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Applied and Environmental Microbiology, April 2005, p. 1737-1744, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1737-1744.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Biocatalytic Production of Perillyl Alcohol from Limonene by Using a Novel Mycobacterium sp. Cytochrome P450 Alkane Hydroxylase Expressed in Pseudomonas putida

Jan B. van Beilen,^1* René Holtackers,¹ Daniel Lüscher,¹ Ulrich Bauer,¹ Bernard Witholt,¹ and Wouter A. Duetz²

Institute of Biotechnology, ETH Hönggerberg, Zürich, Switzerland,¹ Enzyscreen BV, Leiden, The Netherlands²

Received 13 July 2004/ Accepted 28 October 2004

A number of oxygenated monoterpenes present at low concentrations in plant oils have anticarcinogenic properties. One of the most promising compounds in this respect is (–)-perillyl alcohol. Since this natural product is present only at low levels in a few plant oils, an alternative, synthetic source is desirable. Screening of 1,800 bacterial strains showed that many alkane degraders were able to specifically hydroxylate L-limonene in the 7 position to produce enantiopure (–)-perillyl alcohol. The oxygenase responsible for this was purified from the best-performing wild-type strain, Mycobacterium sp. strain HXN-1500. By using N-terminal sequence information, a 6.2-kb ApaI fragment was cloned, which encoded a cytochrome P450, a ferredoxin, and a ferredoxin reductase. The three genes were successfully coexpressed in Pseudomonas putida by using the broad-host-range vector pCom8, and the recombinant converted limonene to perillyl alcohol with a specific activity of 3 U/g (dry weight) of cells. The construct was subsequently used in a 2-liter bioreactor to produce perillyl alcohol on a scale of several grams.

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* Corresponding author. Mailing address: Institute of Biotechnology, ETH Hönggerberg, CH-8093 Zürich, Switzerland. Phone: 41-1-6333444. Fax: 41-1-6331051. E-mail: vanbeilen{at}biotech.biol.ethz.ch.

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Applied and Environmental Microbiology, April 2005, p. 1737-1744, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1737-1744.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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