Integrating carbon-halogen bond formation into medicinal plant metabolism

Abstract

Halogenation, which was once considered a rare occurrence in nature, has now been observed in many natural product biosynthetic pathways. However, only a small fraction of halogenated compounds have been isolated from terrestrial plants. Given the impact that halogenation can have on the biological activity of natural products, we reasoned that the introduction of halides into medicinal plant metabolism would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacological properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metabolism to yield chlorinated alkaloids. A new functional group-a halide-is thereby introduced into the complex metabolism of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore seem to be a viable platform for synthetic biology efforts.

Figure 1

Monoterpene indole alkaloid biosynthesis. A. Tryptophan 1 is decarboxylated by tryptophan decarboxylase to yield tryptamine 2, which reacts with secologanin 3 to form strictosidine 4. After numerous rearrangements, strictosidine 4 is converted into a variety of monoterpene indole alkaloids, such as 19,20-dihydroakuammicine 5, ajmalicine 6, tabersonine 7 and catharanthine 8. These compounds display a variety of pharmacological activities–, . B. RebH and PyrH, along with a partner reductase, halogenate the indole ring of tryptophan 1 to yield chloro-tryptophan. Here we show that after transformation of these enzymes into C. roseus, halogenated tryptophan 1a and 1b can be decarboxylated by tryptophan decarboxylase (C. roseus) to form chlorotryptamine 2a and 2b, and then converted into chlorinated monoterpene indole alkaloids.

Figure 2

Chlorinated alkaloids in C. roseus hairy root culture. A. Liquid chromatography-mass spectrometry (LC-MS) chromatograms showing 12-chloro-19,20-dihydroakuammicine 5a (m/z 359) in RebF/H hairy roots (red trace), contrasted with control cultures transformed with no plasmid (pink trace). An authentic standard of 5a validated the structural assignment (black trace, Supplemetary Fig. 19, 20). B. Chromatograms showing 10-chloroajmalicine 6b in RebF/PyrH/STRvm hairy roots (purple trace), contrasted with control cultures (pink trace). An authentic standard of 6b is shown (black trace). C. Chromatograms showing 15-chlorotabersonine 7b in RebF/PyrH/STRvm hairy roots (purple trace) contrasted with control cultures (pink trace). An authentic standard of 7b is shown (black trace). The other major peak at m/z 371 had an exact mass and UV spectra consistent with a chlorinated catharanthine 8 analog (Supplementary Fig. 18). D. ¹H NMR and ¹H-¹³C HSQC spectra of 5a and 5c.

Figure 3

Extracted LC-MS chromatograms showing the presence of 12-bromo-19,20-dihydroakuammicine 5c (m/z 403) in RebF/H hairy roots. Hairy roots are grown in media supplemented with KBr (0 – 20 mM final concentration) for two weeks prior to alkaloid extractions. 12-bromo-19,20-dihydroakuammicine 5c is not observed in control cultures transformed with no plasmid after incubation in potassium bromide supplemented media. An authentic standard of 12-bromo-19,20-dihydroakuammicine 5c is used to validate the structural assignment (Supplementary Fig. 18).