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Discovery and engineering of colchicine alkaloid biosynthesis

A Publisher Correction to this article was published on 30 July 2020

This article has been updated

Abstract

Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2,3,4,5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.

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Fig. 1: Summary of predicted colchicine biosynthesis.
Fig. 2: Candidate methyltransferase and cytochrome P450 transcripts identified in the public G. superba transcriptome by expression correlation analysis.
Fig. 3: Combined transcriptomics and metabolomics identify notable co-expression of colchicine biosynthetic genes in G. superba.
Fig. 4: Discovery of a pathway for colchicine alkaloid biosynthesis.
Fig. 5: Engineered biosynthesis of N-formyldemecolcine (10) from primary metabolism in N. benthamiana.

Data availability

All reported data in this study are available via database or by request from the corresponding author. Coding DNA sequences for the genes characterized and assessed in this study are provided as FASTA sequences in the Supplementary Information, and are deposited in the National Center for Biotechnology Information (NCBI) GenBank database with the following accessions: GsOMT1 (MT512039), GsNMT (MT512040), GsNMTt (MT512041), GsCYP75A109 (MT512042), GsOMT2 (MT512043), GsOMT3 (MT512044), GsCYP75A110 (MT512045), GsOMT4 (MT512046), GsCYP71FB1 (MT512047), GsTyDC/DDC (MT512048), GsPAL (MT512049), GsCCR (MT512050), GsAER (MT512051), Gs4CL (MT512052), GsC4H (MT512053), GsDAHPS (MT512054), Δ24-CjNCS (MT512055) and Δ29-CjNCS (MT512056). Raw reads from the RNA-seq profiling analysis of G. superba are deposited in the NCBI Sequence Read Archive (SRA) database under the BioProject accession PRJNA634925. The corresponding Transcriptome Shotgun Assembly (TSA) project has been deposited at DDBJ/EMBL/GenBank under accession GIOZ00000000. The version described in this paper is the first version, GIOZ01000000. Gene constructs described in this manuscript will be made available upon request from the corresponding author. Synthetic substrates and purified compounds will be made available upon request from the corresponding author, as possible. Source data are provided with this paper.

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Acknowledgements

We thank K. Smith for his assistance with chemical synthesis and acquisition/interpretation of NMR data; D. Nelson (University of Tennessee) for his assistance in the systematic naming of the cytochrome P450 enzymes characterized in this study, G. Lomonossoff (John Innes Centre) for providing us with the pEAQ-HT expression plasmid, A. Lloyd (The University of Texas at Austin) for sharing a clone of the BvCYP76AD5 coding sequence and T. Kutchan (Danforth Center) for providing us with authentic standards of 7 and 9. We acknowledge the Stanford Center for Genomics and Personalized Medicine (SCGPM) for RNA-seq services and the Stanford Genetics Bioinformatics Service Center for the use of computational resources supported by NIH S10 Instrumentation Grant S10OD023452. This work was supported by an NIH U01 GM110699, an R01 GM121527 and an HHMI-Simons Faculty Scholar award (to E.S.S.). R.S.N. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation.

Author information

Authors and Affiliations

Authors

Contributions

R.S.N., W.L. and E.S.S. conceived experiments. R.S.N. and W.L. analysed transcriptome data, expressed and characterized biosynthetic genes, established the metabolic engineering strategy and synthesized or isolated authentic chemical standards. W.L. performed the RNA-seq experiment and metabolite profiling of G. superba. R.S.N., W.L. and E.S.S. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Elizabeth S. Sattely.

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The authors declare no competing interests.

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Peer review information Nature thanks Russell Cox, Jing-Ke Weng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Characterization of GsOMT1.

a, LC–MS chromatograms demonstrating activity on substrate 1 by protein lysates from N. benthamiana leaves that transiently express GsOMT1. EICs for 1 (m/z 286.1438; left) and the methylated product (indicated by the asterisk) produced in this experiment (m/z 300.1594; right) are shown. This experiment was performed three times, with similar results each time. b, MS/MS fragmentation spectrum of 1, as well as the generated m/z 300.1594 product (*) at a collision energy of 20 V. Fragmentation of both compounds was performed twice, with similar results observed each time. c, Tabulated list and putative structures for ion fragments from the MS/MS analysis of 1. d, Tabulated list and putative structures for ion fragments from the MS/MS analysis of the m/z 300.1594 product. See Supplementary Information for a detailed analysis of MS/MS results. e, Proposed reaction catalysed by GsOMT1, as supported by MS/MS fragmentation and previously published labelling studies. f, Transient expression of GsOMT1 in N. benthamiana with co-infiltrated substrate 1 results in production of the methylated product 2, as shown by the LC–MS chromatograms. This experiment was performed more than three times with similar results observed each time. g, Untargeted metabolite analysis (XCMS) comparing transient expression of GFP (negative control) to that of GsOMT1 with co-infiltrated substrate 1 (n = 3 independent replicates for each experimental condition). The unique mass signatures (P < 0.1 between samples, as determined by XCMS) are shown in ranked order based on their increasing (top) or decreasing (bottom) fold change in abundance between the two conditions. The mass isotopologues (M0 and M1) for the presumed product (m/z 300.1594) are shown in red; the substrate (1, m/z 286.1438) is shown in blue. r.t., retention time.

Source data

Extended Data Fig. 2 Characterization of GsNMT.

a, Co-expression of GsOMT1 and GsNMT in N. benthamiana with co-infiltrated 1 leads to consumption of putative 2 (m/z 300.1594) and the production of a new compound that corresponds to a methylation (m/z 314.1751), as shown by the LC–MS chromatograms. Activity of full-length GsNMT was confirmed in three separate experiments. b, MS/MS fragmentation spectrum of the generated m/z 314.1751 product (*) at a collision energy of 20 V. This experiment was performed twice, with similar results observed each time. c, Tabulated list and putative structures for ion fragments from MS/MS analysis of the m/z 314.1751 product. See Supplementary Information for a detailed analysis of MS/MS results. d, Untargeted metabolite analysis (XCMS) comparing the presence and absence of GsNMT in the transient co-expression system (n = 6 independent replicates for each experimental condition). The unique mass signatures (P < 0.1 between samples, as determined by XCMS) are shown in ranked order based on their increasing (top) or decreasing (bottom) fold change in abundance between the two conditions. The mass isotopologues (M0 and M1) of the presumed product (m/z 314.1751) are shown in red; the mass isotopologues of the presumed substrate (m/z 300.1594) are shown in blue. e, Proposed reaction catalysed by GsNMT as supported by MS/MS fragmentation and previously published labelling studies.

Source data

Extended Data Fig. 3 Characterization of GsCYP75A109.

a, Addition of GsCYP75A109 to the N. benthamiana transient expression system with co-infiltrated 1 leads to the consumption of 3 (m/z 314.1751) and the production of a new compound that corresponds to a hydroxylation (m/z 330.1700), as shown by the LC–MS chromatograms. These results were confirmed in two independent experiments. b, MS/MS fragmentation spectrum of the generated m/z 330.1700 product (*) at a collision energy of 20 V. Consistent results were obtained in three separate experiments. c, Tabulated list and putative structures for ion fragments generated from MS/MS analysis of the m/z 330.1700 product. See Supplementary Information for a detailed analysis of MS/MS results. d, Untargeted metabolite analysis (XCMS) comparing the presence and absence of GsCYP75A109 in the transient co-expression system (n = 6 independent replicates for each experimental condition). The unique mass signatures (P < 0.1 between samples, as determined by XCMS) are shown in ranked order based on their increasing (top) or decreasing (bottom) fold change in abundance between the two conditions. The presumed product (m/z 330.1700) is shown in red; the mass isotopologues (M0, M1) of the presumed substrate (m/z 314.1751) are shown in blue. e, Proposed reaction catalysed by GsCYP75A109 as supported by MS/MS fragmentation and previously published labelling studies. f, An N-terminal truncation of a predicted mitochondrial localization signal from GsNMT (yielding GsNMTt) increases the yield of putative 4 (m/z 330.1700) in the transient co-expression system, as shown by the representative LC–MS chromatograms. g, Quantification of the heterologous production of 3 (m/z 314) or 4 (m/z 330) with the use of GsNMT or GsNMTt in the co-expression system. Filled-in boxes (grey) indicate the presence of a gene in the co-expression experiment; an empty box (white) indicates its absence. For each reaction, data are mean ± s.d. of 3 distinct biological replicates. Statistical comparisons were made using a one-tailed Student’s t-test, with an assumption of unequal variance. n.d., not detected. Direct comparison between the experimental conditions was performed twice with similar results obtained each time. Activity of GsNMTt in pathway engineering was consistent in more than three experiments.

Source data

Extended Data Fig. 4 Characterization of GsOMT2.

a, Addition of GsOMT2 into the N. benthamiana transient co-expression system with co-infiltrated 1 leads to consumption of putative 4 (m/z 330.1700) and the production of a new compound corresponding to both a methylation and a hydroxylation (m/z 360.1805), as shown by the LC–MS chromatograms. This activity was confirmed in more than three independent experiments. b, MS/MS fragmentation spectrum of the generated m/z 360.1805 product (*) at a collision energy of 20 V. MS/MS fragmentation of this peak was performed twice, with similar results each time. c, Tabulated list and putative structures for ion fragments from the MS/MS analysis of the m/z 360.1805 product. See Supplementary Information for a detailed analysis of MS/MS results. d, Untargeted metabolite analysis (XCMS) comparing the presence and absence of GsOMT2 in the transient co-expression system (n = 6 independent replicates for each experimental condition). The unique mass signatures (P < 0.1 between samples, as determined by XCMS) are shown in ranked order based on their increasing (top) or decreasing (bottom) fold change in abundance between the two conditions. The mass isotopologues (M0, M1, and M2) for the presumed product (m/z 360.1805) are shown in red; the mass isotopologues (M0, M1) of the presumed substrate (m/z 330.1700) are shown in blue. e, Proposed reaction catalysed by GsOMT2, and tentatively GsCYP75A109, as supported by MS/MS fragmentation and previously published labelling studies. Note that compound 5 is not observed in our co-expression system, presumably due to its consumption to 6.

Source data

Extended Data Fig. 5 Characterization of GsOMT3.

a, Addition of GsOMT3 into the N. benthamiana transient co-expression system with co-infiltrated 1 leads to consumption of 6 (m/z 360.1805) and the production of a new compound corresponding to a methylation (m/z 374.1962), as shown by the LC–MS chromatograms. The new peak was compared to a racemic standard of autumnaline (7) ((R,S)-autumnaline), which supports the identity of this new compound as 7. This experiment was repeated more than three times with similar results observed each time. b, MS/MS fragmentation spectrum of the generated m/z 374.1962 product (*) compared to that of racemic 7, each at a collision energy of 20 V. This experiment was performed three times, with similar results observed each time. c, Tabulated list and putative structures for ion fragments generated from MS/MS analysis of the m/z 374.1962 product. See Supplementary Information for a detailed analysis of MS/MS results. d, Untargeted metabolite analysis (XCMS) comparing the presence and absence of GsOMT3 in the transient co-expression system (n = 6 independent replicates for each experimental condition). The unique mass signatures (P < 0.1 between samples, as determined by XCMS) are shown in ranked order based on their increasing (top) or decreasing (bottom) fold change in abundance between the two conditions. The mass isotopologues (M0, M1) for the product (m/z 374.1962) are shown in red; the mass isotopologues (M0, M1, and M2) of the presumed substrate (m/z 360.1805) are shown in blue. e, Proposed reaction catalysed by GsOMT3, as supported by MS/MS fragmentation, previously published labelling studies and comparison to a 7 standard.

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Extended Data Fig. 6 Characterization of GsCYP75A110.

a, Addition of GsCYP75A110 into the N. benthamiana transient co-expression system with co-infiltrated 1 leads to consumption of 7 (m/z 374.1962) and the production of three new compounds that each correspond to a loss of two hydrogens (m/z 372.1805), as shown by the LC–MS chromatograms. This experiment was performed more than three times with similar results observed each time. b, MS/MS fragmentation spectrum of the generated m/z 372.1805 product (*) at a collision energy of 20 V. This spectrum is shown because it represents the only peak consumed in downstream biosynthesis (see Extended Data Fig. 7). MS/MS fragmentation of this peak was performed twice, with similar results observed each time. c, Tabulated list and putative structures for ion fragments from the MS/MS analysis of the m/z 372.1805 product. d, Untargeted metabolite analysis (XCMS) comparing the presence and absence of GsCYP75A110 in the transient co-expression system (n = 6 independent replicates for each experimental condition). The unique mass signatures (P < 0.1 between samples, as determined by XCMS) are shown in ranked order based on their increasing (top) or decreasing (bottom) fold change in abundance between the two conditions. The mass signatures for two of the presumed products (m/z 372.1805) are shown in red; the mass signature of the presumed substrate (m/z 374.1962) is shown in blue. e, Expression of GsCYP75A110 individually with substrate (7) co-infiltration, as shown by the LC–MS chromatograms of the substrate (7, m/z 374.1962) and products (m/z 372.1805). The products produced by pathway reconstitution in N. benthamiana are shown for comparison. This experiment was performed once. f, In vitro assays using microsomal protein isolated from yeast expressing GsCYP75A110. LC–MS chromatograms of substrate (7) and products (m/z 372.1805) are shown for comparison to the products produced in the N. benthamiana transient expression system. Peak integrations for the substrate (7) are shown in blue text to demonstrate consumption of the substrate in the presence of GsCYP75A110-containing microsomal protein and NADPH. This experiment was performed once. g, Predicted, alternative phenol coupling isomers may explain the three isomeric peaks detected with m/z 372.1805. h, Proposed reaction catalysed by GsCYP75A110, as supported by MS/MS fragmentation and previously published labelling studies.

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Extended Data Fig. 7 Characterization of GsOMT4.

a, Addition of GsOMT4 into the N. benthamiana transient co-expression system with co-infiltrated 1 leads to consumption of 8 (m/z 372.1805) and the production of a new compound corresponding to a methylation (m/z 386.1962), as shown by the LC–MS chromatograms. Comparison to an O-methylandrocymbine (9) standard purified from C. autumnale plants supports the identity of this compound as 9. This result was confirmed in more than three independent experiments. b, MS/MS fragmentation spectrum of the generated m/z 386.1962 product (*) compared to the purified 9 standard, with both compounds fragmented at a collision energy of 20 V. This was performed twice, with similar results observed each time. c, Tabulated list and putative structures for ion fragments from the MS/MS analysis of the m/z 386.1962 product. d, Untargeted metabolite analysis (XCMS) comparing the presence and absence of GsOMT4 in the transient co-expression system (n = 6 independent replicates for each experimental condition). The unique mass signatures (P < 0.1 between samples, as determined by XCMS) are shown in ranked order based on their increasing (top) or decreasing (bottom) fold change in abundance between the two conditions. The mass signature for the product (9, m/z 386.1962) is shown in red; the mass signature of the presumed substrate (m/z 372.1805) is shown in blue. e, Proposed reaction catalysed by GsOMT4, as supported by MS/MS fragmentation, previously published labelling studies and comparison to an isolated 9 standard.

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Extended Data Fig. 8 Characterization of GsCYP71FB1.

a, Addition of GsCYP71FB1 into the N. benthamiana transient co-expression system with co-infiltrated 1 leads to consumption of 9 (m/z 386.1962) and the production of a new compound with identified masses of m/z 400.1755 [M + H+] and 422.1574 [M + Na]+ as shown by the LC–MS chromatograms. Comparison to an authentic N-formyldemecolcine (10) standard supports formation of this compound. This experiment was performed twice, with similar results observed each time. b, MS/MS fragmentation spectrum of the generated m/z 400.1755 product (*) compared to the 10 standard, with both compounds fragmented at a collision energy of 20 V. This was performed three times, with similar results each time. c, Transient expression of GsCYP71FB1 individually in N. benthamiana with substrate (9) co-infiltration, as shown by the LC–MS chromatograms of the substrate (9, m/z 386.1962) and product (10, m/z 400.1755) in comparison to a 10 standard. This experiment was performed once. d, In vitro assays using microsomal protein isolated from yeast expressing GsCYP71FB1. The LC–MS chromatograms of the substrate (9) and product (10) in comparison to the 10 standard are shown. Peak integrations for the substrate (9) are shown in blue text to demonstrate its consumption in the presence of GsCYP71FB1-containing microsomal protein and NADPH. This experiment was performed once. e, Untargeted metabolite analysis (XCMS) comparing the presence and absence of GsCYP71FB1 in the transient co-expression system (n = 6 independent replicates for each experimental condition). The unique mass signatures (P < 0.1 between samples, as determined by XCMS) are shown in ranked order based on their increasing (top) or decreasing (bottom) fold change in abundance between the two conditions. The mass isotopologues (M0, M1) as well as adducts (+Na, +K) for the product (10, m/z 400.1755) are shown in red; the mass isotopologues (M0, M1) and adducts (2M+Na) of the substrate (9, m/z 386.1962) are shown in blue. f, Proposed reaction catalysed by GsCYP71FB1, as supported by MS/MS fragmentation, previously published labelling studies and comparison to an authentic 10 standard.

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Extended Data Fig. 9 Comparison of intermediates produced in the N. benthamiana co-expression system to G. superba metabolites.

Each biosynthetic product downstream of 1 produced in our co-expression system (black traces) was compared to the equivalent mass ion found in G. superba rhizome extracts (blue traces) or to a verified standard (red traces) by LC–MS analysis. Additionally, MS/MS spectra for co-eluting peaks were compared to demonstrate the chemical similarity between these compounds. Collision energies for all shown MS/MS analyses were 20 V, with the exception of 1, for which fragmentation at 10 V is shown. These LC–MS comparisons were performed once with multiple biological replicates of G. superba metabolite extractions (n = 6 biological replicates from four different tissues: leaf, stem, root and rhizome). The chromatographic traces for G. superba metabolites in this figure are from a representative rhizome extract. Retention time and MS/MS spectra for compounds produced by heterologous expression in N. benthamiana were consistent among individual experiments.

Extended Data Fig. 10 Engineering early metabolites in colchicine biosynthesis.

a, List of module-1 biosynthetic genes and their best BLASTP hit in A. thaliana. Note that all genes except for GsAER seem to have an orthologue in Arabidopsis with >60% identity, suggesting functional equivalence. b, Generalized pathway for the proposed engineered production of 4-HDCA in N. benthamiana. c, LC–MS chromatograms demonstrating that co-expression of module 1 in N. benthamiana leads to production of 4-HDCA, which was detected as the Girard reagent T derivative (m/z 264.1707). Production of 4-HDCA by module-1 proteins was demonstrated three times with similar results each time. d, LC–MS chromatograms (by HILIC analysis) assessing the production of tyramine (left, m/z 138.0913), l-DOPA (middle, m/z 198.0761) and dopamine (right, m/z 154.0863) with individual and co-expression of GsTyDC/DDC and BvCYP76AD5 (module 2). Next to each set of chromatograms is the corresponding relative quantifications of tyramine (m/z 138), l-DOPA (m/z 198) and dopamine (m/z 154) in each reaction. For each reaction, data are mean ± s.d. of 3 distinct biological replicates. Module 2 activity was confirmed in more than three individual experiments. e, Proposed scheme for the engineered biosynthesis of dopamine. f, Comparison of the native function of CjNCS in benzylisoquinoline alkaloid biosynthesis to the putative reaction required in colchicine alkaloid biosynthesis. g, Co-expression of both module-1 and module-2 genes in N. benthamiana leads to concurrent production of the requisite aldehyde (m/z 264.1707, Girard T derivative), as well as dopamine (m/z 154.0863), as shown by the LC–MS chromatograms. Note that dopamine is observed here by C18 chromatography. Co-expression of both modules was performed more than three times, with similar results each time. h, LC–MS chromatograms for the co-expression of module 1 and module 2 with CjNCS in comparison to an authentic 1 standard. These experiments were performed more than three times, with similar results observed each time. i, MS/MS fragmentation comparison between the newly identified m/z 286.1438 peak (*) and the 1 standard. Both were analysed with a collision energy of 10 V. This MS/MS comparison was performed twice. j, Comparison of the function of wild-type, full-length CjNCS to N-terminal truncations of 24 (Δ24-CjNCS) and 29 (Δ29-CjNCS) amino acids. Filled-in boxes (grey) indicate the presence of a gene in the co-expression experiment; an empty box (white) indicates its absence. For each reaction, data are mean ± s.d. of 3 biological replicates.

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Extended Data Fig. 11 Metabolic engineering of colchicine alkaloids in Nicotiana benthamiana.

a, Biosynthetic schematic of the transient metabolic engineering system in N. benthamiana for the production of 2 and 9. b, LC–MS chromatograms for the co-expression of GsOMT1 with module 1, module 2 and Δ24-CjNCS compared to that of GsOMT1 expressed alone with co-infiltration of 1. The EICs for 1 (blue traces, m/z 286.1438) and the production of 2 (red traces, m/z 300.1594) are shown. c, LC–MS chromatograms demonstrating the production of 9 through co-expression of module 3 (without GsCYP71FB1) with module 1, module 2 and Δ24-CjNCS. This is compared to infiltration of 1 (as substrate) with co-expression of module 3 (without GsCYP71FB1), as well as to a standard of O-methylandrocymbine (9). The EICs specific to the exact mass of 9 (m/z 386.1962) are shown. Engineered production of 2 and 9 was demonstrated three times for each molecule. d, Production of two different colchicine alkaloids (2, m/z 300.1594; 10, m/z 400.1755, 422.1574) through metabolic engineering in N. benthamiana when GsAER is either omitted or included. Filled-in boxes (grey) indicate the presence of a module or gene in the co-expression experiment; an empty box (white) indicates its absence. For each reaction, data are mean ± s.d. of 6 biological replicates for each condition. Statistical significance was assessed using a two-tailed Student’s t-test with an assumption of unequal variance. The production of 2 in this context was assessed once; the production of 10 was performed twice with similar results each time. e, Individual dropout of each module-1 and module-2 gene in the engineered production of 10 (m/z 400.1755, 422.1574). For each reaction, data are the mean ± s.d. for each condition. n = 3 for GFP control; n = 5 for PAL, CCR, AER, C4H, TyDC/DDC and BvCYP76AD5 dropouts; n = 6 for 4CL and DAHPS dropouts and for the no-dropout control. All replicates represent independent biological replicates. Statistical comparisons were made using Dunnett’s test (two-tailed) with comparison to the full pathway control (indicated by arrow). *** = P < 0.001. This experiment was performed twice, with similar results each time.

Extended Data Fig. 12 Dropout analysis of module-3 biosynthetic genes.

Metabolic engineering of the full pathway to N-formyldemecolcine (10) in N. benthamiana was compared to transient co-expression systems in which individual module-3 enzymes were removed. a, Accumulation of proposed pathway intermediates in dropout experiments. Grey boxes to the left of the graph indicate biosynthetic genes or modules included in a co-expression experiment; white boxes indicate their absence. For each intermediate, data represent the mean ± s.d. of the extracted ion abundance (n = 3) for the exact ion mass [M + H]+ (for 10, both [M + H]+ and [M + Na]+) at the retention time (r.t.) that corresponds to the compound. b, Dropout of GsOMT1 from the full engineered pathway leads to accumulation of a new compound with a mass equivalent to 2 (m/z 300.1594), as shown by the LC–MS chromatograms. The newly identified peak is indicated by the arrow. c, Transient co-expression of GsOMT1 or GsNMTt with module 1, module 2 and Δ24-CjNCS (for production of 1). The LC–MS chromatograms for the substrate (1, m/z 286.1438), singly methylated products (2a and 2b, m/z 300.1594) and doubly methylated product (3, m/z 314.1751) are shown. d, MS/MS fragmentation spectrum of 2a/2 (collision energy of 20 V), as well as a tabulated list and putative structures for the ion fragments. e, MS/MS fragmentation spectrum of 2b (collision energy of 20 V), as well as a tabulated list and putative structures for the ion fragments. Note that fragment B (m/z 269) supports the placement of the methyl group on the nitrogen. For reference, compare to fragment B of 2a in d. f, Comparative consumption of 1 by GsOMT1 and GsNMTt. Grey boxes indicate the presence of a gene or module in the co-expression experiment; a white box indicates its absence. n = 3 for each reaction; statistical comparisons made using Dunnett’s test with comparison to the module 1/module 2/Δ24-CjNCS control. g, Proposed scheme for the initial methylations of 1. All experiments shown in this figure were performed once.

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This file contains Supplementary Discussion, Supplementary Schemes 1-4, Supplementary Figures 1-8 and Supplementary Tables 1-5.

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Nett, R.S., Lau, W. & Sattely, E.S. Discovery and engineering of colchicine alkaloid biosynthesis. Nature 584, 148–153 (2020). https://doi.org/10.1038/s41586-020-2546-8

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