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.

Keywords: Lignin valorization; Muconic acid; Protocatechuate decarboxylase; Pseudomonas Putida KT2440; cis,cis-Muconate.

Fig. 1

Metabolic pathways for production of muconate from glucose and lignin-derived aromatic compounds. In P. putida KT2440, glucose is metabolized through the Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to produce phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), which can be condensed to enter the shikimate pathway for aromatic amino acid biosynthesis. An intermediate in the shikimate pathway, 3-dehydroshikimate, can be converted to PCA by the action of a 3-DHS dehydratase, such as AsbF from Bacillus cereus used here. Deletion of the genes encoding the PCA dioxygenase, PcaHG, and integration of genes encoding the PCA decarboxylase AroY from Enterobacter cloacae and two associated proteins, EcdB and EcdD, from enables PCA to be converted to catechol rather than entering the β-ketoadipate pathway. Two paralogous dioxygenases, CatA and CatA2, convert catechol to muconate, which accumulates due to deletion of the genes encoding CatB and CatC, two enzymes required for further metabolism of muconate. Lignin-derived aromatic molecules are metabolized through upper pathways to form catechol in the case of phenol or guaiacol while p-coumarate, ferulate, 4-hydroxybenzoate, and vanillate are metabolized to form PCA, which can then be converted to catechol by the action of the PCA decarboxylase for subsequent conversion to muconate.

Fig. 2

Shake-flask evaluations of muconate production from p-coumarate by engineered P. putida KT2440 strains. Cultures were grown in M9 minimal medium containing p-coumarate and fed glucose periodically as a source of carbon and energy for growth and sampled to evaluate culture growth by OD₆₀₀ and the concentration of metabolites in the medium by HPLC. Each value represents the average of three biological replicates. The error bars represent standard deviation of the measurements. (A) KT2440-CJ102, expressing the AroY PCA decarboxylase, (B) KT2440-CJ183, expressing AroY as well as EcdB, and (C) KT2440-CJ184, expressing AroY, EcdB, and EcdD.

Fig. 3

Bioreactor evaluations of muconate production from p-coumarate by engineered P. putida KT2440 strains. Cultures were grown for 72 h in M9 minimal medium by DO-stat control, pulsing p-coumarate and glucose to reach bioreactor concentrations of 1.3 mM and 0.33 mM, respectively. Cultures were sampled to evaluate bacterial growth by OD₆₀₀ and the concentration of metabolites in the medium by HPLC. p-Coumarate values graphed above represent the amount remaining of the total p-coumarate fed by the conclusion of the experiment. Each value represents the average of two biological replicates. The error bars represent the range of the measurements. (A) KT2440-CJ102, expressing the AroY PCA decarboxylase, (B) KT2440-CJ183, expressing AroY as well as EcdB, and (C) KT2440-CJ184, expressing AroY, EcdB, and EcdD.

Fig. 4

Shake-flask evaluations of muconate production from glucose by engineered P. putida KT2440 strains. Cultures were grown for 24 h in M9 minimal medium containing glucose for conversion to muconate and as a source of carbon and energy for growth and sampled to evaluate culture growth by OD₆₀₀ and the concentration of metabolites in the medium by HPLC. Each value represents the average of three biological replicates. The error bars represent standard deviation of the measurements. (A) KT2440-CJ156, expressing the AroY PCA decarboxylase, (B) KT2440-CJ200, expressing AroY as well as EcdB, and (C) KT2440-CJ202, expressing AroY, EcdB, and EcdD.

Fig. 5

Bioreactor evaluations of muconate production from glucose by engineered P. putida KT2440 strains. Cultures were grown in M9 minimal medium and fed glucose at time points as indicated by arrows. Cultures were sampled to evaluate culture growth by OD₆₀₀ and the concentration of metabolites in the medium by HPLC. Glucose values graphed above represent the amount remaining of the total glucose fed by the conclusion of the experiment. Each value represents the average of two biological replicates. The error bars represent the range of the measurements. (A) KT2440-CJ156, expressing the AroY PCA decarboxylase, (B) KT2440-CJ200, expressing AroY as well as EcdB, and (C) KT2440-CJ202, expressing AroY, EcdB, and EcdD.