Silencing of tryptamine biosynthesis for production of nonnatural alkaloids in plant culture

Abstract

Natural products have long served as both a source and inspiration for pharmaceuticals. Modifying the structure of a natural product often improves the biological activity of the compound. Metabolic engineering strategies to ferment "unnatural" products have been enormously successful in microbial organisms. However, despite the importance of plant derived natural products, metabolic engineering strategies to yield unnatural products from complex, lengthy plant pathways have not been widely explored. Here, we show that RNA mediated suppression of tryptamine biosynthesis in Catharanthus roseus hairy root culture eliminates all production of monoterpene indole alkaloids, a class of natural products derived from two starting substrates, tryptamine and secologanin. To exploit this chemically silent background, we introduced an unnatural tryptamine analog to the production media and demonstrated that the silenced plant culture could produce a variety of novel products derived from this unnatural starting substrate. The novel alkaloids were not contaminated by the presence of the natural alkaloids normally present in C. roseus. Suppression of tryptamine biosynthesis therefore did not appear to adversely affect expression of downstream biosynthetic enzymes. Targeted suppression of substrate biosynthesis therefore appears to be a viable strategy for programming a plant alkaloid pathway to more effectively produce desirable unnatural products. Moreover, although tryptamine is widely found among plants, this silenced line demonstrates that tryptamine does not play an essential role in growth or development in C. roseus root culture. Silencing the biosynthesis of an early starting substrate enhances our ability to harness the rich diversity of plant based natural products.

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.

Fig. 2.

Metabolite production in wild-type and tryptophan decarboxylase suppressed lines as evidenced by LC-MS analysis of C. roseus extracts. (A) Alkaloid production monitored in the total ion count of a wild-type and a representative silenced line. (B) Secologanin 7 (m/z 389) production in wild-type and a representative silenced line. An authentic standard of secologanin is also shown.

Fig. 3.

Real time reverse transcriptase PCR of the mRNA encoding tryptophan decarboxylase, anthranilate synthase, strictosidine synthase, strictosidine glucosidase and Orca3. Levels of these genes are normalized to 100% in the wild-type line. The gene expression levels of two representative silenced lines relative to the wild-type levels are also shown. Levels represent the average of three trials, with error bars representing the standard deviation.

Fig. 4.

Levels of ajmalicine 3, serpentine 4, catharanthine 5, and tabersonine 6 production in a representative silenced line supplemented with tryptamine 1 (0, 50, 125, 250, 500, 1,000, 2,500 μM) in the culture media. Levels were estimated in C. roseus extracts by LC-MS.

Fig. 5.

Extracted LC-MS chromatograms showing the presence of fluorinated ajmalicine 3a, serpentine 4a, catharanthine 5a, and tabersonine 6a. These compounds are only present when 5-flourotryptamine 1a is added to the culture media. Results using a concentration of 1.0 mM 5-fluorotryptamine are shown. Authentic standards of fluorinated ajmalicine are serpentine are also shown.

Fig. 6.

Quantification of unnatural alkaloid production in a suppressed and wild-type line fed with 5-fluorotryptamine 1a (250 μM, 500 μM, 1,000 μM and 2,500 μM). Production levels were evaluated in C. roseus extracts by mass spectrometry and represent the average of three measurements. (A) Levels of fluorinated alkaloids produced in a representative silenced line incubated with 5-fluorotryptamine 1a. (B) Levels of fluorinated alkaloids produced in a wild-type line incubated with 5-fluorotryptamine 1a. (C) Levels of natural, nonfluorinated alkaloids produced in a wild-type line incubated with 5-fluorotryptamine 1a.