Next generation sequencing in predicting gene function in podophyllotoxin biosynthesis

Abstract

Podophyllum species are sources of (-)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (-)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (-)-matairesinol into (-)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (-)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.

FIGURE 1.

(−)-Podophyllotoxin (1b) and its derivatives teniposide (2), etopophos (3), and etoposide (4) used in cancer treatment.

FIGURE 2.

Possible biosynthetic pathway and/or grid leading to (−)-podophyllotoxin (1b) and related lignans. Known biosynthetic steps are highlighted in blue, and the reaction catalyzed by CYP719A23 and CYP719A24 described in this work is in green.

FIGURE 3.

Lignans tested as putative substrates in assays for CYP719A23 and CYP719A24 methylenedioxy bridge formation and (−)-haplomyrfolin (40b).

FIGURE 4.

Relative lignan contents in different tissues of P. hexandrum and P. peltatum. Lignans identified included (−)-podophyllotoxin (1b), (−)-α-peltatin (20b), (−)-β-peltatin (27b), podophyllotoxin-glucoside (41), α-peltatin-glucoside (42), β-peltatin-glucoside (43), and 4′-demethylpodophyllotoxin (16) in both P. hexandrum (A) and P. peltatum (B) tissues. The individual lignan amounts are presented as relative peak areas (280 nm) with each given compound having a relative peak area of 100% for the most abundant amount, and the others are reported as a percentage of that value.

FIGURE 5.

Sequence alignment of methylenedioxy bridge-forming cytochrome P450s. CYP719A23 and CYP719A24, cloned from P. hexandrum and P. peltatum, respectively, show ∼68% identity to Coptis japonica (S)-canadine synthase (CYP719A1).

FIGURE 6.

Ultra performance liquid chromatography-mass spectrometry analysis of enzymatic assays. A, ultra performance liquid chromatography chromatogram shows product formation in CYP719A24 and CYP719A23 assays in comparison to negative controls (empty vector and cinnamate-4-hydroxylase, CYP73A107). Positive ion mass spectra of substrate (−)-matairesinol (9b, B) and product (−)-pluviatolide (14b, C) show loss of two mass units.

FIGURE 7.

Fragmentation pattern of (−)-pluviatolide (14b) and (−)-haplomyrfolin (40b). The expected fragments generated by the two isobaric compounds 14b (A) and 40b (B) during LC-ESI-MS analyses are shown (adapted from Schmidt et al. (61)). The fragment at m/z 161 and the absence of a fragment at m/z 163 point to (−)-pluviatolide (14b) as the products of CYP719A23 and CYP719A24.

FIGURE 8.

Reduced CO binding spectra for (−)-pluviatolide synthases. Spectra were obtained with microsomes isolated from S. cerevisiae (strain WAT11) transformed with pYES-DEST52 (empty vector as negative control) (A), pYES-DEST52::CYP719A23 (B), and pYES-DEST52::CYP719A24 (C).

FIGURE 9.

Kinetic parameters for CYP719A23 and CYP719A24. Steady state Michaelis-Menten kinetics derived from initial rates of CYP719A23 (A) and CYP719A24 (B) enriched microsomes with (−)-matairesinol (9b) as substrate. Assays were performed in triplicate.

FIGURE 10.

Phylogenetic analysis of cloned (bold) and known cytochrome P450 enzymes. The phylogenetic tree was generated by sequence alignment using ClustalW (Version 1.4), constructed with neighboring joining clustering algorithm (Version 3.5c), and visualized with Tree view (Version 1.6.6). Amino acid sequences were obtained from UniProtKB, SwissProt, or GenBank^TM with the following accession numbers: AB014459, CYP51G1, A. thaliana; AF212990, CYP701A1, Cucurbita maxima; AB006790, CYP703A1, Petunia × hybrida; NM_202845, CYP707A1, A. thaliana; M32885, CYP71A1, Persea americana; O81971, CYP71D9, Glycine max; NM_129002, CYP710A1, A. thaliana; NP_850074, CYP711A1, A. thaliana; NM_123002, CYP716A1, A. thaliana; Q948Y1, CYP719A1, Coptis japonica; EU882969, CYP719A2 and AB126256, CYP719A3, Eschscholzia californica; EU883001, CYP719A4, Thalictrum flavum; EF451151, CYP719A13, Argemone mexicana; EF451152, CYP719A14, A. mexicana; AB374407, CYP719A18, C. japonica; AB374408, CYP719A19, C. japonica; EF451150, CYP719B1, P. somniferum; L10081, CYP72A1, Catharanthus roseus; Z17369, CYP73A1, Helianthus tuberosus; NP_180607, CYP73A2, A. thaliana; Z22545, CYP75A1, Petunia × hybrida; X71658, CYP76A1, Solanum melongena; X71656, CYP77A1, S. melongena; P48420, CYP78A1, Zea mays; U32624, CYP79A1, Sorghum bicolor; U09610, CYP80A1, Berberis stolonifera; AF014801, CYP80B1, E. californica; AB025030, CYP80B2, C. japonica; AY610509, CYP80B4, T. flavum; AB288053, CYP80G2, C. japonica; P93147, CYP81E1, Glycyrrhiza echinata; AY278229, CYP81E8, Medicago truncatula; BAE48234, CYP81Q, Sesamum indicum; NP_195345, CYP84A3, A. thaliana; P48422, CYP86A1, A. thaliana; AF216313, CYP87A1, H. annuus; U32579, CYP88A1, Z. mays; U61231, CYP89A2, A. thaliana; Q42569, CYP90A1, A. thaliana; NP_850337, CYP98A3, A. thaliana. A 10% change is indicated by the scale bar.