Biocatalytic production of perillyl alcohol from limonene by using a novel Mycobacterium sp. cytochrome P450 alkane hydroxylase expressed in Pseudomonas putida.
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.
van Beilen
Jan B
JB
Institute of Biotechnology, ETH Hönggerberg, CH-8093 Zürich, Switzerland. vanbeilen@biotech.biol.ethz.ch
Holtackers
René
R
Lüscher
Daniel
D
Bauer
Ulrich
U
Witholt
Bernard
B
Duetz
Wouter A
WA
D000595:Amino Acid Sequence; D053138:Cyclohexenes; D042926:Cytochrome P-450 CYP4A / Q000235:genetics* / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods; D000077222:Limonene; D008969:Molecular Sequence Data; D039821:Monoterpenes / Q000378:metabolism*; D009161:Mycobacterium / Q000201:enzymology* / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D013729:Terpenes / Q000378:metabolism*
D053138:Cyclohexenes; D039821:Monoterpenes; D013729:Terpenes; C032208:perillyl alcohol; D003577:Cytochrome P-450 Enzyme System; D000077222:Limonene; D042926:Cytochrome P-450 CYP4A