Catechol biosynthesis from glucose in Escherichia coli anthranilate-overproducer strains by heterologous expression of anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1

Abstract

BackgroundThe aromatic compound catechol is used as a precursor of chemical products having multiple applications. This compound is currently manufactured by chemical synthesis from petroleum-derived raw materials. The capacity to produce catechol is naturally present in several microbial species. This knowledge has been applied to the generation of recombinant Escherichia coli strains that can produce catechol from simple carbon sources.ResultsSeveral strains derived from E. coli W3110 trpD9923, a mutant that overproduces anthranilate, were modified by transforming them with an expression plasmid carrying genes encoding anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. The additional expression of genes encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase and transketolase from E. coli, was also evaluated. Generated strains were characterized in complex or minimal medium in shake-flask and fed-batch bioreactor cultures and incubation temperatures ranging from 37 to 28°C. These experiments enabled the identification of culture conditions for the production of 4.47 g/L of catechol with strain W3110 trpD9923, expressing 1,2-dioxygenase, DAHP synthase and transketolase. When considering the amount of glucose consumed, a yield of 16% was calculated, corresponding to 42% of the theoretical maximum as determined by elementary node flux analysis.ConclusionsThis work demonstrates the feasibility of applying metabolic engineering for generating E. coli strains for the production of catechol from glucose via anthranilate. These results are a starting point to further optimize environmentally-compatible production capacity for catechol and derived compounds.

Identifying Cranberry Juice Consumers with Predictive OPLS‐DA Models of Plasma Metabolome and Validation of Cranberry Juice Intake Biomarkers in a Double‐Blinded, Randomized, Placebo‐Controlled, Cross‐Over Study

“…Catechol, also known as pyrocatechol, naturally occur in fruits and vegetables, but also found to be a metabolite in some bacteria including Escherichia , Mycobacterium , and Pseudomonas . [ 19,20 ] Guaiacol, as a monomethyl ether of catechol, has the property of inducing cell proliferation by being a potent scavenger of reactive oxygen radicals. [ 21 ] Both were exogenous metabolites increased after cranberry consumption.…”

Metabolic Engineering of Pseudomonas putida KT2440 to Produce Anthranilate from Glucose

“…To further optimize the production of oAB in P. putida , feedback insensitive pSEVA234-based ( Silva-Rocha et al, 2013 ) overexpression constructs for trpE S40F G and aroG D146N , or both genes in one operon structure, were transformed to the respective mutants under the IPTG-inducible LacI Q -P trc system. These genes encode feedback insensitive variants of anthranilate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate (DHAP) synthase, respectively, and are known to enhance oAB production in E. coli ( Balderas-Hernandez et al, 2009 , 2014 ; Sun et al, 2013 ). Figure 1 shows the exemplarily oAB production pathway and gives on overview over the metabolic engineering targets investigated in this study.…”

“…The aromatic biosynthesis pathway and the derived compounds of the aromatic acids, such as oAB, have been intensively studied in the last decades ( Bongaerts et al, 2001 ; Ikeda, 2003 ; Kramer et al, 2003 ; Leuchtenberger et al, 2005 ; Pittard and Yang, 2008 ). Microbial production of oAB with engineered Escherichia coli strains was reported by Balderas-Hernandez et al (2009) followed by further publications on oAB-derived compounds such as catechol and muconic acid ( Sun et al, 2013 ; Averesch and Krömer, 2014 ; Balderas-Hernandez et al, 2014 ; Jaeger et al, 2015 ). To enable oAB production in E. coli , Balderas-Hernandez et al (2009 , 2014 ) inserted a point mutation in the oAB phosphoribosyl transferase domain ( trpD ), whereas Sun et al (2013) used the Keio collection deletion strain E. coli BW25113 Δ trp::kan to prevent the conversion of oAB to tryptophan.…”

“…Microbial production of oAB with engineered Escherichia coli strains was reported by Balderas-Hernandez et al (2009) followed by further publications on oAB-derived compounds such as catechol and muconic acid ( Sun et al, 2013 ; Averesch and Krömer, 2014 ; Balderas-Hernandez et al, 2014 ; Jaeger et al, 2015 ). To enable oAB production in E. coli , Balderas-Hernandez et al (2009 , 2014 ) inserted a point mutation in the oAB phosphoribosyl transferase domain ( trpD ), whereas Sun et al (2013) used the Keio collection deletion strain E. coli BW25113 Δ trp::kan to prevent the conversion of oAB to tryptophan. Additional targets to increase the production of oAB in E. coli , for example the overexpression of feedback insensitive variants of the 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase and the anthranilate synthase unit ( trpE S40F G ) were investigated.…”

Benzoate synthesis from glucose or glycerol using engineered Pseudomonas taiwanensis

“…Targeted disruption of this pathway thus allows the synthesis of these derivatives (Figure 1). Due to their potential as bio-based building blocks, the microbial production of these molecules is intensively studied [13][14][15][16][17]. The pathway via l-phenylalanine described in this study is a novel strategy that adds a new aspect to this highly active field of research.…”