PERSPECTIVE ARTICLE published: 26 October 2012 doi: 10.3389/fpls.2012.00239 Quinolizidine biosynthesis: recent advances and future prospects

Somnuk Bunsupa1, MamiYamazaki1 and Kazuki Saito1,2* 1 Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan 2 RIKEN Science Center, Yokohama, Japan

Edited by: Lys-derived , including piperidine, quinolizidine, indolizidine, and lycopodium alka- Gustavo Bonaventure, Max Planck loids, are widely distributed throughout the plant kingdom. Several of these alkaloids have Institute for Chemical Ecology, Germany beneficial properties for humans and have been used in medicine. However, the molecular mechanisms underlying the biosynthesis of these alkaloids are not well understood. In the Reviewed by: Masami Y. Hirai, RIKEN Plant Science present article, we discuss recent advances in our understanding of Lys-derived alkaloids, Center, Japan especially the biochemistry, molecular biology, and biotechnology of quinolizidine alkaloid Taketo Okada, Tokushima Bunri (QA) biosynthesis. We have also highlighted Lys decarboxylase (LDC), the enzyme that University, Japan Kalina Bermudez Torres, Instituto catalyzes the first committed step of QA biosynthesis and answers a longstanding ques- Politécnico Nacional, Mexico tion about the molecular entity of LDC activity in . Further prospects using current *Correspondence: advanced technologies, such as next-generation sequencing, in medicinal plants have also Kazuki Saito, Graduate School of been discussed. Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Keywords: , lysine decarboxylase, lysine-derived alkaloids, o-tigloyltransferase, quinolizidine alkaloids Chiba 260-8675, Japan. e-mail: [email protected]

INTRODUCTION (−)-huperzine A is used in the treatment of Alzheimer’s disease Plant secondary metabolites play multiple roles in the interac- (Decker et al., 1992; Bai et al., 2000; Dwoskin et al., 2000; Dwoskin tion between plants and their environment (Dixon, 2001). Almost and Crooks, 2002). 100,000 secondary metabolites have been discovered from plant Over the past decade, intensive efforts have been made to iden- (Verpoorte, 1998; Hostettmann and Terreaux, 2000; Afendi tify the genes encoding the enzymes involved in the biosynthesis of et al., 2012). Alkaloids are organic nitrogenous bases, usually in a isoquinoline, , tropane, and purine alkaloids (Croteau et al., heterocyclic ring, with characteristic toxicity and marked phar- 2000; Facchini et al., 2004). However, the molecular mechanisms macological effects in humans and animals (De Luca and St underlying the biosynthesis of Lys-derived alkaloids are poorly Pierre, 2000). Alkaloids are derived from the products of primary understood. In this article, we discuss recent advances in our metabolism, with amino acids, such as Phe, Tyr, Trp, Orn, and Lys, understanding of the Lys-derived alkaloids, especially the genes serving as their main precursors (Facchini, 2001). Approximately involved in the quinolizidine alkaloid (QA) biosynthetic path- 20% of plant species accumulate alkaloids and over 12,000 differ- way. We highlight the novel finding of Lys decarboxylase (LDC) ent alkaloids have been described, suggesting higher structural and from plants and discuss how it evolved from primary metabolism biosynthetic diversity compared to other secondary metabolites to alkaloid biosynthesis. Finally, we consider how to apply cur- (Croteau et al., 2000; De Luca and St Pierre, 2000). rent advanced technologies to the study of unidentified alkaloid Alkaloids derived from Lys are widely distributed through- biosynthetic pathways. out the plant kingdom and are subdivided into piperidine, quinolizidine, indolizidine, and lycopodium alkaloids. Piperidine BIOSYNTHESIS OF LYS-DERIVED ALKALOIDS alkaloids are found in plants in the order Piperales (e.g., piperine L-Lys is the homologue of L-Orn, a precursor of pyrrolidine, from Piper nigrum), Myrtales (e.g., (−)-pelletierine from Punica tropane, and pyrrolizidine alkaloids. The extra methylene group granatum), and Asterales (e.g., (−)-lobeline from Lobelia inflata). in Lys participates in forming 6-membered piperidine rings, Quinolizidine (e.g., (−)-lupinine and (−)-sparteine from Lupinus while Orn participates in forming 5-membered pyrrolizidine rings luteus and Cytisus scoparius, respectively) and indolizidine (e.g., (Dewick, 2001; Poupon et al., 2011). Biosynthetic pathways of sev- (−)- from Swainsona canescens) alkaloids are found eral potential Lys-derived alkaloids have been proposed based on mainly in the order (Murakoshi et al., 1975, 1986; Cole- tracer experiments with labeled precursors (Leistner and Spenser, gate et al., 1979; Su and Horvat, 1981; Neuhofer et al., 1993; Felpin 1973 and references therein). Feeding studies demonstrated that and Lebreton, 2004). Lycopodium alkaloids (e.g., (−)-huperzine the first step in piperidine, quinolizidine, and lycopodium alka- A and lycopodine from Huperzia serrata) are found mainly in loid biosynthesis is the decarboxylation of L-Lys into cadaverine by the genus Lycopodium (Figure 1; Liu et al., 1986; Ma and Gang, LDC (EC 4.1.1.18). Oxidative deamination of cadaverine by cop- 2004). Several alkaloids have beneficial properties for humans and per amine oxidase (CuAO, EC 1.4.3.22) yields 5-aminopentanal, are used in medicine. For example, (−)-lobeline is used in the which is spontaneously cyclized to Δ1-piperideine Schiff base treatment of central nervous system disorders and drug abuse and (Leistner and Spenser, 1973; Gupta et al., 1979; Golebiewski and

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FIGURE 1 | Biosynthesis of universal intermediates, skeleton structures, quinolizidine alkaloids. (−)-Swainsonine is an alkaloids. Lycopodine and some Lys-derived alkaloids found in plants. Piperine, (−)-pelletierine, and (−)-huperzine A are lycopodium alkaloids. LDC, Lys decarboxylase; CuAO, and (−)-lobeline are piperidine alkaloids. (−)-Lupinine and (−)-sparteine are copper amine oxidase.

Spenser, 1988; Saito and Murakoshi, 1995; Ma and Gang, 2004). 1995). QAs occur mainly in the family Leguminosae, especially In contrast, indolizidine alkaloids are formed from L-Lys via L- in the genera Lupinus, Baptisia, Thermopsis, Genista, Cytisus, and pipecolic acid which is formed via the aldehyde and Schiff base, Sophora (Ohmiya et al., 1995). More than 500 Lupinus species with retention of the nitrogen of the α-amino acid group (Figure 1; have been described and 200–300 species are distributed in North Harris et al., 1988a,b; Wickwire et al., 1990; Dewick, 2001). Δ1- and South America (Wink et al., 1995). QAs are important as Piperideine and L-pipecolic acid are universal intermediates that potential sources of medicine. They have a broad range of phar- can be modified by condensation, hydrolysis, or methylation to macological properties, including cytotoxic, oxytocic, antipyretic, produce diverse Lys-derived alkaloids (Figure 1; Evans, 2009). antibacterial, antiviral, and hypoglycemic activities, as determined The genes and enzymes involved in the biosynthesis of Lys- by in vivo pharmacological screening (Saito and Murakoshi, 1995). derived alkaloids are not well documented. The best-studied Some QA-containing plants, for example Sophora flavescens, have pathway at the genetic level is the one leading to the formation been used as sources of crude drugs in Chinese–Japanese medicine of QAs, which will be discussed in the following section. One (KAMPO; Tang and Eisenbrand, 1992). other study addresses partially purified piperoyl-CoA: piperidine Quinolizidine alkaloids are synthesized through the cycliza- N-piperoyltransferase (EC 2.3.1.145) from P. nigrum which cat- tion of the cadaverine unit (Golebiewski and Spenser, 1988), alyzes the synthesis of piperine in the presence of piperoyl-CoA which is produced through the action of LDC (Figure 2; Saito and piperidine (Geisler and Gross, 1990). and Murakoshi, 1995). Various QA skeletons are further mod- ified by tailoring reactions (e.g., dehydrogenation, oxygenation, QUINOLIZIDINE ALKALOIDS or esterification) to yield several hundred structurally related Quinolizidine alkaloids are a group of alkaloids possessing a alkaloids (Ohmiya et al., 1995). Lupinus accumulates two types quinolizidine ring or a piperidine ring (Saito and Murakoshi, of QA esters, the derivatives of lupinine and lupanine. QA

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FIGURE 2 | Biosynthetic pathway of quinolizidine alkaloids. Quinolizidine these alkaloids are indicated: LDC, Lys decarboxylase; HMT/HLT, tigloyl-CoA: alkaloids are synthesized by the decarboxylation of Lys, yielding cadaverine, (−)-13α-hydroxymultiflorine/(+)-13α-hydroxylupanine O-tigloyltransferase; which is further modified by various reactions as shown. The metabolites ECT/EFT-LCT/LFT, p-coumaroyl-CoA/feruloyl-CoA: (+)-epilupinine/(−)-lupinine derived from the pathway are shown. Enzymes involved in the synthesis of O-coumaroyl/feruloyltransferase. esters are assumed to be end products of biosynthesis and stor- the two cultivars. Among 71 bitter-specific fragments, three genes age forms (Ohmiya et al., 1995). Two acyltransferases (ATs) exhibited homologies to Orn decarboxylase (ODC, EC 4.1.1.17), involved in QA ester biosynthesis have been identified and charac- CuAO, and AT, representing potential candidates for structural terized: (−)-13α-hydroxymultiflorine/(+)-13α-hydroxylupanine genes encoding QA biosynthetic enzymes (Bunsupa et al., 2011). O-tigloyltransferase (HMT/HLT; EC 2.3.1.93), which cat- Further study showed that the AT might be involved either in alyzes the transfer of the tigloyl group from tigloyl-CoA to the formation of QA esters or N-acylated polyamine conjugates (−)-13α-hydroxymultiflorine or (+)-13α-hydroxylupanine, and (Bunsupa et al., 2011) and ODC was identified as Lys/Orn decar- p-coumaroyl-CoA/feruloyl-CoA: (+)-epilupinine/(−)-lupinine boxylase (L/ODC; see below) (Bunsupa et al., 2012). In addition, O-coumaroyl/feruloyltransferase (ECT/EFT-LCT/LFT), which partial sequences encoding candidate transcription factors that catalyzes the transfer of p-coumaroyl-CoA or feruloyl-CoA to the might regulate the expression of genes in QA biosynthesis were hydroxyl moiety of (+)-epilupinine or (−)-lupinine (Figure 2; obtained; these included DNA binding protein EREBP-3, leucine Saito et al., 1992, 1993; Suzuki et al., 1994). However, only zipper protein, and coronatine-insensitive 1, which provide clues HMT/HLT has been cloned and characterized at the molecular about the regulation of the QA biosynthetic pathway (Bunsupa level (Okada et al., 2005). HMT/HLT belongs to the BAHD super et al., 2011). Genomic PCR of L. angustifolius AT and LDC indi- family, a plant acyl-CoA-dependent AT gene family that transfers cated the presence of at least one copy of both genes in the genomes acyl groups from their CoA esters to various plant metabolites of both the bitter and sweet cultivars. However, transcripts of both (D’Auria, 2006). genes were expressed only in the bitter cultivar. This finding is An investigation of an alkaloid-rich “bitter” cultivar and an interesting in terms of the regulation of the entire QA biosynthetic alkaloid-poor “sweet” cultivar of L. angustifolius using random pathway (Bunsupa et al., 2011, 2012). amplified polymorphic DNA (RAPD) showed that total DNA polymorphisms did not differ substantially between the two cul- A NOVEL LYS DECARBOXYLASE FROM QUINOLIZIDINE tivars (Hirai et al., 2000). A cDNA-amplified fragment length ALKALOIDS PRODUCING PLANTS polymorphism (cDNA-AFLP) was then used to isolate the genes Lys decarboxylase and ODC are key enzymes involved in the for- specifically expressed in the bitter plant; however, no bitter-specific mation of cadaverine and putrescine by the decarboxylation of Lys gene was isolated (Hirai et al., 2000). The PCR-select subtraction and Orn, respectively. Cadaverine, putrescine, and other aliphatic technique was then used to profile differentially expressed genes in polyamines, such as spermidine and spermine, are involved in a

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number of growth and developmental processes (Bagni and Tas- gene transcripts, enzymes, and metabolites (Ziegler and Facchini, soni, 2001). Unlike plant ODCs, plant LDCs have not been well 2008). The majority of QAs are synthesized in shoot tissues. Of studied at the molecular level. There are several studies on crude the QAs that accumulate in , half are synthesized in situ and protein extracts or partially purified protein extracts from Glycine half are translocated primarily by phloem (Lee et al., 2007). The max (Kim et al., 1998; Ohe et al., 2009) and L. polyphyllus (Hart- concentration of QAs in seeds decreases during germination and mann et al., 1980). There was speculation that a member of group development and increases as soon as new leaves are formed (Wink IV of the PLP-dependent amino acid decarboxylases might be and Witte, 1985). The biosynthesis of QAs occurs mainly in the responsible for the production of cadaverine from Lys (Sandmeier green parts of plants where the enzymes for Lys, cadaverine, and et al., 1994) and that ODC may also have LDC activity in plants lupanine synthesis are present (Wink and Hartmann, 1979, 1982; that make cadaverine-derived alkaloids (Sinclair et al., 2004). Wink and Mende, 1987). La-L/ODC is localized in chloroplasts, Recently, we isolated a cDNA of L/ODC from L. angus- while HMT/HLT localized in mitochondria. ECT is assumed to tifolius (La-L/ODC) using differential transcript screening in be present in another organelle, but not in mitochondria (Suzuki QA-producing and non-producing cultivars (Bunsupa et al., 2011, et al., 1996; Bunsupa et al., 2012). 2012). We also obtained L/ODCs cDNAs from S. flavescens and Echinosophora koreensis, which also produce QAs. Recombinant ENGINEERING IN QA-PRODUCING PLANTS L/ODCs from QA-producing plants almost equally catalyzed the The concentration of QAs in cell suspension cultures of Lupinus decarboxylation of L-Lys and L-Orn unlike the known ODCs and related plants is approximately 100–1000 times lower than that only accept Orn (Boeker and Fischer, 1983; Bunsupa et al., that of concentration in leaves of differentiated plants (Hartmann 2012). In vivo experiments of La-L/ODC expression in Arabidop- et al., 1980; Hernandez et al., 2011). In cell suspension cultures, sis thaliana and Nicotiana tabacum provided evidence that this the storage tissue is probably repressed; thus, the QAs cannot be enzyme is involved in the first step of QA biosynthesis (Bunsupa stored in large quantities and are degraded rapidly either inside et al., 2012). This is the first plant LDC identified at the molecular the cells or in the cell culture medium (Wink, 1987). In callus, the level by cDNA cloning and the second QA biosynthesis enzyme concentration of QAs is correlated to the amount of chlorophyll for which the cDNA has been identified. in the cells (Saito et al., 1989a,b). Identification of La-L/ODC answers a longstanding question Lupinus spp. are the major grain legumes in Australia and have about the molecular entity of LDC activity in plants. We found been used as feed supplements. In the past decade, they have LDCs in plant species that produce QAs. A point mutation from gained attention as ingredients for human food because of their His-344 to Phe-344 (La-L/ODC numbering) is key for La-L/ODC high percentage of protein (34–43% of dry matter) and acceptable to exhibit substrate promiscuity for Lys and Orn. This amino acid content of essential amino acids (Sujak et al., 2006). To genetically substitution has presumably occurred via adaptive evolution of engineer plants successfully, a routine transformation system is LDC within the QA-producing plants to expand substrate accessi- needed. Thus, it is necessary to develop a transformation system bility to Lys (Bunsupa et al., 2012). These results reveal how plants for Lupinus lines to confer desired traits, such as herbicide resis- have been opportunistic in adapting primary metabolic processes tance, resistance to certain pathogens, increased pod and set, to permit the diversification of secondary metabolism, in this case, and improved seed quality (Pigeaire et al., 1997). Many transfor- substrate promiscuity. Accordingly, the mutation in this position mation systems for Lupinus sp. have been developed; however, only may be found not only in QA-producing Leguminosae but also in a few have been successful. For example, shoot regeneration using plants that produce cadaverine-derived alkaloids. This finding is a Agrobacterium-mediated gene transfer to preorganized meristem- breakthrough in the understanding of both QA biosynthesis and atic tissue combined with axillary regeneration has been successful LDC occurrence and function in plants. in L. angustifolius (Pigeaire et al., 1997), L. luteus (Li et al., 2000), A major safety issue of lupine-based foods is the presence of and L. mutabilis (Babaoglu et al., 2000). However, transformation QAs, which are toxic to human and mammals. Genetic mapping of efficiency is poor and transformation in some Lupinus species L. angustifolius and L. albus has been completed, which enables the and even in different cultivars of the same species is difficult. In mapping of the genes controlling key domestication traits, includ- L. mutabilis, the transformed roots synthesize isoflavones, but not ing alkaloid production, water permeability, flowering time, pod QAs (Babaoglu et al., 2004). Genetic engineering of L. angustifolius shattering, pigment production in seeds, and anthracnose resis- by over expressed sun flower seed albumin gene has succeeded to tance (Boersma et al., 2005; Nelson et al., 2006; Phan et al., 2006). enhance methionine levels and increase nutritive value of seeds of This information will be useful not only to the Lupinus research transgenic Lupinus plants (Molvig et al., 1997). community but also to other legume research communities study- ing molecular breeding and evolution in leguminous plants. The FUTURE PROSPECTS identified La-L/ODC provides a good candidate enzyme as an In the past few years, progress has been made in the elucidation of entry-point enzyme of QA biosynthesis for metabolic engineering alkaloid biosynthetic pathways and their regulation. However, only to manipulate the production of QAs. two genes, HMT/HLT and L/ODC, have been isolated and char- acterized so far. Rapid progress in the development of integrative COMPARTMENTALIZATION AND SUBCELLULAR approaches, such as genomics, transcriptomics, proteomics, and LOCALIZATION metabolomics, has provided essential information for the under- The subcellular compartmentalization of alkaloid biosynthetic standing of many complex biological processes at different levels enzymes is as complex as the cell type-specific localization of (Fukushima et al., 2009; Yonekura-Sakakibara and Saito, 2009).

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Comparative and integrative analyses of such “omics” data will transcription factors involved in the production of plant secondary facilitate the determination of gene function and pinpoint rate lim- metabolites. Such publicly accessible databases will provide novel iting steps and factors in the biosynthetic pathway of secondary information for the research community and ultimately accelerate metabolites. Transcriptomic and metabolomic approaches, par- the progress of the study of plant science. ticularly transcriptome coexpression network analysis, have been The predicted function or in vitro characterization of alkaloid used to identify genes involved in specific pathways not only in biosynthetic enzymes and other components must be confirmed Arabidopsis but also in crops and medicinal plants. Such strategies in vivo (Ziegler and Facchini, 2008). However, there is no reli- have proven to be useful in fundamental plant biology and applied able and highly efficient transformation protocol for Lupinus and biotechnology (Ziegler et al., 2006; Hirai et al., 2007; Saito et al., related plants. Virus induced gene silencing (VIGS), a technology 2008: Seki et al., 2008; Yonekura-Sakakibara et al., 2008, 2012; Lis- that exploits an RNA-mediated antiviral defense mechanism, is combe et al., 2009; Okazaki et al., 2009; Saito and Matsuda, 2010; a fast method for the generation of transient transformed plants Okazaki and Saito, 2012). (Lu et al., 2003). VIGS has been used widely in plants for analysis Advances in sequencing technology, next-generation sequenc- of gene function and has been adapted for high-throughput func- ing, and computer technology for processing of large data sets tional genomics (Burch-Smith et al., 2004). Thus, VIGS might are opening a new era in plant science. It is estimated that the be a powerful tool for assessing and characterizing the func- cost of DNA sequencing drops at a faster rate than the cost tion of candidate genes in the QA biosynthetic pathway. Further of computer data processing (Ehrhardt and Frommer, 2012). investigations on inter- and intracellular transport of pathway Therefore, the development of sequencing techniques with high- intermediates and products and on cellular and subcellular local- throughput will likely be applied to alkaloid-producing plants ization of enzymes are also important because they are crucial and provide the basis for many sequence-based approaches factors in controlling the accumulation of target metabolites in that are now limited to model plants such as Arabidopsis and plants (Facchini et al., 2004). rice (Ziegler and Facchini, 2008). Today, many researchers are making progress in plant genome and transcriptome sequenc- CONCLUSION ing. For instance, the 1KP project aims to generate large-scale Knowledge of the biosynthetic pathway of Lys-derived alkaloids is gene sequence information for 1000 different plants, includ- limited. The best-studied pathway at the genetic level is QA biosyn- ing a wide variety of species from algae to land and aquatic thesis; however, only two genes have been identified. Application plants, to understand the vast biodiversity that has barely been of current technologies, such as next-generation sequencing, high- touched by genomics to date1. In addition, the medicinal plant end proteomics, and metabolomics approaches, is needed to consortium project focuses on providing transcriptomic and study secondary metabolic pathways for a better understanding metabolic resources for 14 key medicinal plants, for instance of metabolic networks. Such information will aid in the develop- Atropa belladonna and Camptotheca acuminata, to the worldwide ment of successful strategies in genetic engineering of secondary research community for the advancement of drug production metabolites in plants. and development2. Recently, we started the deep transcriptome sequencing of Japanese medicinal plants and both cultivars of L. angustifolius to obtain more information about the enzymes and ACKNOWLEDGMENT This study was supported in part by the Grants-in-Aid for Sci- 1http://www.onekp.com/project.html entific Research (KAKENHI) from The Ministry of Education, 2http://medicinalplantgenomics.msu.edu/index.shtml Culture, Sports, Science, and Technology (MEXT).

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