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

Abstract

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.The production of (Ϫ)-perillyl alcohol from L-limonene is of interest because of the limited availability of (Ϫ)-perillyl alcohol in nature and its proven anticarcinogenic properties; phase II trials to evaluate perillyl alcohol for the treatment of breast, pancreatic, and colorectal cancer are in progress (37). The only microbial enzyme system described thus far that transforms limonene to perillyl alcohol was found in Bacillus stearothermophilus BR388. However, this enzyme system is not sufficiently regiospecific; significant quantities of carveol, carvone, and terpineol are also produced (25). Removal of these side products is difficult as their boiling points and hydrophobicities are almost identical, and expensive purification methods (for example, chromatography) would be required to obtain sufficiently pure perillyl alcohol. Therefore, the industrial production of perillyl alcohol with this Bacillus enzyme system is not attractive. The conversion of limonene to perillic acid by a Pseudomonas putida strain expressing a cymene monooxygenase was described by Mars et al. (21) and could be interesting as perillyl alcohol is likely to be an intermediate in the production of perillic acid. Other literature concerning limonene biotransformations was reviewed recently (5).The approach that we used to find strains capable of regiospecific hydroxylation of limonene consisted of screening a collection of 1,800 bacterial strains grown on a range of relatively reduced substrates, such as toluene, naphthalene, and various alkanes. Using this approach, we anticipated that we would find oxygenases involved in catabolic pathways that would accept L-limonene as a substrate. Previous work has demonstrated that many catabolic oxygenases accept a wide range of unnatural substrates. Toluene dioxygenases, for example, have been shown to oxygenate more than 100 substrates (reference 11 and references therein). Another example is ...

Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases

“…PCR with degenerate CYP153 primers, developed to clone the Mycobacterium sp. strain HXN-1500 alkane hydroxylase (35), yielded PCR products of the expected length from most of these strains (designated "PCR" in Table 2). These include all mycobacteria, all R. erythropolis isolates, and several of the Proteobacteria.…”

“…Mycobacterium sp. strain HXN-1500, a strain able to convert limonene to perillyl alcohol (35), was found to contain a soluble alkane hydroxylase that is closely related to the Acinetobacter sp. strain EB104 hexane hydroxylase (CYP153A1), the first member of a new cytochrome P450 family (26).…”

“…strain HXN-1500 CYP153A6 gene and flanking regions. The CYP153A6 gene was subsequently functionally expressed in Pseudomonas putida GPo12 by using the broad-host-range vector pCom8, and the recombinant was used for the production of perillyl alcohol from limonene (35).…”

CYP153A6, a Soluble P450 Oxygenase Catalyzing Terminal-Alkane Hydroxylation

“…P. putida GPo12 (pGEc47⌬B) containing a broad-host-range expression vector with a polycistronic construct of the CYP153A6 P450 enzyme, ferredoxin, and ferredoxin reductase from Mycobacterium sp. HXN-1500 was described previously (pCom8-PFR A6 ) (41). The recombinant strain was grown on minimal E2 agar medium (3.5 g/liter NaNH 4 HPO 4 · 4H 2 O, 7.5 g/liter K 2 HPO 4 · 3H 2 O, 3.7 g/liter KH 2 PO 4 , 2 mM MgSO 4 , 15 g/liter Difco agar) with n-octane provided as vapor.…”

“…Sphingomonas sp. HXN-200, which contains five different CYP153 enzymes, was shown to hydroxylate piperidines, pyrrolidines, and azetidines to useful pharmaceutical intermediates (19,41). This broad substrate specificity and the ability to hydroxylate the terminal, most unreactive carbon atom of aliphatic alkanes make the CYP153 enzymes an interesting P450 family for the synthesis of pharmaceutical intermediates but also from an environmental perspective.…”