Engineered Production of Strictosidine and Analogues in Yeast

Abstract

Monoterpene indole alkaloids (MIAs) are an expansive class of plant natural products, many of which have been named on the World Health Organization's List of Essential Medicines. Low production from native plant hosts necessitates a more reliable source of these drugs to meet global demand. Here, we report the development of a yeast-based platform for high-titer production of the universal MIA precursor, strictosidine. Our fed-batch platform produces ∼50 mg/L strictosidine, starting from the commodity chemicals geraniol and tryptamine. The microbially produced strictosidine was purified to homogeneity and characterized by NMR. Additionally, our approach enables the production of halogenated strictosidine analogues through the feeding of modified tryptamines. The MIA platform strain enables rapid access to strictosidine for reconstitution and production of downstream MIA natural products.

Keywords: metabolic engineering; monoterpene indole alkaloids; strictosidine; synthetic biology; yeast.

Figure 1.

Biosynthetic pathway of strictosidine. GES: geraniol synthase; G8H: geraniol 8-hydroxylase; GOR: 8-hydroxygeraniol oxidoreductase; ISY: iridoid cyclase; MLPL: major latex protein-like; IO: iridoid oxidase; 7DLGT: 7-deoxyloganetic acid transferase; 7DLH: 7-deoxyloganic acid hydroxylase; LAMT: loganic acid O-methyltransferase; SLS: secologanin synthase; STR: strictosidine synthase. CPR: cytochrome P450 Reductase; CYB5 cytochrome b5; CYPADH: cytochrome P450 alcohol dehydrogenase; UDP-Glc; uridine diphosphate glucose; SAM: S-adenosylmethionine.

Figure 2.

Optimizing expression of pathway accessory enzymes. (A) 7-deoxyloganic acid production titers between strains expressing plasmids harboring different combinations of accessory enzymes; (B) Extracted ion chromatograms (EIC) of pathway intermediates of their characteristic m/z signals from LC/MS and their structures. The retention times match to standards; (C) Production titers of 7-deoxyloganic acid, loganic acid and loganin in yJM009 and yJM010 cotransformed with pJB152 and pJB040. Bars indicate the mean of biological triplicates with the error bars representing the standard error.

Figure 3.

Comparison of strictosidine platforms. (A) Strictosidine titers of platform strains starting from nepetalactol; (B) Strictosidine titers from varied copy numbers of plasmids expressing pathway P450s. HC: high-copy vector, LC: low-copy vector; (C) Strictosidine titers of platform strains starting from geraniol; (D) Growth curves of untransformed, plasmid-less strains compared against the wild-type; (E) Growth curves of transformed strictosidine production strains compared against the wild-type. Bars indicate the mean of biological triplicates with the error bars representing the standard error.

Figure 4.

NMR spectra of purified (−)-strictosidine from yeast. (A) ¹H NMR at 500 MHz in in methanol-d₄; (B) ¹³C NMR at 125 MHz in methanol-d₄. Strictosidine is purified in the salt form as a result of acidic chromatographic conditions. For detailed assignment of peaks see Table S2 and Figures S5–S7.

Figure 5.

Production of halogenated strictosidine derivatives. (A) Structures of tryptamines successfully incorporated into strictosidine in vivo; (B) Extracted ion chromatogram (EIC) of characteristic m/z [M+H]⁺ signal for different strictosidine analogs from LC/MS analysis; (C) Tandem mass spectrometry (MS/MS) fragmentation patterns from QTOF-LC/MS for strictosidine (black), 7-fluorostrictosidine (red), and 7-chlorostrictosidine (blue) and corresponding predominant product ion structures.