title: Maximization of cell viability rather than biocatalyst activity improves whole-cell ω-oxyfunctionalization performance.
abstract: It is a common misconception in whole-cell biocatalysis to refer to an enzyme as the biocatalyst, thereby neglecting the structural and metabolic framework provided by the cell. Here, the low whole-cell biocatalyst stability, that is, the stability of specific biocatalyst activity, in a process for the terminal oxyfunctionalization of renewable fatty acid methyl esters was investigated. This reaction, which is difficult to achieve by chemical means, is catalyzed by Escherichia coli featuring the monooxygenase system AlkBGT and the uptake facilitator AlkL from Pseudomonas putida GPo1. Corresponding products, that is, terminal alcohols, aldehydes, and acids, constitute versatile bifunctional building blocks, which are of special interest for polymer synthesis. It could clearly be shown that extensive dodecanoic acid methyl ester uptake mediated by high AlkL levels leads to whole-cell biocatalyst toxification. Thus, cell viability constitutes the primary factor limiting biocatalyst stability and, as a result, process durability. Hence, a compromise had to be found between low biocatalyst activity due to restricted substrate uptake and poor biocatalyst stability due to AlkL-mediated toxification. This was achieved by the fine-tuning of heterologous alkL expression, which, furthermore, enabled the identification of the alkBGT expression level as another critical factor determining biocatalyst stability. Controlled synthesis of AlkL and reduced alkBGT expression finally enabled an increase of product titers by a factor of 4.3 up to 229 g Lorg-1in a two-liquid phase bioprocess setup. Clearly, ω-oxyfunctionalization process performance was determined by cell viability and thus biocatalyst stability rather than the maximally achievable specific biocatalyst activity. Biotechnol. Bioeng. 2017;114: 874-884. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Solar Materials, Helmholtz Centre for Environmental Research-UFZ, Permoserstr. 15, 04318, Leipzig, Germany.
authors.forename: Marvin
authors.initials: M
authors.lastname: Kadisch
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: Department of Solar Materials, Helmholtz Centre for Environmental Research-UFZ, Permoserstr. 15, 04318, Leipzig, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Schrewe
authors.affiliation: Department of Molecular Systems Biology, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Jehmlich
authors.affiliation: Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Scheer
authors.affiliation: Department of Molecular Systems Biology, Helmholtz Centre for Environmental Research-UFZ, Leipzig, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: von Bergen
authors.affiliation: Department of Solar Materials, Helmholtz Centre for Environmental Research-UFZ, Permoserstr. 15, 04318, Leipzig, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
authors.affiliation: Department of Solar Materials, Helmholtz Centre for Environmental Research-UFZ, Permoserstr. 15, 04318, Leipzig, Germany.
authors.forename: Bruno
authors.initials: B
authors.lastname: Bühler
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001711:Biotransformation; D002470:Cell Survival / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007218:Industrial Microbiology; D007850:Lauric Acids / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D006899:Mixed Function Oxygenases / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D007850:Lauric Acids; D006899:Mixed Function Oxygenases
keywords: alkane monooxygenase; fatty-acid methyl ester; industrial biotechnology; metabolic and reaction engineering; oxyfunctionalization; whole-cell biotransformation