Metabolic Engineering of Pseudomonas putida KT2440 to Produce Anthranilate from Glucose

Abstract

The Pseudomonas putida KT2440 strain was engineered in order to produce anthranilate (oAB, ortho-aminobenzoate), a precursor of the aromatic amino acid tryptophan, from glucose as sole carbon source. To enable the production of the metabolic intermediate oAB, the trpDC operon encoding an anthranilate phosphoribosyltransferase (TrpD) and an indole-3-glycerol phosphate synthase (TrpC), were deleted. In addition, the chorismate mutase (pheA) responsible for the conversion of chorismate over prephenate to phenylpyruvate was deleted in the background of the deletion of trpDC to circumvent a potential drain of precursor. To further increase the oAB production, a feedback insensitive version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase encoded by the aroGD146N gene and an anthranilate synthase (trpES40FG) were overexpressed separately and simultaneously in the deletion mutants. With optimized production conditions in a tryptophan-limited fed-batch process a maximum of 1.54 ± 0.3 g L-1 (11.23 mM) oAB was obtained with the best performing engineered P. putida KT2440 strain (P. putida ΔtrpDC pSEVA234_aroGD146N_trpES40FG).

Microbial production of methyl anthranilate, a grape flavor compound

“…Next, further metabolic engineering approaches were taken to increase the level of ANT and consequently to improve MANT production in C. glutamicum. Although reported in E. coli (12,13) and P. putida (21), ANT overproduction has never been explored in C. glutamicum. To increase ANT production in C. glutamicum, several target genes were manipulated: e.g., aroG, aroB, and aroK known as potential limiting steps in the shikimate pathway (Fig.…”

Metabolic Engineering of Pseudomonas putida KT2440 for the Production of para-Hydroxy Benzoic Acid

“…On a side note, it was earlier postulated by Kuepper et al (2015) after screening more than a thousand colonies for pheA knockout mutants on complex medium that the presence of the homogentisate degradation pathway might hamper the positive identification of positive colonies and the addition of precursor PPY in minimal medium was helpful to screen for this mutant (Kuepper et al, 2015). We successfully identified pheA mutants on rich medium when looking for tiny colonies that could be easily overlooked amongst the big colonies that did not contain the knockout.…”

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

“…Examples of production of added-value aromatic molecules in P. putida are illustrated by cinnamic acid (Nijkamp et al, 2005) and phenol (Wierckx et al, 2005;Wynands et al, 2018)achieved, among other manipulations, by introducing a phenylalanine-ammonia lyase from Rhodosporidium toruloides and the tyrosine phenol lyase from Pantoea agglomerans. Other strains producing derivatives of these aromatic compounds were developed, producing, for example p-hydroxystyrene (Verhoef et al, 2009), phydroxybenzoate , anthranilate (Kuepper et al, 2015), vanillate (Graf and Altenbuchner, 2014) and p-coumaric acid (Calero et al, 2016). Furthermore, a number of molecules are being synthesized in industrial-scale fermentations using engineered P. putida strains.…”