of AroY-C iso , but the effect of additional coexpression of Aro-D was dependent on plasmid design, and no conclusive statement could be made about its functional relevance (23). Similar contradictory results have been obtained with the AroY-D homologue EcdD from E. cloacae (24). In the results for a feeding experiment (Fig.…”

High value‐added monomer chemicals and functional bio‐based materials derived from polymeric components of lignocellulose by organosolv fractionation

“…Moreover, being similar to the chemicals produced from glucose by chemical catalysis, xylose can be converted into various important precursors like polyols such as 1,3‐propanediol, 2,3‐butanediol, and organic diacids such as succinic acid, itaconic acid, fumaric acid, muconic acid (further to adipic acid), and other functional acids such as lactic acid, and 3‐hydroxypropionic acid (3‐HPA) (Fig. ).…”

Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals

“…Important to this outcome is the exploitation of non‐model organisms that can naturally depolymerize and metabolize lignin to directly produce high value aromatic products. Key to this will be further understanding of the involved pathways and their regulation, as well as the continued identification and elimination of associated pathway bottlenecks . Thus, further discovery of non‐conventional microbes and development of genetic tools for their subsequent engineering will be critical to achieving this goal.…”