title: Improved production of adipate with Escherichia coli by reversal of β-oxidation.
abstract: The linear C6dicarboxylic acid adipic acid is an important bulk chemical in the petrochemical industry as precursor of the polymer nylon-6,6-polyamide. In recent years, efforts were made towards the biotechnological production of adipate from renewable carbon sources using microbial cells. One strategy is to produce adipate via a reversed β-oxidation pathway. Hitherto, the adipate titers were very low due to limiting enzyme activities for this pathway. In most cases, the CoA intermediates are non-natural substrates for the tested enzymes and were therefore barely converted. We here tested heterologous enzymes in Escherichia coli to overcome these limitations and to improve the production of adipate via a reverse β-oxidation pathway. We tested in vitro selected enzymes for the efficient reduction of the enoyl-CoA and in the final reaction for the thioester cleavage. The genes encoding the enzymes which showed in vitro the highest activity were then used to construct an expression plasmid for a synthetic adipate pathway. Expression of paaJ, paaH, paaF, dcaA, and tesB in E. coli BL21(DE3) resulted in the production of up to 36 mg/L of adipate after 30 h of cultivation. Beside the activities of the pathway enzymes, the availability of metabolic precursors may limit the synthesis of adipate, providing another key target for further strain engineering towards high-yield production of adipate with E. coli.
authors.affiliation: IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany. n.kallscheuer@fz-juelich.de.
authors.forename: Nicolai
authors.identifier: http://orcid.org/0000-0003-4925-6923
authors.initials: N
authors.lastname: Kallscheuer
authors.affiliation: IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany.
authors.forename: Jochem
authors.initials: J
authors.lastname: Gätgens
authors.affiliation: Institute of Bioorganic Chemistry, Heinrich-Heine-Universität, Düsseldorf, 52425, Jülich, Germany.
authors.forename: Marvin
authors.initials: M
authors.lastname: Lübcke
authors.affiliation: Institute of Bioorganic Chemistry, Heinrich-Heine-Universität, Düsseldorf, 52425, Jülich, Germany.
authors.forename: Jörg
authors.identifier: http://orcid.org/0000-0002-9819-889X
authors.initials: J
authors.lastname: Pietruszka
authors.affiliation: IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany.
authors.forename: Michael
authors.identifier: http://orcid.org/0000-0002-4701-8254
authors.initials: M
authors.lastname: Bott
authors.affiliation: IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany. t.polen@fz-juelich.de.
authors.forename: Tino
authors.identifier: http://orcid.org/0000-0002-0065-3007
authors.initials: T
authors.lastname: Polen
mesh_terms: D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D000272:Adipates / Q000378:metabolism*; D004746:Enoyl-CoA Hydratase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000272:Adipates; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C029900:adipic acid; D010088:Oxidoreductases; D000102:Acetyl-CoA C-Acyltransferase; C443987:3-oxoadipyl-coenzyme A thiolase; D004746:Enoyl-CoA Hydratase; C577533:PaaF protein, E coli
keywords: Adipic acid, adipate; Carbon chain elongation; Escherichia coli; Metabolic engineering; Reverse β-oxidation