Metabolic Engineering of Pseudomonas Putida KT2440 for the Production of Compounds Derived from the Shikimic Acid Pathway

We show an engineered biosynthetic pathway for aromatic substances based on the simple sugar glucose in Pseudomonas putida KT2440. P. putida KT2440 is the best characterized strain of the saprophytic Pseudomonads and has been certified as a biosafety host for cloning of foreign genes[1]. Chromosomal changes were implemented using a method for markerless gene insertion and deletion, based on homologous recombination and counterselection with the antimetabolite 5-fluorouracil in an upp mutant strain [2]. Making use of the inherent shikimate pathway [1,3] we aimed to accumulate the intermediate chorismate as building block for further biosynthesis. Chorismate is the branching point in the biosynthesis of a diverse set of aromatic compounds [4-6]. The chorismate-utilizing reactions for the aromatic amino acid biosynthesis were blocked by constructing pheA and trpE deletion mutants [7]. To channel carbon flow into the shikimate pathway, the gene aroF for the (tyrosine-sensitive) DAHP synthase (AroF) of E. coli [8] was chromosomally integrated into the P. putida KT2440 genome. Additionally, genes aroB and aroL, for the DHQ synthase (AroB) and shikimate kinase (AroL) were chromosomally integrated to increase known rate limiting reactions [9,10]. The availability of PEP and erythrose-4-phosphate (precursors for DAHP synthesis) was enhanced through changes in the central glucose catabolism.

In shake flask experiments, up to 2 mM of chorismate (from 50 mM glucose) were measured in the supernatant of the constructed strains. Depletion of accumulated chorismate was observed during the course of cultivation, indicating its subsequent consumption. Based on the chorismate producing strains, further genes were added on newly created expression plasmids to obtain chorismate-derived products. Examples will be given.

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