Metabolite Diversity in Alkaloid Biosynthesis: A Multilane (Diastereomer) Highway for Camptothecin Synthesis in Camptotheca acuminata

Abstract

Camptothecin is a monoterpene indole alkaloid (MIA) used to produce semisynthetic antitumor drugs. We investigated camptothecin synthesis in Camptotheca acuminata by combining transcriptome and expression data with reverse genetics, biochemistry, and metabolite profiling. RNAi silencing of enzymes required for the indole and seco-iridoid (monoterpene) components identified transcriptional crosstalk coordinating their synthesis in roots. Metabolite profiling and labeling studies of wild-type and RNAi lines identified plausible intermediates for missing pathway steps and demonstrated nearly all camptothecin pathway intermediates are present as multiple isomers. Unlike previously characterized MIA-producing plants, C. acuminata does not synthesize 3-α(S)-strictosidine as its central MIA intermediate and instead uses an alternative seco-iridoid pathway that produces multiple isomers of strictosidinic acid. NMR analysis demonstrated that the two major strictosidinic acid isomers are (R) and (S) diastereomers at their glucosylated C21 positions. The presence of multiple diastereomers throughout the pathway is consistent with their use in synthesis before finally being resolved to a single camptothecin isomer after deglucosylation, much as a multilane highway allows parallel tracks to converge at a common destination. A model "diastereomer" pathway for camptothecin biosynthesis in C. acuminata is proposed that fundamentally differs from previously studied MIA pathways.

Figure 1.

Proposed Pathway for Camptothecin Biosynthesis in C. acuminata. Camptothecin (12) is synthesized from the central precursor strictosidinic acid (5) derived from condensation of tryptamine (4) and the iridoid secologanic acid (3), a monoterpenoid glycoside. In vivo labeling experiments with deuterated [α,α,β,β-d₄]-tryptamine resulted in labeling of strictosidinic acid and downstream metabolites with the number and the likely positions of deuterium (D) indicated in red. Enzyme activities are indicated. TDC1 and CYC1 were identified and characterized in this study. 7-DLS, 7-deoxyloganetic acid synthase; 7-DLGT, 7-deoxyloganetic acid glucosyltransferase; 7-DLH, 7-deoxyloganic acid hydroxylase; SLAS, secologanic acid synthase; STRAS, strictosidinic acid synthase; DH, dehydration; RD, reduction.

Figure 2.

Tissue Distribution Profiles of Proposed Camptothecin Pathway Metabolites in Wild-Type C. acuminata. Tissues were collected from wild-type plants that had been under greenhouse cultivation in soil for 8 months and 70% acetonitrile extracts were analyzed using a 15-min gradient elution method for UHPLC/MS. Multiple isomers were detected for strictosidinic acid (Figure 1, compound 5) and poststrictosidinic acid metabolites (Figure 1, compounds 6, 10, 11, and 12). Average values are shown with sd (n = 3) for the most abundant and quantifiable isomers. SA, shoot apex; YL, young leaf; ML, mature leaf.

Figure 3.

Structures of the Two Major Strictosidinic Acid Isomers Isolated from C. acuminata Leaf Tissue. NMR analyses (Table 2) show that the two major strictosidinic acid isomers (isomers 2 and 3) differ in stereochemical configuration at position C21, the site of glucosylation.

Figure 4.

Formation of Iridoid Diastereomers. Equilibrium between the open and closed ring conformations of 7-deoxyloganetic acid yields diastereomers at the C2 hydroxyl group.

Figure 5.

Heat Map of Expression Data of Candidate Genes for the Seco-Iridoid Branch of Camptothecin Biosynthesis. Hierarchical clustering of 25,725 transcripts was generated based on average linkage of Pearson correlation coefficients of log₂-transformed FPKM expression values from the MPGR website (http://medicinalplantgenomics.msu.edu/) with MeV v4.9 (Saeed et al., 2006). A subcluster encompassing 23 genes is shown with GenBank accession numbers listed in parentheses. The color scale depicts transcript abundance (expressed as log₂-transformed FPKM). Red dots indicate the position of candidates for the seco-iridoid pathway, and for comparison, the expression profile (expressed as log₂-transformed FPKM) of TDC1 is shown (yellow dot). CYC1 and TDC1, both highlighted in bold, have been characterized in this study. Note that lists of candidate genes, GenBank accession numbers, MPGR transcript identifiers, and FPKM values are given in Supplemental Data Sets 2 and 3. HMTD, heavy metal transport/detoxification; GUF, gene of unknown function.

Figure 6.

Iridoid Synthase Assay with Recombinant CYC1 and CYC2 Enzymes. Purified recombinant proteins were assayed for iridoid synthase activity in reaction mixtures with 8-oxogeranial (Figure 1, compound 1) in the absence or presence of NADPH. Assays were extracted with dichloromethane and analyzed by GC/MS. The respective total ion chromatograms are shown in comparison to that obtained for a control assay with C. roseus iridoid synthase (a to g, reaction products; h and i, 8-oxoneral and 8-oxogeranial substrates, respectively). In the presence of NADPH, CYC1 and C. roseus iridoid synthase catalyzed conversion of the substrate to reduction products while no products were detectable in assays with CYC2. Incubation of the proteins in reaction mixtures lacking NADPH did not result in detectable products; CYC1 minus NADPH is shown as a representative trace.

Figure 7.

Correlation Analyses between Stem Transcript Abundance of the RNAi-Targeted Gene with the Mature Leaf Camptothecin Content of Transgenic RNAi Plants and Wild-Type Plants. Data are shown for TDC1-RNAi plants (A) and for CYC1-RNAi plants (B) in comparison to wild-type plants (filled triangles). Each circle represents one RNAi plant from an independent transformation event, with filled circles indicating lines that were selected for further in-depth analyses. For the wild type, three independent lines were taken through tissue culture and regeneration. Stems and mature leaves were collected from plants that had been under greenhouse cultivation in soil for approximately 4 months. Note that the values are plotted on double log₁₀ scale.

Figure 8.

Expression Analyses of the RNAi-Targeted Gene in Root, Young Stems, and Young Leaves of TDC1-RNAi Plants and CYC1-RNAi Plants Relative to the Wild Type. Tissues were collected from plants that had been under greenhouse cultivation for 8 months. Average transcript copy numbers were normalized to ACTIN6 mRNA (n = 5 and 3 for RNAi and wild-type plants, respectively) and are shown for TDC1-RNAi plants (A) and CYC1-RNAi plants (B) relative to the wild type. Note that the y axis is discontinuous to allow depiction of the significant differences in RNAi target gene copy numbers between wild-type and RNAi lines. Asterisks indicate significant differences in RNAi plants (unpaired t test; *P < 0.05; **P < 0.001) relative to the wild type.

Figure 9.

Levels of Tryptamine, Loganic Acid, Secologanic Acid, and Camptothecin in Different Tissues of Wild-Type C. acuminata and CYC1-RNAi and TDC1-RNAi Plants. Tissues were collected from plants that had been under greenhouse cultivation for 8 months. Average levels of tryptamine, loganic acid, secologanic acid, and camptothecin (Figure 1, compounds 4, 2, 3, and 12, respectively) are shown with sd for wild-type (n = 3), CYC1-RNAi (n = 5), and TDC1-RNAi lines (n = 5). Asterisks indicate significantly different metabolite levels in CYC1-RNAi lines and TDC1-RNAi (unpaired t test; *P < 0.05; **P < 0.001) relative to the wild type.

Figure 10.

Root and Stem Expression of Pathway Genes Not Targeted by RNAi in TDC1-RNAi and CYC1-RNAi Plants in Comparison to the Wild Type. Tissues were collected from RNAi plants that had been under greenhouse cultivation for 8 months. mRNA levels for CYC1, CYC2, and G8O in roots (A) and stems (B) of TDC1-RNAi and wild-type plants are shown. Root (C) and stem (D) transcript levels, respectively, for TDC1, CYC2, and G8O in CYC1-RNAi plants compared with the wild type. Average transcript copy numbers were normalized to ACTIN6 mRNA for RNAi and wild-type plants (n = 5 and 3, respectively). Asterisks indicate significant differences in RNAi plants in comparison to the wild type (unpaired t test; *P < 0.05). Note that the y axis scales for CYC2 and G8O are different from CYC1 and TDC1.