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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.

SUBMITTER: Johnson CW

PROVIDER: S-EPMC5779730 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Publications

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

Johnson Christopher W CW Salvachúa Davinia D Khanna Payal P Smith Holly H Peterson Darren J DJ Beckham Gregg T GT

Metabolic engineering communications 20160422

The conversion of biomass-derived sugars and aromatic molecules to <i>cis</i>,<i>cis</i>-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 ar ...[more]

PMID: 29468118

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Project description:Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.

Dataset Information

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

Publications

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

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