Identification of Fungal Limonene-3-Hydroxylase for Biotechnological Menthol Production

Abstract

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and β-pinene to verbenol and pinocarveol, respectively.IMPORTANCE (-)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (-)-menthol production from renewable (+)-limonene.

Keywords: CYP; P450; biotechnology; fungal enzyme; fungal enzymes; hydroxylation; limonene; menthol; oxidation; terpenes.

FIG 1

Biotechnological-chemical processes for (−)-menthol production (top; green and red) versus the native pathway in peppermint plants (Mentha × piperita) (bottom; brown) (adapted from reference 9). L5H, limonene-5-hydroxylase; L3H, limonene-3-hydroxylase; IPD, (−)-trans-isopiperitenol dehydrogenase; IPR, (−)-isopiperitenone reductase; IPI, (+)-cis-isopulegone isomerase; PR, (+)-pulegone reductase; MR, (−)-menthone reductase.

FIG 2

trans-Isopiperitenol production with different Dothideomycetes. (A) trans-Isopiperitenol production over time of different Dothideomycetes. Culture samples of different fungi were taken over a period of 5 h (once per hour) after addition of 1 g liter⁻¹ (+)-limonene. Samples were extracted with ethyl acetate, and the trans-isopiperitenol amount was determined by GC-MS analysis. The negative control was medium without cells. The data points and error bars represent the mean values and standard deviations for three biological replicates. (B) GC-MS analysis of biotransformation of (+)-limonene by A. pullulans. The chromatogram of ethyl acetate extract from (a) A. pullulans culture after addition of 1 g liter⁻¹ limonene (0 h) is compared to chromatograms of (b) A. pullulans culture 24 h after addition of limonene and (c) isopiperitenol reference substance [2:1 mixture of (+)-trans-/cis-isopiperitenol]. trans-Isopiperitenol (peak 2; RT, 14 min) was identified as the main product of (+)-limonene (peak 1) biotransformation by comparison of retention time and (C) mass spectrum to those of (D) the isopiperitenol reference substance.

FIG 3

Clustering of cytochrome P450 monooxygenases of A. pullulans CBS 100280 (EXF-150) and of H. carpetanum CBS 115712 (only those with the highest identity to L3H.Ap were selected). The proteins annotated as cytochrome P450 monooxygenases were analyzed for their amino acid sequence identity and subdivided into protein families. CYP candidate genes of A. pullulans chosen for expression in S. cerevisiae are marked with blue dots. Limonene-3-hydroxylase enzymes from A. pullulans (L3H.Ap = CYP65FA1) and H. carpetanum (L3H.Hc = CYP65EZ1) as well as other tested CYP65 candidates from A. pullulans are marked in red, green, and yellow, respectively. For accession numbers, see Table 4. Clustering was performed with the software Geneious using default settings (ClustalW alignment; Geneious tree builder; resampling method, bootstrap; number of replicates, 1,000). Branch labels show percent consensus support.

FIG 4

trans-Isopiperitenol concentrations achieved in a (+)-limonene biotransformation experiment with different S. cerevisiae CEN-PK2-1C strains. Concentrated cell suspensions were used. Empty vectors were yeast strains harboring empty pPK245 (and pPK448) vectors; CYP reductase.Ap refers to cells expressing a putative CYP reductase gene from A. pullulans CBS 100280 (EXF-150) from the pPK448 vector and containing empty pPK245. CYP532A30.Ap to CYP65EZ1.Hc indicate cells expressing different putative CYPs from A. pullulans CBS 100280 (EXF-150, Ap) or H. carpetanum CBS 115712 (Hc) from the pPK245 vector together with putative CYP reductase from A. pullulans from pPK448 plasmid (left) or without reductase.Ap (right). trans-Isopiperitenol concentrations were calculated from peak areas, which were determined by GC-MS analysis, and normalized to the peak area of the internal standard (3-carene) and the number of cells corresponding to 150 OD units. The data points and error bars represent the mean values and standard deviations for three biological replicates (n = 3).

FIG 5

Hydroxylation of limonene with P. pastoris X-33 strains expressing L3H enzymes. (A) trans-Isopiperitenol peak areas achieved in (+)-limonene biotransformation experiments with different P. pastoris X-33 strains (concentrated cell suspensions). pBSYA1Z, episomal, constitutive gene expression; pPICZA, genome integrated, methanol-induced gene expression; empty, P. pastoris with empty expression vector; L3H.Ap, with CYP65FA1 of A. pullulans; PM17, with limonene-3-hydroxylase of Mentha × piperita. (B) Product peak areas achieved in biotransformation experiments with (+)- or (−)-limonene with P. pastoris X-33 strains (concentrated cell suspensions) harboring empty pBSYA1Z plasmid or expressing limonene-3-hydroxylase enzymes from A. pullulans (L3H.Ap) or Mentha × piperita (PM17) from pBSYA1Z backbone. While with (−)-limonene several different products were formed with all strains (see Fig. S3), only peak areas of the main (by-)products are shown for comparison. For chromatograms, see panel C; also see Fig. S2 and S3. For panels A and B, peak areas were determined by GC-MS analysis and normalized to the peak area of the internal standard (3-carene) and the number of cells corresponding to (A) 150 or (B) 225 OD units. The data points and error bars represent the means and standard deviations for six (A) or three (B) biological replicates. (C) GC-MS analysis of (+)-limonene biotransformation experiment shown in panel B. The chromatogram of the negative control with the empty pBSYA1Z expression plasmid (blue) is compared to those of strains harboring PM17 from Mentha × piperita (brown) and L3H.Ap (red) and an isopiperitenol reference substance [2:1 mixture of (+)-trans-/cis-isopiperitenol] (black). trans-Isopiperitenol (peak 2; RT, 10.3 min) was identified as the main product (>90%) of (+)-limonene biotransformation. As a side product (<10%), p-1,8-menthadien-4-ol (peak 3; RT, 9.8 min) was formed by cells expressing L3H.Ap. (D and E) Substrates and main products of bioconversion reactions catalyzed by L3H.Ap. 1, (+)-limonene; 2, (+)-trans-isopiperitenol; 3, p-1;8-menthadien-4-ol; 4, (−)-limonene; 5, (−)-trans-isopiperitenol; 6, carveol (for the MS spectrum, see Fig. S4).

FIG 6

Product peak areas and trans-isopiperitenol concentrations achieved in (+)-limonene biotransformation experiments with P. pastoris X-33 strains expressing limonene-3-hydroxylase L3H.Ap under different conditions. Concentrated cell suspensions were used. Peak areas were determined by GC-MS analysis and normalized to the peak area of the internal standard (3-carene) and the number of cells corresponding to 225 OD units. trans-Isopiperitenol concentrations were calculated from normalized peak areas. The data points and error bars represent the mean values and standard deviations of three biological replicates. (A) Investigation of the influence of buffer pH and glucose feeding on product formation. Empty, P. pastoris strain with empty pBSYA1Z expression vector; L3H.Ap, strain expressing CYP65FA1 of A. pullulans from the pBSYA1Z backbone. For biotransformation, 100 mM potassium phosphate buffer at pH 6.0, 7.4, or 8.0 was used. “+Gluc” indicates that the cell suspension contained 2% glucose. When glucose was added, the pH of the biotransformation suspension decreased to less than 5 during the experiment. (B) Investigation of the influence of reaction vessel volume on product formation at pH 8.0. For the experiment, a P. pastoris strain expressing L3H.Ap from pBSYA1Z backbone was used. Biotransformation was carried out in reaction vessels with a total volume of 100 ml or 2 ml with cell suspension volumes of 7.5 ml or 750 μl, respectively. For bars labeled “100 ml vessel + DMSO” and “100 ml vessel + Lim,” samples were taken at different time points (2, 5, 20, 28, and 51 h after DMSO or limonene addition). For bars labeled “100 ml vessel + Lim_closed” and “2 ml vial + Lim,” reaction vessels remained closed during the entire experiment, and samples were taken only at 28 h.

FIG 7

Hydroxylation of monoterpene substrates with P. pastoris X-33 expressing L3H.Ap. (A) Relative quantitative composition of products formed in biotransformation experiments of α-pinene, β-pinene, or 3-carene with P. pastoris X-33 cells (concentrated cell suspension) expressing L3H.Ap from the pBSYA1Z backbone. If possible, products were identified via comparison of retention times and mass spectra with those of chemically synthesized reference substances, or by comparison of mass spectra to those of the NIST mass spectral library (v14) (for MS spectra, see Fig. S4). For chromatograms, see Fig. S5 to S7. The data points and error bars represent the mean values and standard deviations for three biological replicates (n = 3). (B) Substrates and main products of bioconversion reactions catalyzed by L3H.Ap.