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).
Highlights
IntroductionAnthranilate (oAB, ortho-aminobenzoate) is an aromatic acid used as a platform chemical for the production of food ingredients (Raffensperger and Vogt, 1961), dyes, perfumes (Wiklund and Bergman, 2006), crop protection compounds (Askham, 1992; Yadav and Krishnan, 1998; Chambers et al, 2013), pharmaceutical compounds (Bahia et al, 2011; Shafiq et al, 2011; Haynes et al, 2012; Gao et al, 2013; Loque and Weniger, 2013; Walsh et al, 2013), and plastics such as nylon (Sun et al, 2013)
Contrary to other established production hosts, the pheA deletion only requires phenylalanine to complement growth since P. putida can convert phenylalanine to tyrosine (Molina-Henares et al, 2009)
Microbial production of oAB under industrial relevant conditions from glucose as sole carbon source was achieved in P. putida KT2440 via the biosynthesis pathway of aromatic amino acids
Summary
Anthranilate (oAB, ortho-aminobenzoate) is an aromatic acid used as a platform chemical for the production of food ingredients (Raffensperger and Vogt, 1961), dyes, perfumes (Wiklund and Bergman, 2006), crop protection compounds (Askham, 1992; Yadav and Krishnan, 1998; Chambers et al, 2013), pharmaceutical compounds (Bahia et al, 2011; Shafiq et al, 2011; Haynes et al, 2012; Gao et al, 2013; Loque and Weniger, 2013; Walsh et al, 2013), and plastics such as nylon (Sun et al, 2013) It is currently produced in energy intensive chemical processes from petroleumbased precursors, like phthalamic acid (Klipper and Gripper, 1981; Berg, 2009). The development and application of green production processes is accelerated by an environmental and political interest to be less dependent on fossil resources.
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