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Applied and Environmental Microbiology, November 2008, p. 6697-6702, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00925-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification, Cloning, and Characterization of a Novel Ketoreductase from the Cyanobacterium Synechococcus sp. Strain PCC 7942{triangledown}

Kathrin Hölsch,1 Jan Havel,1,{dagger} Martin Haslbeck,2 and Dirk Weuster-Botz1*

Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany,1 Lehrstuhl Biotechnologie, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany2

Received 23 April 2008/ Accepted 4 September 2008

A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-[acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44°C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 ± 2.15 U mg–1) and 2',3',4',5',6'-pentafluoroacetophenone (8.57 ± 0.49 U mg–1) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2',3',4',5',6'-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% [S] versus 43.3% [S]).

* Corresponding author. Mailing address: Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany. Phone: 49 (089) 28915713. Fax: 49 (089) 28915714. E-mail: d.weuster-botz{at}lrz.tum.de

{triangledown} Published ahead of print on 12 September 2008.

{dagger} Present address: TÜV SÜD Product Service GmbH, MHS2—Nonactive Medical Devices, Ridlerstr. 65, 80339 Munich, Germany.

Applied and Environmental Microbiology, November 2008, p. 6697-6702, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00925-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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