Divergent Camptothecin Biosynthetic Pathway in Ophiorrhiza Pumila
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Yang et al. BMC Biology (2021) 19:122 https://doi.org/10.1186/s12915-021-01051-y RESEARCH ARTICLE Open Access Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila Mengquan Yang2†, Qiang Wang1,3†, Yining Liu2, Xiaolong Hao1, Can Wang1, Yuchen Liang2, Jianbo Chen3, Youli Xiao2ˆ and Guoyin Kai1* Abstract Background: The anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined. Results: In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)- strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila,weshowedthat3α-(S)-strictosidine, not 3-(S), 21- (S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis. Conclusions: In our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata.Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata,andN. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches. Keywords: Biosynthesis, Camptothecin, In vivo labelling, Ophiorrhiza pumila, Strictosidine Background (TCM) [1]. CPT chemotype also appears sporadically in The alkaloid camptothecin (CPT) was first isolated in multiple taxa within the superasterids, with a total of 43 1966 from the bark of the tree Camptotheca acuminata plant species [2]. Of those, C. acuminata, Nothapodytes (“Xi-Shu” in Chinese, which translates to happy tree). C. nimmoniana, and Ophiorrhiza pumila are the three acuminata is a deciduous tree native to southern China main representative CPT-producing plants (Fig. 1a). In that is extensively used in traditional Chinese medicine 1994, the US Food and Drug Administration (FDA) ap- proved the therapeutic use of two well-known antitu- * Correspondence: [email protected] mour CPT derivatives, irinotecan and topotecan, which †Mengquan Yang and Qiang Wang contributed equally to this work. ˆYouli Xiao is deceased. inhibit the replication, growth, and reproduction of can- 1Laboratory of Medicinal Plant Biotechnology, College of Pharmacy, Zhejiang cer cells by inhibiting DNA topoisomerase I [3]. The Chinese Medical University, Hangzhou 310053, Zhejiang, China clinical applications of these two CPT-derived drugs to Full list of author information is available at the end of the article the treatment of cancer have greatly increased demand, raising issues about the sustainable production of CPT © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yang et al. BMC Biology (2021) 19:122 Page 2 of 16 A B SUPERASTERIDS LAMIIDS Camptotheca acuminata ASTERIDS Nothapodytes nimmoniana CAMPANULIDS Ophiorrhizapumila Fig. 1 Distribution of CPT in the plant kingdom and possible biosynthesis of alkaloids through strictosidine or strictosidinic acid. a Phylogenetic tree of the major lineages of land plants summarizing the known taxonomic distribution of CPT-producing species. Orders that contain at least one CPT- producing species are indicated with red diamonds. The three CPT-producing plants used in this study are shown on the right and belong to the Cornales (C. acuminata), Icacinales (N. nimmoniana), and Gentianales (O. pumila)orders.b The biosynthesis of monoterpene indole alkaloids (MIAs) through strictosidine or strictosidinic acid derived from tryptamine and secoiridoids. STR, strictosidine synthase; STRAS, strictosidinic acid synthase [4]. Because chemical synthesis of CPT on an industrial secologanin via the iridoid pathway [6]. The biosynthesis scale is hindered by the complexity of its unique penta- of secologanin involves the successive action of the en- cyclic pyrroloquinoline scaffold, the sourcing of CPT still zymes geranyl diphosphate synthase (GPS), geraniol syn- depends heavily on extraction from its resource plants thase (GE), geraniol 10-hydroxylase (G10H), 10- [5, 6]. However, the few plants that naturally produce hydroxygeraniol oxidoreductase (10-HGO), iridoid syn- CPT grow slowly and will not meet this increasing mar- thase (IS), iridoid oxidase (IO), 7-deoxyloganetic acid ket demand, necessitating an alternative approach to UDP-glucosyltransferase (7-DLGT), 7-deoxyloganic acid raise CPT production [6, 7]. One such promising hydroxylase (7-DLH, catalyzed by CYP72A224), secolo- method involves the introduction of key biosynthetic ganin synthase (SLS), and cytochrome P450 reductase genes or regulators through metabolic engineering or (CPR), finally forming secologanin [14]. In the shi- the reconstruction of the CPT biosynthetic pathway in kimate pathway, several enzymes participate in multi- heterologous microbial systems by synthetic biology. step reactions from chorismic acid to tryptamine, This method will require the dissection and thorough which is another precursor for CPT biosynthesis. understanding of CPT biosynthesis, which is currently These enzymes include anthranilate synthase (ASA), lacking [7–11]. phosphoribosyl diphosphate anthranilate transferase More than 2000 monoterpene indole alkaloids (MIAs) (PRT), tryptophan synthase α (TSA), tryptophan syn- are thought to originate from the common intermediate thase β (TSB), and tryptophan decarboxylase (TDC). strictosidine, which separates the MIA biosynthetic TDC is considered to be a rate-limiting enzyme in pathway into prestrictosidine and poststrictosidine path- CPT biosynthetic pathway [18]. Subsequently, trypta- ways or steps (Fig. 1b) [12–14]. In plants, the biosyn- mine and secologanin are condensed into strictosidine thesis of strictosidine has been well studied [6, 15–17]. by strictosidine synthase (STR). However, multiple en- The methylerythritol 4-phosphate (MEP) and mevalonic zymatic steps are still missing from our understanding acid (MVA) pathways provide basic terpene precursors of the poststrictosidine pathway, and only a few of such as isopentenyl pyrophosphate (IPP) to form the metabolite intermediates in the CPT biosynthetic Yang et al. BMC Biology (2021) 19:122 Page 3 of 16 pathway, such as pumiloside and deoxypumiloside, Results have been identified [19, 20]. Two parallel pathways of CPT biosynthesis were Based on our current understanding of CPT biosyn- proposed by metabolite profiling of intermediates in O. thesis, strictosidine is commonly considered the key pumila intermediate in the biosynthesis of MIAs such as vin- The distribution of the bioactive compound CPT var- blastine and vincristine. However, although CPT belongs ies across O. pumila tissues [23]. To assess the diver- to the MIA family, no strictosidine was detected in the sity and abundance of putative intermediates in the CPT-producing plant C. acuminata [15]. Furthermore, CPT biosynthetic pathway in O. pumila, we collected loganic acid, secologanic acid, and strictosidinic acid different tissues (leaves, stems, roots, and hairy roots) were all detected by metabolite profiling, whereas loga- for metabolite profiling (Additional file 1:Fig.S1). nin, secologanin, and strictosidine were all undetectable We subjected total methanolic extracts to ultra-high- in C. acuminata, leading to the conclusion that strictosi- performance liquid chromatography (UHPLC) dinic acid was the sole intermediate for CPT biosyn- followed by mass spectrometry (MS) for untargeted thesis (Fig. 1b) [15]. In contrast, in N. nimmoniana, only metabolic analysis. We annotated 15 metabolites in secologanin was detected [21], indicating that strictosi- the CPT biosynthetic pathway based on accurate mass dine, and not strictosidinic