Regioselective aromatic hydroxylation of quinaldine by water using quinaldine 4-oxidase in recombinant Pseudomonas putida

Abstract

Biocatalytic hydrocarbon oxyfunctionalizations are typically accomplished using oxygenases in living bacteria as biocatalysts. These processes are often limited by either oxygen mass transfer, cofactor regeneration, and/or enzyme instabilities due to the formation of reactive oxygen species. Here, we discuss an alternative approach based on molybdenum (Mo)-containing dehydrogenases, which produce, rather than consume, reducing equivalents in the course of substrate hydroxylation and use water as the oxygen donor. Mo-containing dehydrogenases have a high potential for overcoming limitations encountered with oxygenases. In order to evaluate the suitability and efficiency of a Mo-containing dehydrogenase-based biocatalyst, we investigated quinaldine 4-oxidase (Qox)-containing Pseudomonas strains and the conversion of quinaldine to 4-hydroxyquinaldine. Host strain and carbon source selection proved to be crucial factors influencing biocatalyst efficiency. Resting P. putida KT2440 (pKP1) cells, grown on and induced with benzoate, showed the highest Qox activity and were used for process development. To circumvent substrate and product toxicity/inhibition, a two-liquid phase approach was chosen. Without active aeration and with 1-dodecanol as organic carrier solvent a productivity of 0.4 g l (tot) (-1) h(-1) was achieved, leading to the accumulation of 2.1 g l (tot) (-1) 4-hydroxyquinaldine in 6 h. The process efficiency compares well with values reported for academic and industrially applied biocatalytic oxyfunctionalization processes emphasizing the potential and feasibility of the Qox-containing recombinant cells for heteroaromatic carbon oxyfunctionalizations without the necessity for active aeration.

Integrated organic–aqueous biocatalysis and product recovery for quinaldine hydroxylation catalyzed by living recombinant Pseudomonas putida

“…To investigate benzoate metabolism and induction with benzoate, three different exponential feeding regimes for benzoate and ammonium, targeted at growth rates of 0.20, 0.35, and 0.50 h -1 , were applied during fed-batch cultivation. For 2LP biotransformations, cells grown at a growth rate of 0.35 h -1 were harvested, resuspended, and used with 1-dodecanol as the organic carrier solvent at an organic:aqueous phase ratio of 1:5, as described previously [37]. Samples from the organic and aqueous phases were prepared for gas chromatography (GC) analysis using known protocols [6].…”

“…Fed-batch cultivation and 2LP biotransformation A 3-L bioreactor (KLF, Bioengineering, Wald, Switzerland) was used for batch and fed-batch cultivation of P. putida KT2440 (pKP1) as described previously [37]. To investigate benzoate metabolism and induction with benzoate, three different exponential feeding regimes for benzoate and ammonium, targeted at growth rates of 0.20, 0.35, and 0.50 h -1 , were applied during fed-batch cultivation.…”

“…The cell pellet was then washed twice with 50 mM potassium phosphate buffer (pH 7.4) and resuspended in the same buffer supplemented with 1% (w/v) glucose as the energy source. Resting cell assays were performed with a cell dry weight (CDW) concentration of 0.5 g CDW L -1 as described previously [37].…”

“…We recently described quinaldine 4-oxidase (Qox)-containing Pseudomonas putida as a biocatalyst for regioselective hydroxylation of quinaldine to 4-hydroxyquinaldine, both of which are poorly soluble in water and toxic to cells even at low concentrations [37]. This study constituted the first biotechnological in vivo application of Qox, highlighting the potential of hydroxylating dehydrogenases for achieving carbon oxyfunctionalizations without active aeration and reducing limitations encountered with other oxidoreductases (e.g., cofactor regeneration) in technical applications.…”

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In an earlier study, biocatalytic carbon oxyfunctionalization with water serving as oxygen donor, e.g., the bioconversion of quinaldine to 4-hydroxyquinaldine, was successfully achieved using resting cells of recombinant Pseudomonas putida, containing the molybdenumenzyme quinaldine 4-oxidase, in a two-liquid phase (2LP) system (Ü tkür et al J Ind Microbiol Biotechnol 38:1067-1077, 2011. In the study reported here, key parameters determining process performance were investigated and an efficient and easy method for product recovery was established.

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