Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast

Abstract

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

Fig. 1. De novo alstonine and serpentine production in yeast.

a, Integration of plant biosynthetic pathways with native yeast metabolic pathways to produce alstonine and serpentine. IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPPS, GPP synthase; FPS^N144W, FPP synthase N144W variant; CPR, NADPH-cytochrome P450 reductase; CYB5, cytochrome b5; GES, geraniol synthase; G8H, geraniol 8-hydroxylase; 8HGO, 8-hydroxygeraniol oxidoreductase; ISY, iridoid synthase; IO, iridoid oxidase; CYPADH, alcohol dehydrogenase 2; 7DLGT, 7-deoxyloganetic acid glucosyl transferase; 7DLH, 7-deoxyloganic acid hydroxylase; LAMT, loganic acid O-methyltransferase; TDC, tryptophan decarboxylase; SLS, secologanin synthase; STR, strictosidine synthase. b, Screen of AS candidates in YPD cultivation medium. Gene candidates are linked to strain identifiers as follows: RteAS1 (Sc87), RteAS2 (Sc88), RteAS3 (Sc90), RteAS4 (Sc92), RteAS5 (Sc94), CroAS_nat (Sc96), RseSBE_nat (Sc97), GseSBE_nat (Sc98), CroAS2_nat (Sc100), CroAS2 (Sc101), CroAS (Sc102), RseSBE (Sc103), GseSBE (Sc104) and CroSS_nat (Sc157) and a negative-control strain (Sc86). c,d, Representative production profiles for alstonine (c), serpentine (d) and pathway intermediates using a small-scale fed-batch process for strains Sc112 and Sc85, respectively, cultivated in 1 ml of 3× SC medium supplemented with 3 mM tryptophan. For b, n = 3, and error bars represent 1 s.d. from the mean, with data points overlaid as black dots. Source data

Fig. 2. Bioactivity testing of alstonine in yeast and mammalian cells.

a, Dose–response curves of ADRA2A with the agonist epinephrine (left; R² = 0.988) and antagonist activities of yohimbine and alstonine (right; R² = 0.965 and 0.980, respectively). Data are reported in relative luminescence units (RLU) based on nano-luciferase (NanoLuc) readouts normalized to the maximum observed luminescence. Data report triplicate biological replicates (n = 3) measured on yeast cells heterologously expressing the ADRA2A receptor; [epinephrine], concentration of epinephrine; [antagonist], concentration of antagonist; [serotonin], concentration of serotonin; [alstonine], concentration of alstonine. b, Dose–response curves of 5-HT_2C with the agonist serotonin (left; R² = 0.892) and alstonine with and without 0.5 µM competing serotonin (right; R² = 0.792 and 0.913). cAMP levels were monitored using BRET in COS7 cells, and data are reported for biological duplicates (n = 2). For all data presented, each replicate is shown, and a non-linear regression model was applied using the least squares method. The dashed lines in a and b (left) indicate 50 µM and 0.5 µM, respectively, which were the agonist concentrations used in the respective competitor assays (right). Source data

Fig. 3. Biosynthesis of halogenated alstonine derivatives in engineered yeast.

a, Representative liquid chromatography–MS/MS (LC–MS/MS) traces of the chemical standard alstonine and the peaks corresponding to halogenated alstonines after feeding 0.25 mM secologanin and 100 mg liter^–1 corresponding haloindole derivatives with a single halogen atom at C4 (green), C5 (red), C6 (gold) or C7 (blue). b, Representative MS/MS spectra of alstonine standard and fluoroalstonine, chloroalstonine and bromoalstonine. c, Peak assignments for alstonine MS/MS spectra shown in b. d, Progress of the given substitution through the MIA pathway is indicated by the corresponding colored lines. The presence of the colored box indicates direct detection of the halogenated compound after haloindole derivative feeding to cells. Abbreviated enzyme names are stated above the catalyzed reaction. Source data

Fig. 4. Secologanin is the global limiting substrate and TDC is the limiting enzyme for new-to-nature MIA production in engineered yeast.

a, Schematic of the natural and derivative MIA pathways competing for the secologanin pool. Bottleneck reactions catalyzed by CroSLS and CroSTR are highlighted in red. b, Secologanin levels in broth following 144 h of cultivation of Sc154 supplemented with 100 mg liter^–1 fluoroindole with and without 0.25 mM secologanin. c, Fluoroalstonine levels in broth following 144 h of cultivation of Sc154 supplemented with 100 mg liter^–1 fluoroindole with and without 0.25 mM secologanin. For b and c, data represent mean values plotted with standard deviation, data points are overlaid as black dots, and statistical significance was calculated using two-tailed Student’s t-tests; ***P < 0.001; NS, not significant. Data are shown as means (n = 3). Source data

Fig. 5. De novo synthesis of chloroalstonine in yeast.

a, Schematic outline of the de novo chloroalstonine biosynthetic pathway in yeast. b, Peak areas for chlorinated MIA masses for halogenase-expressing strains and strain Sc161 (control). Data are shown as mean ± s.d., and data points are overlaid as black dots; n = 3. Significance was calculated by using a two-tailed Student’s t-test; *P < 0.05. c, Detection of LaeRebH aggregation with the 4×UAS-SSA1p–365:mKate2 aggregation biosensor. Green fluorescent protein (GFP; left) and yellow fluorescent protein m4 (YFPm4; middle) strains were used as controls for the expression of soluble and aggregation-prone proteins, respectively. Histograms show reporter signals from uninduced (2% glucose) and induced (2% galactose) expression of the candidate proteins. Histograms show all data from n = 4 × 4,000 events. Geometric means are reported in the top left. d, MS chromatogram traces for alstonine standard and chloroalstonine from ScH125 broth cultured at 25 °C. e, Comparison of the MS/MS spectra of an alstonine standard and the chloroalstonine peak in d shows a similar fragmentation pattern and a mass shift consistent with a hydrogen-to-chlorine substitution. Source data

Extended Data Fig. 1. De novo alstonine and serpentine production in yeast.

a. Representative chromatogram from LC-MS analysis of spent medium from yeast (MIA-CM-3) expressing CroTHAS1+RseSGD and CroHYS+RseSGD when cultivated in synthetic complete (SC) medium supplemented with secologanin and tryptamine. Controls include tetrahydroalstonine and ajmalicine standards, as well as a negative control (MIA-CM-3). b. De novo production of alstonine produced by CroTHAS1+RteAS2 (Sc77), CroTHAS1+GseSBE_nat (Sc78), and CroTHAS1+CroSS_nat (Sc112), and de novo production of serpentine by yeast strain expressing CroHYS+CroSS_nat (Sc85), when cultivated in 3 x SC medium supplemented with 3 mM tryptophan. The strains are based on MIA-CM-3, with a genome integrated copy of RseSGD. c, d. Carbon feedstock (c) and alstonine (d) levels during the 20 h glucose batch followed by a pulsed-ethanol fed-batch process of de novo alstonine-producing strain ScH144 in 2 ltr bioreactor. For b, n = 3 and error bars represent one standard deviation from the mean. Source data

Extended Data Fig. 2. Alstonine titers in yeast strains expressing individual alstonine synthase variants when cultivated in deep-well plates.

Titers of alstonine (μg/L) after 144 h cultivations of 6 biological replicates of strains Sc138-153 with 1 mM tryptamine and 0.2 mM secologanin in SC media, + 1 mM Trp. Gene candidates are linked to strain identifiers as follows: RteAS1 (Sc87), RteAS2 (Sc88), RteAS3 (Sc90), RteAS4 (Sc92), RteAS5 (Sc94), CroAS_nat (Sc96), RseSBE_nat (Sc97), GseSBE_nat (Sc98), CroAS2_nat (Sc100), CroAS2 (Sc101), CroAS (Sc102), RseSBE (Sc103), GseSBE (Sc104), CroSS_nat (Sc157) as well as negative control strain (Sc86). The background strain for this screen was MIA-B0, with an integrated pathway from tryptamine and secologanin to THA, allowing for feeding of these close precursors. Mean, n = 6, with error bars indicating SD. Source data

Extended Data Fig. 3. Alstonine production of strain Sc112 in 2 ltr bioreactor.

Titers of alstonine (mg/L) during 144 h cultivation in 2 ltr bioreactor with 60 g/ltr glucose feeding. Single measurements. Source data

Extended Data Fig. 4. Chromatograms from alstonine purification.

a. Chromatogram of alstonine purification on DAC LC50 system. Alstonine compound is eluted with a retention time of about 40 min after using washing solvent. b. Chromatogram of alstonine purification on a semi-preparative system. Alstonine compound is eluted with a retention time of 332 min after using washing solvent. c. Chromatogram of purified alstonine at 250 nm. d. Maxplot chromatogram of purified alstonine.

Extended Data Fig. 5. NMR spectra for alstonine produced in yeast.

a. ¹H NMR with water suppression full range in MeOH-d₃ alstonine standard (red), and SPE purified alstonine from alstonine-producing yeast (blue). b. ¹H NMR with water suppression aromatic range in MeOH-d₃ alstonine standard (red), and SPE purified alstonine from enzymatic experiment. c. ¹H NMR with water suppression aliphatic range in MeOH-d₃ alstonine standard (red), and SPE purified alstonine from enzymatic experiment. Gray bars indicate impurities.

Extended Data Fig. 6. Comparison of alstonine and serpentine standards and producers.

a–c, LCMS analysis of Profile of MS/MS transitions for alstonine standard (a), broth of alstonine producer Sc77 (b) and serpentine standard (c). Transitions characteristic for alstonine and serpentine: 262.9 (gray), 316.9 (blue), and 234.9 (green). d–g, HRMS analysis of alstonine standard (d), broth of alstonine producer Sc112 (e), serpentine standard (f), and broth of serpentine producer Sc85 (g). Source data

Extended Data Fig. 7. Biosynthesis of halo-strictosidine by MIA-CM3 and MIA-CM10.

MIA-CM3 and MIA-CM10 were cultivated (see Methods) with 100 mg/L haloindole and 0.25 mM secologanin and the broth at 144 hours analyzed using HRMS. a. Representative chromatograms for MIA standards and fluorinated, chlorinated, brominated and difluorinated derivatives after feeding the corresponding haloindole. b. Representative MS-MS spectra extracted from the corresponding peaks in panel a. c,d, Indole to strictosidine pathways expressed in MIA-CM3 (c) and MIA-CM10 (d) with the detection of halogenated derivatives overlaid. The presence of a colored or gray box indicates identification of the derivative with a halo-substitution(s) at the indicated carbon (indole numbering). Enzyme abbreviation: TRP5 - tryptophan synthase 5, TDC - tryptophan synthase, STR - strictosidine synthase. Source data

Extended Data Fig. 8. Isotopic profiles of chlorinated and brominated alstonine and serpentine.

(Top and Middle panels) Measured isotopic profiles of chlorinated (left) and brominated (right) serpentine and alstonine. (Bottom panels) Predicted isotopic profiles of chlorinated (left) and brominated (right) serpentine and alstonine.

Extended Data Fig. 9. Mean peak areas of halogenated MIA intermediates in Sc161 broth.

Sc161 was cultivated in triplicate for 144 h with 100 mg/L halo-indole and 0.25 mM secologanin and the broth analyzed with HRMS. Mean peak areas are plotted with standard deviation and data points overlaid as black dots. Legends indicate the position of halo-substitution(s) according to indole carbon numbering. Source data

Extended Data Fig. 10. Biosynthesis of halo-serpentines by ScH125.

ScH125 was cultivated with 100 mg/L halo-indole and 0.25 mM secologanin. Broth samples were taken at 144 hours and analyzed using HRMS. a. Representative chromatograms for serpentine standard and halo-serpentines after feeding the equivalent halo-indole. b. Representative MS-MS spectra extracted from the peaks in panel a. c. Serpentine and halo-serpentine peak assignment for MS/MS spectra in panel b. d. Indole to serpentine pathway expressed in ScH125 with the detection of halogenated derivatives overlaid. The presence of a colored or gray box indicates identification of the derivative with a halo-substitution(s) at the indicated carbon (indole numbering). Enzyme abbreviation: TRP5 - tryptophan synthase 5, TDC - tryptophan synthase, STR - strictosidine synthase, SGD - strictosidine deglycosylase, HYS - heteroyohimbine synthase, SS - serpentine synthase. Source data