An artificial TCA cycle selects for efficient α‐ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli

Eleni Theodosiou; Marina Breisch; Mattijs K. Julsing; Francesco Falcioni; Bruno Bühler; Andreas Schmid

doi:10.1002/bit.26281

journal article Mar 16, 2017

An artificial TCA cycle selects for efficient α‐ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli

Eleni Theodosiou Marina Breisch Mattijs K. Julsing Francesco Falcioni Bruno Bühler

Andreas Schmid

Biotechnology and Bioengineering Vol. 114 No. 7 pp. 1511-1520 · Wiley

View at Publisher Save 10.1002/bit.26281

Abstract

ABSTRACTAmino 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.

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Details

Published: Mar 16, 2017
Vol/Issue: 114(7)
Pages: 1511-1520
License: View

Authors

E

Eleni Theodosiou

Department of Solar Materials Helmholtz Centre for Environmental Research‐UFZ Permoserstr. 15 Leipzig 04318 Germany

M

Marina Breisch

Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering TU Dortmund University Dortmund Germany

M

Mattijs K. Julsing

Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering TU Dortmund University Dortmund Germany

F

Francesco Falcioni

Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering TU Dortmund University Dortmund Germany

B

Bruno Bühler

Department of Solar Materials Helmholtz Centre for Environmental Research‐UFZ Permoserstr. 15 Leipzig 04318 Germany

A

Andreas Schmid

Department of Solar Materials Helmholtz Centre for Environmental Research‐UFZ Permoserstr. 15 Leipzig 04318 Germany

Funding

Mercator Research Center Ruhr Award: Pr-2013-0010

EU-FP7 Oxygreen Project Award: 212281

Cite This Article

Eleni Theodosiou, Marina Breisch, Mattijs K. Julsing, et al. (2017). An artificial TCA cycle selects for efficient α‐ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli. Biotechnology and Bioengineering, 114(7), 1511-1520. https://doi.org/10.1002/bit.26281

An artificial TCA cycle selects for efficient α‐ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli

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