Solution of the multistep pathway for assembly of corynanthean, strychnos, iboga, and aspidosperma monoterpenoid indole alkaloids from 19 E-geissoschizine

Abstract

Monoterpenoid indole alkaloids (MIAs) possess a diversity of alkaloid skeletons whose biosynthesis is poorly understood. A bioinformatic search of candidate genes, combined with their virus-induced gene silencing, targeted MIA profiling and in vitro/in vivo pathway reconstitution identified and functionally characterized six genes as well as a seventh enzyme reaction required for the conversion of 19E-geissoschizine to tabersonine and catharanthine. The involvement of pathway intermediates in the formation of four MIA skeletons is described, and the role of stemmadenine-O-acetylation in providing necessary reactive substrates for the formation of iboga and aspidosperma MIAs is described. The results enable the assembly of complex dimeric MIAs used in cancer chemotherapy and open the way to production of many other biologically active MIAs that are not easily available from nature.

Keywords: Catharanthus roseus monoterpeoind indole alkaloids; catharanthine assembly; multiple-pathway gene function; tabersonine assembly; virus-induced gene silencing.

Fig. 1.

VIGS silencing of GO, Redox1, Redox2, SAT, HL1, HL2, and HL3 results in accumulation of intermediates that reveal their involvement in the assembly of catharanthine and tabersonine. (A) Representative LC-chromatograms (280 nm) of leaf surface MIAs extracted in chloroform and mean relative expression levels by qRT-PCR of respective genes of VIGS plants silenced for geissoschizine oxidase (GO), Redox1, Redox2, and stemmadenine-O-acetyltransferase (SAT), compared with empty vector (EV) controls. (B) Representative LC-chromatograms (280 nm) of whole leaf MIAs extracted in ethyl acetate and mean relative expression levels by qRT-PCR of respective genes of VIGS plants silenced for Hydrolases 1–3 (HL1–HL3) compared with EV controls. (C–G) MIA contents in leaves of VIGS–GO, –Redox1, –Redox2, –SAT, and –HL1, -2, and -3 compared with the EV controls. Biological replicates = 5. Error bars represent the SD. Peak identification: 1, catharanthine; 2, vindoline; 3, ajmalicine; 4, 19E-geissoschizine; 5, 19E-geissoschizine methanol adducts that disappear when dissolved in acetonitrile; 6, 16R,19E-isositsirikine; 7, pericyclivine; 8, perivine; 9, short-lived MIA of m/z 369; 10, akuammicine; 11, 16S-deshydroxymethylstemmadenine (DHS); 12, 16R-DHS; and 13, stemmadenine that shows structural fluctuation (splitting peaks) when analyzed by LC-MS. See Fig. 3 for respective structures.

Fig. 2.

Functional expression and establishment of biochemical function of GO, Redox1, Redox2, SAT, HL1, and HL2. (A) MIA formation when using 19E-geissoschizine and various combinations of yeast microsomes containing GO and recombinant enzymes purified from E. coli. (B) Conversion of stemmadenine to O-acetylstemmadenine (OAS) using recombinant SAT purified from E. coli. (C) Formation of catharanthine or tabersonine using OAS, crude leaf proteins of C. roseus, and recombinant HL1/HL2 purified from E. coli. (D) Formation of catharanthine or tabersonine using OAS, crude leaf proteins of T. litoralis, and recombinant HL1/HL2 purified from E. coli. Each trace shows the selected ion chromatogram (SIC). Peak identification: 14, O-acetylstemmadenine that shows structural fluctuation (splitting peaks) when analyzed by LC-MS and 15, tabersonine. See Fig. 3 for respective structures.

Fig. 3.

The formation of catharanthine and tabersonine from 4,21 dehydrogeissoshizine involves GS, GO, Redox1, Redox2, SAT, an NADPH requiring reaction, HL1, and HL2. Pathway intermediates are involved in the elaboration of four major MIA skeletons: (i) corynanthean (geissoschizine and isositsirikine), (ii) strychnos (akuammicine, stemmadenine, OAS, and DHS), (iii) iboga (catharanthine), and (iv) aspidosperma (tabersonine). Pink arrows indicate the major biosynthetic route in C. roseus, and dashed arrows indicate hypothetical steps.