A Papaver somniferum 10-Gene Cluster for Synthesis of the Anticancer Alkaloid Noscapine

  1. Thilo Winzer1
  2. Valeria Gazda1
  3. Zhesi He1
  4. Filip Kaminski1
  5. Marcelo Kern1
  6. Tony R. Larson1
  7. Yi Li1
  8. Fergus Meade1
  9. Roxana Teodor1
  10. Fabi·n E. Vaistij1
  11. Carol Walker2
  12. Tim A. Bowser2
  13. Ian A. Graham1,*

Abstract

Noscapine is an antitumor alkaloid from opium poppy that binds tubulin, arrests metaphase, and induces apoptosis in dividing human cells. Elucidation of the biosynthetic pathway will enable improvement in commercial production of noscapine and related bioactive molecules. Transcriptomic analysis revealed the exclusive expression of 10 genes encoding five distinct enzyme classes in a high noscapine-producing poppy variety, HN1. Analysis of an F2 mapping population indicated the genes are tightly linked in HN1, and bacterial artificial chromosome sequencing confirmed they exist as a complex gene cluster for plant alkaloids. Virus-induced gene silencing resulted in accumulation of pathway intermediates allowing gene function to be linked to noscapine synthesis and a novel biosynthetic pathway to be proposed.

Noscapine was first characterized from opium poppy (Papaver somniferum) by the distinguished French chemist Pierre Jean Robiquet in 1817 (1) but unlike codeine, which he also discovered, and several other opiates, noscapine is non-painkilling and non-addictive. Noscapine has been used as a human cough suppressant for decades with its effect on the cough reflex and bronchial muscles being reported as early as 1954 (2), and the bioavailability and pharmacokinetics of the orally administered form being more recently established (3). The demonstration in 1998 that noscapine acts as a potent antitumor agent that binds to tubulin, affecting its polymerisation, arresting cell division and inducing apoptosis (4) was followed by its antitumor activity being demonstrated in various forms of cancer (58). The history of safe use as an anti-tussive, rapid absorption after oral administration and apoptosis-inducing effect on a number of cancer cell lines, may place noscapine at an advantage over other tubulin binding anticancer natural products such as the well established taxanes (9).

Noscapine belongs to the phthalideisoquinoline subclass of the structurally diverse isoquinoline alkaloids whereas codeine, morphine, thebaine and oripavine belong to the morphinan subclass (10). While the biosynthesis of morphinans has been elucidated at the molecular level (1116) our knowledge of noscapine biosynthesis has not advanced significantly since the demonstration using isotope labeling in the 1960s, that it is derived from scoulerine (17). Understanding the biochemical genetics underpinning noscapine biosynthesis should enable improved production of this important pharmaceutical and related molecules both in poppy and other systems.

We metabolite profiled capsule extract from three opium poppy varieties developed in Tasmania for alkaloid production that we designate as High Morphine 1 (HM1), High Thebaine 1 (HT1) and High Noscapine 1 (HN1) on the basis of the most abundant alkaloid in each case (Fig. 1A). We found noscapine to be unique to HN1 relative to HM1 and HT1 as were 53 other low abundance compounds, some of which are candidate intermediates in the noscapine biosynthetic pathway (table S1). We performed Roche 454 pyrosequencing on cDNA libraries derived from stem and capsule tissue from all three varieties. Analysis of Expressed Sequence Tag (EST) abundance led to the discovery of a number of previously uncharacterized genes that are expressed in the HN1 variety but are completely absent from the HM1 and HT1 EST libraries (Fig. 1B). We putatively identified the corresponding genes as three O-methyltransferases (PSMT1, PSMT2, PSMT3, fig. S1), four cytochrome P450s (CYP82X1, CYP82X2, CYP82Y1 and CYP719A21, fig. S2), an acetyltransferase (PSAT1, fig. S3), a carboxylesterase (PSCXE1, fig. S4) and a short-chain dehydrogenase/reductase (PSSDR1, fig. S5). In contrast a number of other functionally characterized genes associated with benzylisoquinoline alkaloid synthesis, including Berberine Bridge Enzyme (BBE), Tetrahydroprotoberberine cis-N-MethylTransferase (TNMT), Salutaridine Reductase (SalR), Salutaridinol 7-O-AcetylTransferase (SalAT) and Thebaine 6-O-demethylase (T6ODM) were expressed in all three varieties (Fig. 1B). PCR analysis revealed that the genes exclusively expressed in the HN1 variety are present as expected in the genome of HN1 but absent from the genomes of the HM1 and HT1 varieties (Fig. 1B and fig. S6).

View larger version:

Fig. 1

Identification of genes exclusively present in the genome of a noscapine producing poppy variety, HN1 (High Noscapine 1). (A) Relative abundance of the major alkaloids extracted from the capsules of three commercial varieties of poppy, HM1 (High Morphine 1), HT1 (High Thebaine 1) and HN1. M = morphine, C = codeine, T = thebaine, O = oripavine and N = Noscapine. Rt = retention time, (s) = seconds (B) EST libraries from stem and capsule were generated by pyrosequencing and unique contiguous sequences assembled as described (18). The first ten genes are represented only in EST libraries from the HN1 variety while the last five genes are present in EST libraries from all three varieties. All genes are represented at consistently higher levels in stem compared to capsule as shown in color code. PCR on genomic DNA from all three varieties revealed that the ten HN1-specific genes are absent from the genomes of the HM1 and HT1 varieties and the five other functionally characterized genes are present in all three varieties (fig. S6).

We generated an F2 mapping population of 271 individuals using HN1 and HM1 as parents. Genotyping of the field grown F2 population revealed that the HN1-specific genes are tightly linked and associated with the presence of noscapine suggesting they occur as a gene cluster involved in noscapine biosynthesis (Fig. 2). Analysis of noscapine levels in field grown F2 capsules revealed that individuals containing this putative gene cluster fall into two classes. The first, containing 150 individuals, has relatively low levels of noscapine and the second containing 63 individuals exhibits the high noscapine trait of the parental HN1 variety. The 58 F2 individuals that lack the putative gene cluster contain undetectable levels of noscapine (Fig. 2B). F3 family analysis confirmed that F2 individuals exhibiting the high noscapine trait were homozygous for the gene cluster while those exhibiting the low noscapine trait were heterozygous (table S2). Noscapine levels in both the glasshouse grown F1 population and the heterozygous F2 class are much lower than the intermediate levels expected for a semi-dominant trait, suggesting involvement of some form of repression. A number of other HN1-specific metabolites were similarly decreased (table S3) suggesting global downregulation of this branch of alkaloid metabolism when the gene cluster is in the heterozygous state. However, qRT-PCR analysis did not detect any transcriptional repression in the heterozygous state on what we determine below as two genes, PSMT1 and PSMT2, which encode the first step and an intermediate step respectively in the noscapine pathway (fig. S7).

View larger version:

Fig. 2

Segregation analysis of noscapine content in an F2 mapping population demonstrates requirement for the noscapine gene cluster. (A) Box plot depiction of noscapine levels as percentage dry weight (DW) in glasshouse grown parental lines HN1 and HM1 and the F1 generation. (B) F2 generation segregates into three classes of zero, low and high noscapine (N). F2 GC- and F2 GC+ indicate the absence and presence respectively of the noscapine gene cluster. Numbers in brackets indicate number of individuals in each class. (C) Total major morphinans as percentage capsule dry weight in the F2 zero, low and high noscapine classes (T = thebaine, O = oripavine, C = codeine, M = morphine).

The morphinan branch of alkaloid metabolism on the other hand remains largely unaffected in F1 (table S4) and heterozygous F2 material, only showing a decrease in capsules producing high levels of noscapine presumably due to substrate competition (Fig. 2C). The large step change to high noscapine in the homozygous F2 class suggests this trait is linked to the gene cluster locus rather than spread quantitatively among other loci.

To further characterize the putative noscapine gene cluster we prepared a Bacterial Artificial Chromosome (BAC) library from genomic DNA isolated from HN1 and identified six overlapping BACs containing genes from the cluster. Next Generation and Sanger sequencing was used to generate a high quality assembly of 401 Kb confirming the arrangement of the 10 genes in a cluster spanning 221 Kb (Fig. 3 and table S5) (18). The homology and intron-exon structure of the CYP82 and PSMT genes suggest tandem gene duplication after genome re-organization of the progenitor genes. A similar case can be made for PSCXE1 since a second homolog, PSCXE2, is present in the region flanking the gene cluster (Fig. 3, fig. S4) but this gene is not represented in any of our EST libraries. However, on only one occasion are members of the same gene family adjacent in the cluster and in that case, CYP82X1 and CYP82X2, are inverted. CYP82Y1 is separated from CYP82X2 by a gap of 45 Kb containing PSAT1 and PSMT2. PSMT3 and PSMT2 are separated by a gap of 73 Kb containing CYP82Y1. Interspersed among the ten genes are both retrotransposon and DNA transposable element (TE) sequences (Fig. 3 and table S5), which may have some function in gene rearrangement for cluster formation as thought to be the case for the thalianol and marneral clusters from A. thaliana (19). A search of the PLACE database of plant cis-acting DNA elements (20) revealed a number of short motifs (4-5 bases) present in the 1 Kb predicted promoter regions (upstream of the open reading frames) of the 10 genes, among which the WRKY elements are noteworthy (21, 22) (table S6).

View larger version:

Fig. 3

The HN1 gene cluster. The structure and position of the ten HN1-specific genes expressed in stems and capsule tissues are shown above the central black line which represents 401 Kb of genomic sequence. Exons are represented by filled grey boxes and introns by fine black lines. Arrows indicate the 5′ to 3′ orientation of each gene. Additional open reading frames depicted below the central black line are as defined by the key. None of these ORFs are represented in the stem and capsule EST libraries. The location and annotation of all ORFs in the 401 Kb sequence are detailed in table S5.

In order to functionally characterize the genes in the HN1 cluster we performed Virus Induced Gene Silencing (VIGS) on poppy seedlings using established methods (18). VIGS in poppy seedlings persists through to mature plant stages (23) and we therefore routinely assayed both leaf latex and capsule extract (Fig. 4). We managed to silence six of the eight genes we tested by VIGS as determined by mRNA abundance in infected leaf tissue (fig. S8). Silencing PSMT1, which, shows high homology with Scoulerine-9-O-MethylTransferase from Coptis japonica (24) (fig. S1A), resulted in accumulation of scoulerine in both latex and capsules and also low levels of reticuline in latex (Fig. 4A). We expressed the PSMT1 gene product in Saccharomyces cerevisiae and, consistent with the VIGS data, this converts scoulerine to tetrahydrocolumbamine at high efficiency (fig. S9). We therefore conclude that PSMT1 is responsible for the first committed step in the pathway to noscapine synthesis.

View larger version:

Fig. 4

Functional characterization using virus induced gene silencing of 6 genes from the HN1 gene cluster. Results from both leaf latex (left hand bar graphs) and capsules (right hand bar graphs) are consistent with each of these genes encoding enzymes involved in noscapine biosynthesis (A to F). All compounds that accumulate, apart from scoulerine, have been putatively identified on the basis of mass spectra as detailed in fig. S10. The mass-to-charge (m/z) value (M) followed by retention time (T) in seconds is shown for each compound on the horizontal axis. Metabolites showing a >2 fold positive change (VIGS/controls) and >0.05% total alkaloid profile are shown as percentage total metabolites. A complete list of all metabolites that were significantly changed in the VIGS experiments is shown in table S7. (G) Proposed pathway for noscapine biosynthesis based on VIGS data. Solid arrows depict steps supported by VIGS data, dotted arrows depict additional proposed steps. Italics depict those reactions that await gene assignment. The noscapine structure is numbered according to the IUPAC convention. Labeling of the 3-OH secoberbine intermediates is based on the numbering of the noscapine structure. For the secoberbine intermediates that accumulate in VIGS experiments, R1 = H or OH, R2 = H or OH and R3 = CH2OH or CHO or COOH (fig. S10). The proposed pathway assumes R1 = H, R2 = H and R3 = CHO in the secoberbine intermediates. The crossed out PSMT2 depicts silencing of this gene product resulting in narcotoline accumulation as described in the text.

The product of PSMT1, tetrahydrocolumbamine, accumulated in latex and capsules that were silenced for CYP719A21 indicating that this gene is responsible for the second step in the pathway (Fig. 4B and fig. S10). CYP719A21 shows high homology to cytochrome P450 oxidases that act as methylenedioxy bridge-forming enzymes and we therefore propose that CYP719A21 encodes a canadine synthase (25, 26) (fig. S2B). We propose that canadine is methylated to form N-methylcanadine, which in turn is converted to secoberbine intermediates (Fig. 4G). Consistent with this, canadine and N-methylcanadine are HN1-specific metabolites (table S1). The product of TNMT has previously been shown to specifically N-methylate protoberberine alkaloids, including canadine (27). TNMT is present and expressed in HN1, HM1 and HT1 (Fig. 1B) and does not appear to be associated with the HN1 gene cluster (Fig. 3). Three other open-reading frames with TNMT homology are present in the flanking region of the HN1 gene cluster but these are not expressed in stems or capsules further implicating a role for TNMT in the pathway.

Silencing of a second cytochrome P450 gene, CYP82X2, resulted in accumulation of several secoberbine intermediates some of which may represent side products to the main synthetic pathway (Fig. 4C). The fragmentation pattern of intermediate 1 (fig. S10) is consistent with the compound being narcotolinol (R1 = OH, R2 = H and R3 = CH2OH) implying CYP82X2 hydroxylates at the R2 position. The production of these secoberbine intermediates from N-methylcanadine requires breakage of the berberine bridge and ring opening as depicted in Fig. 4G. Silencing of the carboxylesterase gene PSCXE1 resulted in accumulation of up to 20% total alkaloid content of the acetylated compound papaveroxine (Fig. 4D and fig. S10). Synthesis of papaveroxine from secoberbine intermediates requires hydroxylation and methylation at the position equivalent to the C4′ position of noscapine as well as acetylation of the hydroxyl group at the C3 position (Fig. 4G). The accumulation of papaveroxine in material silenced for PSCXE1 implies that the corresponding enzyme removes an acetyl group from this compound to produce narcotinehemiacetal. That narcotinehemiacetal is an intermediate in the pathway to noscapine synthesis is substantiated by the fact that it accumulates upon silencing of the short-chain dehydrogenase/reductase PSSDR1 (Fig. 4E and fig. S10). Conversion of narcotinehemiacetal to noscapine requires dehydrogenation and we therefore conclude that PSSDR1 is involved in this final synthetic step (Fig. 4G).

The VIGS data for PSCXE1 and PSSDR1 therefore support a biosynthetic route to noscapine that involves O-methylation of secoberbine intermediates at the position equivalent to the C4′ hydroxyl group of noscapine (Fig. 4G). However, silencing PSMT2 did not show any impact on secoberbine intermediates but rather accumulation of narcotoline at up to 20% total alkaloids (Fig. 4F). These results suggest that narcotoline is an end product of a desmethyl pathway that accumulates when PSMT2 mediated methylation at the 4′OH group of secoberbine intermediates is compromised. As for the noscapine pathway, the production of narcotoline via a desmethyl pathway is expected to require acetyltransferase, carboxylesterase and dehydrogenase activities.

This work has therefore provided evidence for the involvement of six of the ten genes from the HN1 gene cluster in noscapine biosynthesis. The remaining unaccounted for oxidation and acetyltransferase steps in the proposed pathway could be encoded by the CYP82X1, CYP82Y1 and PSAT1 genes, which remain to be characterized.

This discovery extends the involvement of gene clusters to the alkaloid class of secondary metabolites in higher plants. Noscapine has been reported in a number of Papaver species and it will be interesting to establish if it has evolved as a single event prior to speciation of P. somniferum or independently multiple times as recently reported for the glucosinolate gene cluster in Lotus japonicus (28). As with the other plant gene clusters reported to date (19, 2831) donor sequences could be recruited from genes encoding related plant enzymes in a process involving gene duplication and neofunctionalization. The arrangement of the HN1 cluster suggests genome re-organization is an ongoing process, occurring in some cases before duplication as evidenced by the small gene families (PSCXE, PSCYP82 and PSMT) or after duplication as evidenced by the single copy genes (PSSDR1, PSAT1, CYP719A21). The selective advantage to drive cluster evolution in this way could come from co-inheritance of favorable combinations of alleles and coordinate regulation of gene expression at the level of chromatin (32). This work provides the platform for the improved production of noscapine and related bioactive molecules through the molecular breeding of commercial poppy varieties or engineering of new production systems.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1220757/DC1

Materials and Methods

Figs. S1 to S10

Tables S1 to S8

References (3348)

  • Received for publication 20 February 2012.
  • Accepted for publication 18 May 2012.

References and Notes

  1. 1.µ
    1. 1.P. J. Robiquet

, Observations sur le mémoire de M. Sertuerner relatif à l’analyse de l’opium. Annales de Chimie et de Physique 5, 275 (1817).

  1. 2.µ
  2. 1.H. Konzett,
  3. 2.E. Rothlin

, Zur Wirkung von Narkotin auf den Hustenreflex und auf die Bronchialmuskulatur [The effect of narcotine on cough reflex and on bronchial musculature]. Experientia 10, 472 (1954).

CrossRefMedline

  1. 3.µ
  2. 1.M. O. Karlsson,
  3. 2.B. Dahlström,
  4. 3.S.-Å. Eckernäs,
  5. 4.M. Johansson,
  6. 5.A. T. Alm

, Pharmacokinetics of oral noscapine. Eur. J. Clin. Pharmacol. 39, 275 (1990).

CrossRefMedlineWeb of Science

  1. 4.µ
  2. 1.K. Ye
  3. 2.et al

., Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells. Proc. Natl. Acad. Sci. U.S.A. 95, 1601 (1998).

Abstract/FREE Full Text

  1. 5.µ
  2. 1.Y. Ke
  3. 2.et al

., Noscapine inhibits tumor growth with little toxicity to normal tissues or inhibition of immune responses. Cancer Immunol. Immunother. 49, 217 (2000).

CrossRefMedlineWeb of Science

  1. 6.
  2. 1.J. Zhou
  3. 2.et al

., Paclitaxel-resistant human ovarian cancer cells undergo c-Jun NH2-terminal kinase-mediated apoptosis in response to noscapine. J. Biol. Chem. 277, 39777 (2002).

Abstract/FREE Full Text

  1. 7.
  2. 1.M. Mahmoudian,
  3. 2.P. Rahimi-Moghaddam

, The anti-cancer activity of noscapine: A review. Recent Pat. Anti-Cancer Drug Discov. 4, 92 (2009).

CrossRef

  1. 8.µ
  2. 1.J. W. Landen
  3. 2.et al

., Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma. Cancer Res. 62, 4109 (2002).

Abstract/FREE Full Text

  1. 9.µ
  2. 1.D. G. I. Kingston

, Tubulin-interactive natural products as anticancer agents. J. Nat. Prod. 72, 507 (2009).

MedlineWeb of Science

  1. 10.µ
  2. 1.J. Ziegler,
  3. 2.P. J. Facchini

, Alkaloid biosynthesis: Metabolism and trafficking. Annu. Rev. Plant Biol. 59, 735 (2008).

CrossRefMedline

  1. 11.µ
  2. 1.R. Lenz,
  3. 2.M. H. Zenk

, Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase from Papaver somniferum plant cell cultures: The enzyme catalyzing the formation of thebaine in morphine biosynthesis. J. Biol. Chem. 270, 31091 (1995).

Abstract/FREE Full Text

  1. 12.
  2. 1.B. Unterlinner,
  3. 2.R. Lenz,
  4. 3.T. M. Kutchan

, Molecular cloning and functional expression of codeinone reductase: The penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum. Plant J. 18, 465 (1999).

CrossRefMedlineWeb of Science

  1. 13.
  2. 1.T. Grothe,
  3. 2.R. Lenz,
  4. 3.T. M. Kutchan

, Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum. J. Biol. Chem. 276, 30717 (2001).

Abstract/FREE Full Text

  1. 14.
  2. 1.J. Ziegler
  3. 2.et al

., Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis. Plant J. 48, 177 (2006).

CrossRefMedlineWeb of Science

  1. 15.
  2. 1.A. Gesell
  3. 2.et al

., CYP719B1 is salutaridine synthase, the C-C phenol-coupling enzyme of morphine biosynthesis in opium poppy. J. Biol. Chem. 284, 24432 (2009).

Abstract/FREE Full Text

  1. 16.µ
  2. 1.J. M. Hagel,
  3. 2.P. J. Facchini

, Dioxygenases catalyze the O-demethylation steps of morphine biosynthesis in opium poppy. Nat. Chem. Biol. 6, 273 (2010).

CrossRefMedlineWeb of Science

  1. 17.µ

A. R. Battersby, M. Hirst, D. J. McCaldin, R. Southgate, J. Staunton, Alkaloid biosynthesis. XII. The biosynthesis of narcotine. J. Chem. Soc. Perkin 1 17, 2163 (1968).

  1. 18.µSee supplementary materials on Science Online.
  2. 19.µ
  3. 1.B. Field
  4. 2.et al

., Formation of plant metabolic gene clusters within dynamic chromosomal regions. Proc. Natl. Acad. Sci. U.S.A. 108, 16116 (2011).

Abstract/FREE Full Text

  1. 20.µ
  2. 1.K. Higo,
  3. 2.Y. Ugawa,
  4. 3.M. Iwamoto,
  5. 4.T. Korenaga

, Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res. 27, 297 (1999).

Abstract/FREE Full Text

  1. 21.µ
  2. 1.N. R. Apuya
  3. 2.et al

., Enhancement of alkaloid production in opium and California poppy by transactivation using heterologous regulatory factors. Plant Biotechnol. J. 6, 160 (2008).

CrossRefMedlineWeb of Science