De novo production of the plant-derived alkaloid strictosidine in yeast

Abstract

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.

Keywords: Catharanthus roseus; Saccharomyces cerevisiae; monoterpene indole alkaloid; secologanin; strictosidine.

Fig. 1.

Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.

Fig. 2.

The reconstituted S. cerevisiae strictosidine biosynthetic pathway. C. roseus genes (orange) were integrated into the S. cerevisiae genome to allow for strictosidine production. To increase flux through the pathway, second copies of native S. cerevisiae genes (gray) were integrated into the genome. The gray line indicates MAF1 is a repressor of tRNA biosynthesis. The gray X represents genes that were deleted to reduce the consumption of pathway intermediates involved in other biosynthetic pathways. ERG20 was deleted and replaced by AgGPPS2 (A. grandis), which exclusively produces geranyl pyrophosphate, and mFPS144 (G. gallus), which favors the production of geranyl pyrophosphate over farnesyl pyrophosphate, an essential S. cerevisiae off-pathway compound. The arrows with question marks are catalyzed by IO, but may also require helper enzymes. National Center for Biotechnology Information accession numbers for all integrated genes are located in Table S1. Abbreviations: AgGPPS2, Abies grandis geranyl pyrophosphate synthase; CPR, cytochrome P450 reductase; CYB5, cytochrome b₅; 7-DLGT, 7-deoxyloganetic acid glucosyl transferase; 7-DLH, 7-deoxyloganic acid hydroxylase; DMAPP, dimethylallyl pyrophosphate; G8H, geraniol 8-hydroxylase; GES, geraniol synthase; GOR, 8-hydroxygeraniol oxidoreductase; IDI1, isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase; IO, 7-deoxyloganetic acid synthase/iridoid oxidase; IPP, isopentenyl pyrophosphate; ISY, iridoid synthase; LAMT, loganic acid O-methyltransferase; mFPS144, Gallus gallus mutant farnesyl pyrophosphate synthase N144W; SAM2, S-adenosylmethionine synthetase; SLS, secologanin synthase; STR, strictosidine synthase; TDC, tryptophan decarboxylase; tHMGR, truncated 3-hydroxy-3-methyl-glutaryl-CoA reductase; ZWF1, glucose-6-phosphate dehydrogenase.

Fig. 3.

S. cerevisiae produces strictosidine de novo. (A) LC-MS chromatograms of extracellular metabolites (multiple reaction monitoring for strictosidine/vincoside only) of strain 0 grown with strictosidine pathway intermediates. The bottom trace shows the retention times of authentic standards of strictosidine and its diastereomer vincoside. (B) LC-MS chromatogram for strictosidine/vincoside produced extracellularly by strain 0 expressing codon-optimized G8H on a plasmid. The observed peak coelutes with a strictosidine authentic standard (bottom trace). (C) MS-MS chromatogram and exact mass data of the strictosidine peak produced in strain 1 containing a high-copy number plasmid expressing codon-optimized G8H.

Fig. 4.

CYPADH increases strictosidine production. (A) The proposed three-step mechanism to convert nepetalactol to 7-deoxyloganetic acid (Top) compared with the similar three step mechanism to convert amorphadiene to artemisinic acid. (B) Amount of strictosidine produced in strain 0 containing plasmids encoding ALDH1, ADH1, or ADH2 relative to strain 0 harboring empty plasmid when incubated with the indicated pathway intermediates. Data are mean ± SD of three independent experiments.