Oxidosqualene cyclases involved in the biosynthesis of triterpenoids in Quercus suber cork

Abstract

Cork is a water-impermeable, suberin-based material harboring lignin, (hemi)cellulose, and extractable small molecules (primarily triterpenoids). Extractables strongly influence the properties of suberin-based materials. Though these previous findings suggest a key role for triterpenoids in cork material quality, directly testing this idea is hindered in part because it is not known which genes control cork triterpenoid biosynthesis. Here, we used gas chromatography and mass spectrometry to determine that the majority (>85%) of non-polar extractables from cork were pentacyclic triterpenoids, primarily betulinic acid, friedelin, and hydroxy-friedelin. In other plants, triterpenoids are generated by oxidosqualene cyclases (OSCs). Accordingly, we mined Quercus suber EST libraries for OSC fragments to use in a RACE PCR-based approach and cloned three full-length OSC transcripts from cork (QsOSC1-3). Heterologous expression in Saccharomyces cerevisiae revealed that QsOSC1-3 respectively encoded enzymes with lupeol synthase, mixed α- and β-amyrin synthase, and mixed β-amyrin and friedelin synthase activities. These activities together account for the backbone structures of the major cork triterpenoids. Finally, we analyzed the sequences of QsOSC1-3 and other plant OSCs to identify residues associated with specific OSC activities, then combined this with analyses of Q. suber transcriptomic and genomic data to evaluate potential redundancies in cork triterpenoid biosynthesis.

Figure 1

Non-polar extractable compounds from Quercus suber cork tissue. (A) Comparison of a published mass spectrum of hydroxy-friedelin (Moiteiro et al. 2006) and the mass spectrum of the major non-polar extractable compound in cork tissue, identified as hydroxy-friedelin. (B) Abundance of each wax component detected in cork in μg per mg dry tissue. Wax compounds are grouped according to their biosynthetic relationships. Bar heights and error bars represent the average and standard deviation of n = 4 biologically independent measurements. Significant differences (p < 0.01) were determined using a one-way ANOVA and subsequent Tukey Honest Significant Difference tests. (C) Biosynthetic routes to the triterpenoid wax compounds found in cork wax, predicted in analogy to other species (Xu et al., 2004). 2,3-Oxidosqualene, the precursor to triterpenoid compounds, is synthesized from six acetyl-CoA-derived isopentenyl diphosphate units supplied by the cytosolic mevalonate pathway. This compound can then be cyclized by oxidosqualene cylases to form diverse tetra- and pentacyclic structures.

Figure 2

Expression of candidate OSCs in Quercus suber throughout the growing season as determined by RT-qPCR. The relative abundance of OSC transcripts was calculated by normalization using tubulin as the reference. Bar heights and error bars represent the mean and standard deviation of n = 4 biologically independent measurements. (A) QsOSC1, (B) QsOSC2, (C) QsOSC3. Significant differences (p < 0.05) within each time series were determined using a one-way ANOVA and subsequent Tukey Honest Significant Difference tests.

Figure 3

Expression of Quercus suber OSCs in Saccharomyces cerevisiae. A, B, D, G, I Total ion chromatograms of TLC-purified triterpenoid extracts of galactose-induced yeast harboring empty pYES vector (A), pYES::QsOSC1 (B) and pYES::QsOSC2 (D), total ion chromatograms of crude extracts of pYES::QsOSC3 (G), and of commercial triterpenoid standards (I). C, E, F, H Mass spectra of the major peak in B and the lupeol standard (C), peak 1 in D and the β-amyrin standard (E), peak 2 in D and the α-amyrin standard (F), peak 3 in G and the friedelin standard (H). The mass spectra from peak 1 in D and peak 1 in G were indistinguishable. Other minor peaks (4-7) in G were identified as taraxerol, isomultiflorenol, lupeol, and multiflorenol, respectively, by comparison with the mass spectra of authentic standards. The peak marked with a star in G is an endogenous tetracyclic yeast triterpenoid (Fig. S7).

Figure 4

Structural analysis of Quercus suber oxidosqualene cyclases. (A) Sections of a multiple amino acid sequence alignment of OSCs from Q. suber and other plants. The tracks show the levels of consensus at each position in the alignment between sequences coding for specific OSC activity characterized here and sequences coding for other OSC activities. Grey highlights indicate residues associated with catalytic initiation, salmon, blue and yellow highlights indicate residues associated with lupeol, amyrin, and friedelin synthase activity, respectively. (B) Predicted tertiary structure of the Q. suber friedelin synthase QsOSC3 with residues of interest highlighted by colors corresponding to those in (A). Substrate (lanosterol) co-crystallized with the OSC template structure of human lanosterol synthase shown in black. (C) Active-site view of the image in B. (LUP = lupeol synthase; AMY = amyrin synthase; FRS = friedelin synthase).

Figure 5

Selected amino acid motifs, scaffold locations, and expression profiles of characterized Quercus suber OSCs and additional predicted OSCs. (A) Neighbor-joining tree built using the nucleotides underlining amino acid residues shown in the adjacent alignment subsets – the positions that were associated with specific OSC activities (see Fig. 4). (B) Position of Q. suber OSCs on scaffolds of a Q. suber genome assembly. Black, solid lines indicate contiguous scaffolds. Features on the plus and minus strands are drawn above and below the scaffold lines, respectively. Grey rectangles immediately adjacent to scaffold lines denote boundaries of annotated genes. White (uncharacterized) or colored (characterized) rectangles above or below gene boundary markers indicate annotated mRNAs, and black rectangles superimposed on top of mRNA rectangles indicate exons. (C) Expression of Q. suber OSCs in various tissues. Rows correspond to genes and columns to Sequence Read Archive samples and both are clustered according to expression profile (z-scores calculated from transcripts per million values). Colored tree tips indicate the activity of characterized Q. suber OSCs (salmon = QsOSC1/lupeol synthase, blue = QsOSC2/amyrin synthase, yellow = QsOSC3/friedelin synthase). For detailed information on the SRA samples used see Table S3.

Figure 6

Model of triterpenoid biosynthesis in Quercus suber cork tissue. From squalene epoxide (in center), the Q. suber lupeol synthase QsOSC1 (salmon) produces lupeol, which can then be used by P450 enzyme(s) (grey) to generate the betulinic acid detected in cork wax. The Q. suber amyrin synthase QsOSC2 (blue) generates α and β-amyrin, which P450 enzyme(s) (grey) then convert into uvaol, ursolic acid, erythrodiol, and oleanolic acid. The Q. suber friedelin synthase QsOSC3 (yellow) generates β-amyrin and friedelin, and the latter can then be converted into 23-hydroxy-friedelin by a P450 enzyme. Greyed structures have not been detected in cork.