title: An artificial TCA cycle selects for efficient α-ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli.
abstract: Amino acid hydroxylases depend directly on the cellular TCA cycle via their cosubstrate α-ketoglutarate (α-KG) and are highly useful for the selective biocatalytic oxyfunctionalization of amino acids. This study evaluates TCA cycle engineering strategies to force and increase α-KG flux through proline-4-hydroxylase (P4H). The genes sucA (α-KG dehydrogenase E1 subunit) and sucC (succinyl-CoA synthetase β subunit) were alternately deleted together with aceA (isocitrate lyase) in proline degradation-deficient Escherichia coli strains (ΔputA) expressing the p4h gene. Whereas, the ΔsucCΔaceAΔputA strain grew in minimal medium in the absence of P4H, relying on the activity of fumarate reductase, growth of the ΔsucAΔaceAΔputA strictly depended on P4H activity, thus coupling growth to proline hydroxylation. P4H restored growth, even when proline was not externally added. However, the reduced succinyl-CoA pool caused a 27% decrease of the average cell size compared to the wildtype strain. Medium supplementation partially restored the morphology and, in some cases, enhanced proline hydroxylation activity. The specific proline hydroxylation rate doubled when putP, encoding the Na+/l-proline transporter, was overexpressed in the ΔsucAΔaceAΔputA strain. This is in contrast to wildtype and ΔputA single-knock out strains, in which α-KG availability obviously limited proline hydroxylation. Such α-KG limitation was relieved in the ΔsucAΔaceAΔputA strain. Furthermore, the ΔsucAΔaceAΔputA strain was used to demonstrate an agar plate-based method for the identification and selection of active α-KG dependent hydroxylases. This together with the possibility to waive selection pressure and overcome α-KG limitation in respective hydroxylation processes based on living cells emphasizes the potential of TCA cycle engineering for the productive application of α-KG dependent hydroxylases. Biotechnol. Bioeng. 2017;114: 1511-1520. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Department of Solar Materials, Helmholtz Centre for Environmental Research-UFZ, Permoserstr. 15, Leipzig 04318, Germany.
authors.forename: Eleni
authors.initials: E
authors.lastname: Theodosiou
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Breisch
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany.
authors.forename: Mattijs K
authors.initials: MK
authors.lastname: Julsing
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany.
authors.forename: Francesco
authors.initials: F
authors.lastname: Falcioni
authors.affiliation: Department of Solar Materials, Helmholtz Centre for Environmental Research-UFZ, Permoserstr. 15, Leipzig 04318, Germany.
authors.forename: Bruno
authors.identifier: 0000-0001-6458-5739
authors.initials: B
authors.lastname: Bühler
authors.affiliation: Department of Solar Materials, Helmholtz Centre for Environmental Research-UFZ, Permoserstr. 15, Leipzig 04318, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
mesh_terms: D002384:Catalysis; D002952:Citric Acid Cycle / Q000235:genetics*; D004926:Escherichia coli / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D006909:Hydroxyproline / Q000096:biosynthesis* / Q000235:genetics; D007656:Ketoglutaric Acids / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods
chemical_list: D007656:Ketoglutaric Acids; D006899:Mixed Function Oxygenases; D006909:Hydroxyproline
keywords: TCA cycle engineering; l-proline transporter; proline hydroxylation; whole-cell biocatalysis; α-KG dependent dioxygenase