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Applied and Environmental Microbiology, June 1999, p. 2324-2332, Vol. 65, No. 6
Institute of Biotechnology, Swiss Federal
Institute of Technology Zurich, CH-8093 Zurich, Switzerland
Received 14 January 1999/Accepted 23 March 1999
Membrane-located monooxygenase systems, such as the
Pseudomonas putida mt-2-derived xylene oxygenase, are
attractive for challenging transformations of apolar compounds,
including enantiospecific epoxidations, but are difficult to synthesize
at levels that are useful for application to biotechnological
processes. In order to construct efficient biocatalysis strains, we
utilized the alkane-responsive regulatory system of the OCT
plasmid-located alk genes of Pseudomonas oleovorans GPo1, a very attractive system for recombinant
biotransformation processes. Determination of the nucleotide sequence
of alkS, whose activated gene product positively regulates
the transcription of the structural genes alkBFGHJKL, on a
3.7-kb SalI-HpaI OCT plasmid fragment was
completed, and the N-terminal amino acid sequence of an AlkS-LacZ
fusion protein was found to be consistent with the predicted DNA
sequence. The alkS gene and the alkBp promoter were assembled into a convenient alkane-responsive genetic expression cassette which allowed expression of the xylene oxygenase genes in a
recombinant Escherichia coli strain at a specific activity of 91 U per g (dry weight) of cells when styrene was the substrate. This biocatalyst was used to produce (S)-styrene oxide in
two-liquid-phase cultures. Volumetric productivities of more than
2 g of styrene oxide per h per liter of aqueous phase were
obtained; these values represented a fivefold improvement compared with
previous results.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
An Alkane-Responsive Expression System for the
Production of Fine Chemicals
*
Corresponding author. Mailing address: Institut
für Biotechnologie, ETH Zürich, Hönggerberg HPT,
CH-8093 Zürich, Switzerland. Phone: 41-1-633 32 86. Fax: 41-1-633 10 51. E-mail: bw{at}biotech.biol.ethz.ch.
Present address: DSM Biotech GmbH, Jülich, Germany.
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