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Silencing of Tryptamine Biosynthesis for Production of Nonnatural Alkaloids in Plant Culture

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Silencing of Tryptamine Biosynthesis for Production of Nonnatural Alkaloids in Plant Culture Silencing of tryptamine biosynthesis for production of nonnatural alkaloids in plant culture Weerawat Runguphan, Justin J. Maresh1, and Sarah E. O’Connor2 Department of Chemistry, Massachusetts Institute of Technology, 18-592, 77 Massachusetts Avenue, Cambridge, MA 02139 Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved July 1, 2009 (received for review March 26, 2009) Natural products have long served as both a source and inspiration tryptamine analogs to plant cell culture (Fig. 1). Precursor for pharmaceuticals. Modifying the structure of a natural product directed biosynthesis with wild-type C. roseus plants and tissue often improves the biological activity of the compound. Metabolic culture has already demonstrated that tryptamine analogs can be engineering strategies to ferment ‘‘unnatural’’ products have been taken up and processed by the plant into a wide variety of enormously successful in microbial organisms. However, despite alkaloids (10, 11). However, if tryptamine 1 is required for plant the importance of plant derived natural products, metabolic engi- growth or survival, silenced lines would not be viable. No studies neering strategies to yield unnatural products from complex, of down-regulating or suppressing tryptamine biosynthesis in lengthy plant pathways have not been widely explored. Here, we plants have been described, and little information is available in show that RNA mediated suppression of tryptamine biosynthesis the literature to support a definitive role for tryptamine, a in Catharanthus roseus hairy root culture eliminates all production metabolite that is found throughout the plant kingdom (12). of monoterpene indole alkaloids, a class of natural products Overproduction of tryptamine has been shown to deter insect derived from two starting substrates, tryptamine and secologanin. feeding (13), but this role is nonessential in the controlled To exploit this chemically silent background, we introduced an environment of plant cell culture. Additionally, 5-hydroxytryp- unnatural tryptamine analog to the production media and dem- tamine (serotonin), which may be produced by hydroxylation of onstrated that the silenced plant culture could produce a variety of tryptamine, has been implicated in certain physiological roles novel products derived from this unnatural starting substrate. The such as flower development (14). Notably, tryptamine may also be involved in auxin biosynthesis (15). novel alkaloids were not contaminated by the presence of the CHEMISTRY Despite the uncertainty concerning the function of tryptamine natural alkaloids normally present in C. roseus. Suppression of in plants, suppression of tryptophan decarboxylase nevertheless tryptamine biosynthesis therefore did not appear to adversely provided an attractive entry point for introduction of new affect expression of downstream biosynthetic enzymes. Targeted substrate analogs. In prokaryotic pathways, the gene of interest suppression of substrate biosynthesis therefore appears to be a is usually deleted or ‘‘knocked out’’ using homologous recom- viable strategy for programming a plant alkaloid pathway to more bination based strategies. However, although some success has effectively produce desirable unnatural products. Moreover, al- been achieved with homologous recombination in higher plants though tryptamine is widely found among plants, this silenced line (16), strategies to down-regulate or knockdown specific gene demonstrates that tryptamine does not play an essential role in expression levels using RNA silencing are extremely well estab- PLANT BIOLOGY growth or development in C. roseus root culture. Silencing the lished for plants and these lines can be constructed on a relatively biosynthesis of an early starting substrate enhances our ability to short time scale (Ϸ6–9 months) (17–19). harness the rich diversity of plant based natural products. Here, we successfully demonstrate RNA silencing of trypto- phan decarboxylase to suppress the biosynthesis of tryptamine in metabolic engineering ͉ natural products C. roseus culture. Alkaloid production almost completely disap- peared in silenced cultures and could be rescued by the addition enetic manipulation of natural product biosynthetic path- of exogenous tryptamine to the culture medium. A representa- Gways has emerged as a powerful strategy to rapidly access tive unnatural tryptamine analog, 5-fluorotryptamine 1a, was complex novel molecules (1). In one approach, the producer also incorporated into the pathway to yield several unnatural, organism is supplemented with analogs of the naturally occur- fluorinated alkaloids. Disruption of this early biosynthetic gene ring starting materials where, ideally, these nonnatural starting did not impact the expression of known downstream alkaloid materials are converted by the enzymes of the biosynthetic biosynthetic enzymes, indicating that changes can be made in the pathway into the corresponding unnatural products (2). The early part of this pathway without adversely affecting the alkaloid yields and purity of these unnatural products is typically im- biosynthetic machinery. Moreover, these experiments demon- proved if the biosynthesis of the natural starting material is strate that tryptamine and monoterpene indole alkaloids do not play an essential role in growth or development in C. roseus hairy genetically blocked, and the producing organism is forced to use root culture. exogenously supplied precursors for product biosynthesis. This strategy, termed mutasynthesis, was first applied several decades Results ago to yield novel antibiotics in the soil bacterium Streptomyces Alkaloid Production in Tryptophan Decarboxylase Silenced Lines. fradiae (3). Although mutasynthesis has proven to be highly Tryptophan decarboxylase, a pyridoxal phosphate dependent successful in microbial systems (4, 5), the strategy has not been enzyme that produces tryptamine 1 from tryptophan 2 (8, 9), was applied in more complex eukaryotes, notably plants. Here, we describe mutasynthesis of the monoterpene indole alkaloids in the medicinal plant Madagascar periwinkle (Catharanthus Author contributions: W.R. and J.J.M. designed research; W.R. and J.J.M. performed roseus). research; W.R. and S.E.O. analyzed data; and W.R. and S.E.O. wrote the paper. Tryptamine 1 is the starting substrate for hundreds of mono- The authors declare no conflict of interest. terpene indole alkaloids produced by C. roseus (Fig. 1) (6, 7). This article is a PNAS Direct Submission. This substrate is produced enzymatically from tryptophan 2 by 1Present address: Department of Chemistry, DePaul University, 1110 W. Belden Avenue, tryptophan decarboxylase, an enzyme that serves as a key link Chicago, IL 60614. between primary metabolism and natural product metabolism 2To whom correspondence should be addressed. E-mail: [email protected]. (8, 9). If tryptamine biosynthesis was blocked, alkaloid biosyn- This article contains supporting information online at www.pnas.org/cgi/content/full/ thesis could be rescued by introducing exogenous tryptamine or 0903393106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903393106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 COOH Tryptophan Decarboxylase NH2 NH2 N N H H L-tryptophan 2 RNA silencing tryptamine 1 exogenously added endogenously produced F CHO NH2 O- -Glc N H O 1a MeO2C 7 R R N H N N H O N H H H CO2Me 3 R = H N MeO2C H 5 R = H 3a R = F R 5a R = F R N H N O H CO Me N 6 R = H 2 H H 4 R = H 6a R = F 4a R = F MeO2C Fig. 1. Tryptamine 1 is produced by enzymatic decarboxylation of tryptophan 2. Trypamine 1 reacts with secologanin 7 to yield a variety of monoterpene indole alkaloids. Ajmalicine 3, serpentine 4, catharanthine 5, and tabersonine 6 are produced in Catharanthus roseus hairy root culture. targeted for gene silencing (19–22) (Fig. 1). The plasmid de- sis, suggesting that suppression of a single metabolic enzyme can signed to suppress tryptophan decarboxylase (pTDCi) was trans- sometimes affect other pathway enzymes (28–30). Finally, the formed into Agrobacterium rhizogenes, which was then used to gene encoding the alpha subunit of anthranilate synthase (31), infect C. roseus seedlings to generate hairy root culture as an enzyme involved in tryptophan 2 biosynthesis, showed modest described in refs. 23 and 24. Hairy root lines harboring the decreases in expression levels in the silenced lines (Fig. 3). pTDCi silencing plasmid were cultured in liquid media, and Increased concentrations of tryptophan inhibit anthranilate alkaloids were extracted from the plant tissue according to synthase expression (32). Therefore, in the silenced lines, where standard protocols. Gratifyingly, production of all major tryptophan is no longer diverted to tryptamine biosynthesis, tryptamine derived alkaloids ajmalicine 3, serpentine 4, catha- concentrations of tryptophan could increase, resulting in the ranthine 5 and tabersonine 6 was substantially decreased in the decreased expression of anthranilate synthase. A full transcrip- five silenced lines that were examined (Fig. 2A and SI). Sup- tional analysis of the monoterpene indole alkaloid pathway was pression of alkaloid production appears to be stable, with little not performed because the majority of the pathway enzymes are change in alkaloid production levels after seven subcultures not known at the genetic level. performed thus far. Unnatural Alkaloid Production in Suppressed Lines. Alkaloid biosyn- Gene Expression in Silenced Lines. Real time reverse transcription
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