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Glyceollin biosynthesis in a plant chassis engineered for isoflavone production

Nature Chemical Biology volume 21, pages 1497–1508 (2025)Cite this article

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Abstract

Glyceollins are structurally complex potent antimicrobial isoflavonoid phytoalexins produced by the crop soybean (Glycine max), yet their biosynthesis remains elusive, making it impossible to carry out synthetic biology-based production and engineering for further development. Here, via assembling synergistic engineering strategies, we successfully rewired the metabolic fluxes in Nicotiana benthamiana leaves for high-yield production of isoflavonoid precursor daidzein (7.04 g kg−1 dry weight (dw)), allowing for efficient screening and identification of six cytochrome P450 monooxygenases, namely glyceollin synthases, that furnish the pyrano/furano E ring and complete the 15-step biosynthetic pathways of diverse glyceollins. We establish that purified glyceollins are important for plant defense as they can effectively suppress the growth of Phytophthora sojae in vitro. Our engineered plant chassis can provide facile access to bioactive isoflavonoids, as manifested by the de novo total biosynthesis of glyceollins (for example, I, II, III and VII at up to 5.9 g kg−1, dw) and medicarpin (0.72 g kg−1, dw) for enhanced pathogen resistance and medicinal value.

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Fig. 1: Overview of glyceollin biosynthetic pathways and proposed modular isoflavonoid pathway reconstitution in N. benthamiana.
Fig. 2: Flux engineering strategies. Stage I: identification of high turnover bottleneck enzymes for isoflavonoid synthesis in N. benthamiana.
Fig. 3: Flux boosting with 2A peptide-synchronized co-expression and flux remodeling with TFs for enhanced daidzein and genistein synthesis in N. benthamiana.
Fig. 4: Identification and characterization of GSs complete the biosynthesis of diverse glyceollins.
Fig. 5: De novo biosynthesis of bioactive isoflavonoids using the engineered N. benthamiana chassis.

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Data availability

The Glycine max RNA-seq database used for analysis is available at https://plantrnadb.com. Twelve candidate genes encoding cytochrome P450 monooxygenases were selected for the functional characterization (NCBI accession codes: NM_001317483.2, XM_003537409.4, NM_001317501.1, XM_003546576.4, NM_001254191.2, XM_003548157.5, NM_001255341.3, NM_001254151.3, XM_003537846.4, NM_001251012.2, XM_003538926.5 and NM_001255757.3). Two other functional homologous P450 GSs are GmGS2b (XM_003543475.5) and GmGS3 (NM_001253148.1). The corresponding gene IDs and loci for the biosynthetic genes used in this study can be found in Supplementary Table 2. Coding sequences for the genes used in this study were retrieved from the NCBI database. All other data are available in the main text or the supplementary materials. Data are available from the corresponding author upon request. Source data are provided with this paper.

References

  1. Ahuja, I., Kissen, R. & Bones, A. M. Phytoalexins in defense against pathogens. Trends Plant Sci. 17, 73–90 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Veitch, N. C. Isoflavonoids of the leguminosae. Nat. Prod. Rep. 24, 417–464 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Vitale, D. C., Piazza, C., Melilli, B., Drago, F. & Salomone, S. Isoflavones: estrogenic activity, biological effect and bioavailability. Eur. J. Drug. Metab. Pharmacokinet. 38, 15–25 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Liang, J. et al. Daidzein as an antioxidant of lipid: effects of the microenvironment in relation to chemical structure. J. Agric. Food Chem. 56, 10376–10383 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Verdrengh, M., Jonsson, I. M., Holmdahl, R. & Tarkowski, A. Genistein as an anti-inflammatory agent. Inflamm. Res. 52, 341–346 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Zhou, Y. X., Zhang, H. & Peng, C. Puerarin: a review of pharmacological effects. Phytother. Res. 28, 961–975 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Dixit, M. et al. Medicarpin, a natural pterocarpan, heals cortical bone defect by activation of Notch and Wnt canonical signaling pathways. PLoS ONE 10, e0144541 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Keung, W. M. & Vallee, B. L. Daidzin and its antidipsotropic analogs inhibit serotonin and dopamine metabolism in isolated mitochondria. Proc. Natl Acad. Sci. USA 95, 2198–2203 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Choi, Y. R., Shim, J. & Kim, M. J. Genistin: a novel potent anti-adipogenic and anti-lipogenic agent. Molecules 25, 2042–2057 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tay, K. C. et al. Formononetin: a review of its anticancer potentials and mechanisms. Front. Pharmacol. 10, 820–838 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yu, C., Zhang, P., Lou, L. & Wang, Y. Perspectives regarding the role of biochanin A in humans. Front. Pharmacol. 10, 793–803 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Pham, T. H., Lecomte, S., Efstathiou, T., Ferriere, F. & Pakdel, F. An update on the effects of glyceollins on human health: possible anticancer effects and underlying mechanisms. Nutrients 11, 79–102 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ng, T. B. et al. Glyceollin, a soybean phytoalexin with medicinal properties. Appl. Microbiol. Biotechnol. 90, 59–68 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Graham, T. L. Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates. Plant Physiol. 95, 594–603 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wang, Q. et al. Soy isoflavone: the multipurpose phytochemical. Biomed. Rep. 1, 697–701 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ebel, J. & Grisebach, H. Defense strategies of soybean against the fungus Phytophthora megasperma f. sp. glycinea: a molecular analysis. Trends Biochem. Sci. 12, 23–27 (1988).

    Article  Google Scholar 

  17. Huang, J.-S. & Barker, K. R. Glyceollin I in soybean-cyst nematode interactions: spatial and temporal distribution in toots of resistant and susceptible soybeans. Plant Physiol. 96, 1302–1307 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Jahan, M. A. et al. Glyceollin transcription factor GmMYB29A2 regulates soybean resistance to phytophthora sojae. Plant Physiol. 183, 530–546 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lygin, A. V. et al. Glyceollin is an important component of soybean plant defense against Phytophthora sojae and Macrophomina phaseolina. Phytopathology 103, 984–994 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Yu, J., Tu, X. & Huang, A. C. Functions and biosynthesis of plant signaling metabolites mediating plant–microbe interactions. Nat. Prod. Rep. 39, 1393–1422 (2022).

    Article  CAS  PubMed  Google Scholar 

  21. Jeandet, P. et al. Deciphering the role of phytoalexins in plant–microorganism interactions and human health. Molecules 19, 18033–18056 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Vogt, T. Phenylpropanoid biosynthesis. Mol. Plant 3, 2–20 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Dastmalchi, M. & Dhaubhadel, S. Soybean chalcone isomerase: evolution of the fold, and the differential expression and localization of the gene family. Planta 241, 507–523 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. Uchida, K., Akashi, T. & Aoki, T. Functional expression of cytochrome P450 in Escherichia coli: an approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis. Plant Biotechnol. 32, 205–213 (2015).

    Article  CAS  Google Scholar 

  25. Fischer, D., Ebenau-Jehle, C. & Grisebach, H. Phytoalexin synthesis in soybean: purification and characterization of NADPH: 2′-hydroxydaidzein oxidoreductase from elicitor-challenged soybean cell cultures. Arch. Biochem. Biophys. 276, 390–395 (1990).

    Article  CAS  PubMed  Google Scholar 

  26. Paiva, N. L., Edwards, R., Sun, Y., Hrazdina, G. & Dixon, R. A. Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis. Plant Mol. Biol. 17, 653–667 (1991).

    Article  CAS  PubMed  Google Scholar 

  27. Tiemann, K., Inze, D., Van Montagu, M. & Barz, W. Pterocarpan phytoalexin biosynthesis in elicitor-challenged chickpea (Cicer arietinum L.) cell cultures. Purification, characterization and cDNA cloning of NADPH: isoflavone oxidoreductase. Eur. J. Biochem. 200, 751–757 (1991).

    Article  CAS  PubMed  Google Scholar 

  28. Paiva, N. L., Sun, Y., Dixon, R. A., VanEtten, H. D. & Hrazdina, G. Molecular cloning of isoflavone reductase from pea (Pisum sativum L): evidence for a 3R-isoflavanone intermediate in (+)-pisatin biosynthesis. Arch. Biochem. Biophys. 312, 501–510 (1994).

    Article  CAS  PubMed  Google Scholar 

  29. Uchida, K., Akashi, T. & Aoki, T. The missing link in Leguminous pterocarpan biosynthesis is a dirigent domain-containing protein with isoflavanol dehydratase activity. Plant Cell Physiol. 58, 398–408 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lining Guo, R. A. D. & Nancy, L. Paiva. The ‘pterocarpan synthase’ of alfalfa association and co-induction of vestitone reductase and 7,2-dihydroxy-4-methoxy-isoflavanol DMI dehydratase the two final enzymes in medicarpin biosynthesis. FEBS J. 356, 221–225 (1994).

    Article  Google Scholar 

  31. Guo, L. & Paiva, N. L. Molecular cloning and expression of alfalfa (Medicago sativa L.) vestitone reductase, the penultimate enzyme in medicarpin biosynthesis. Arch. Biochem. Biophys. 320, 353–360 (1995).

    Article  CAS  PubMed  Google Scholar 

  32. Schopfer, C. R., Kochs, G., Lottspeich, F. & Ebel, J. Molecular characterization and functional expression of dihydroxypterocarpan 6a-hydroxylase, an enzyme specific for pterocarpanoid phytoalexin biosynthesis in soybean (Glycine max L.). FEBS J. 432, 182–186 (1998).

    Article  CAS  Google Scholar 

  33. Akashi, T., Sasaki, K., Aoki, T., Ayabe, S. & Yazaki, K. Molecular cloning and characterization of a cDNA for pterocarpan 4-dimethylallyltransferase catalyzing the key prenylation step in the biosynthesis of glyceollin, a soybean phytoalexin. Plant Physiol. 149, 683–693 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sukumaran, A., McDowell, T., Chen, L., Renaud, J. & Dhaubhadel, S. Isoflavonoid-specific prenyltransferase gene family in soybean: GmPT01, a pterocarpan 2-dimethylallyltransferase involved in glyceollin biosynthesis. Plant J. 96, 966–981 (2018).

    Article  CAS  PubMed  Google Scholar 

  35. De La Pena, R. & Sattely, E. S. Rerouting plant terpene biosynthesis enables momilactone pathway elucidation. Nat. Chem. Biol. 17, 205–212 (2021).

    Article  PubMed  Google Scholar 

  36. Jiang, B. et al. Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III. Science 383, 622–629 (2024).

    Article  CAS  PubMed  Google Scholar 

  37. Huang, A. C. et al. A specialized metabolic network selectively modulates Arabidopsis root microbiota. Science 364, eaau6389 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Reed, J. et al. Elucidation of the pathway for biosynthesis of saponin adjuvants from the soapbark tree. Science 379, 1252–1264 (2023).

    Article  CAS  PubMed  Google Scholar 

  39. Dudley, Q. M. et al. Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana. Commun. Biol. 5, 949–960 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Liu, C.-J., Blount, J. W. & Dixon, R. A. Bottlenecks for metabolic engineering of isoflavone glycoconjugates in Arabidopsis. Proc. Natl Acad. Sci. USA 99, 14578–14583 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Pandey, A. et al. Co-expression of Arabidopsis transcription factor, AtMYB12, and soybean isoflavone synthase, GmIFS1, genes in tobacco leads to enhanced biosynthesis of isoflavones and flavonols resulting in osteoprotective activity. Plant Biotechnol. J. 12, 69–80 (2014).

    Article  CAS  PubMed  Google Scholar 

  42. Yao, X. et al. Engineering the expression of plant secondary metabolites–genistein and scutellarin through an efficient transient production platform in Nicotiana benthamiana L. Front. Plant Sci. 13, 994792 (2022).

    Google Scholar 

  43. Zhou, Q. et al. Insight into neofunctionalization of 2,3-oxidosqualene cyclases in B,C-ring-opened triterpene biosynthesis in quinoa. New Phytol. 241, 764–778 (2024).

    Article  CAS  PubMed  Google Scholar 

  44. Huang, A. C. et al. Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution. Proc. Natl Acad. Sci. USA 114, E6005–E6014 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhang, C. et al. Phenylalanine ammonia-lyase2.1 contributes to the soybean response towards Phytophthora sojae infection. Sci. Rep. 7, 7242–7254 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Dastmalchi, M., Bernards, M. A. & Dhaubhadel, S. Twin anchors of the soybean isoflavonoid metabolon: evidence for tethering of the complex to the endoplasmic reticulum by IFS and C4H. Plant J. 85, 689–706 (2016).

    Article  CAS  PubMed  Google Scholar 

  47. Gutierrez-Gonzalez, J. J. et al. Differential expression of isoflavone biosynthetic genes in soybean during water deficits. Plant Cell Physiol. 51, 936–948 (2010).

    Article  CAS  PubMed  Google Scholar 

  48. Dhaubhadel, S., Gijzen, M., Moy, P. & Farhangkhoee, M. Transcriptome analysis reveals a critical role of CHS7 and CHS8 genes for isoflavonoid synthesis in soybean seeds. Plant Physiol. 143, 326–338 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Mameda, R., Waki, T., Kawai, Y., Takahashi, S. & Nakayama, T. Involvement of chalcone reductase in the soybean isoflavone metabolon: identification of GmCHR5, which interacts with 2-hydroxyisoflavanone synthase. Plant J. 96, 56–74 (2018).

    Article  CAS  PubMed  Google Scholar 

  50. Jung, W. et al. Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes. Nat. Biotechnol. 18, 208–212 (2000).

    Article  CAS  PubMed  Google Scholar 

  51. Akashi, T., Aoki, T. & Ayabe, S.-I. Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis. Plant Physiol. 137, 882–891 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Sainsbury, F., Thuenemann, E. C. & Lomonossoff, G. P. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol. J. 7, 682–693 (2009).

    Article  CAS  PubMed  Google Scholar 

  53. Shimada, N. et al. A cluster of genes encodes the two types of chalcone isomerase involved in the biosynthesis of general flavonoids and legume-specific 5-deoxy(iso)flavonoids in Lotus japonicus. Plant Physiol. 131, 941–951 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Cheng, A.-X. et al. Identification of chalcone isomerase in the basal land plants reveals an ancient evolution of enzymatic cyclization activity for synthesis of flavonoids. N. Phytol. 217, 909–924 (2018).

    Article  CAS  Google Scholar 

  55. Li, J. et al. Diversion of metabolic flux towards 5-deoxy(iso)flavonoid production via enzyme self-assembly in Escherichia coli. Metab. Eng. Commun. 13, e00185 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ryan, M. D., King, A. M. Q. & Thomas, G. P. Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J. Gen. Virol. 72, 2727–2273 (1991).

    Article  CAS  PubMed  Google Scholar 

  57. Donnelly, M. L. L. et al. The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences. J. Gen. Virol. 82, 1027–1041 (2001).

    Article  CAS  PubMed  Google Scholar 

  58. Donnelly, M. L. L., Luke, G., Mehrotra, A., Gani, D. & Ryan, M. D. Analysis of the aphthovirus 2A/2B polyprotein cleavage mechanism indicates not a proteolytic reaction but a novel translational effect: a putative ribosomal skip. J. Gen. Virol. 82, 1013–1025 (2001).

    Article  CAS  PubMed  Google Scholar 

  59. Lee, A. R. et al. Increased sesqui- and triterpene production by co-expression of HMG-CoA reductase and biotin carboxyl carrier protein in tobacco (Nicotiana benthamiana). Metab. Eng. 52, 20–28 (2019).

    Article  CAS  PubMed  Google Scholar 

  60. Deng, Y. & Lu, S. Biosynthesis and regulation of phenylpropanoids in plants. Crit. Rev. Plant Sci. 36, 257–290 (2017).

    Article  Google Scholar 

  61. Liu, J., Osbourn, A. & Ma, P. MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Mol. Plant 8, 689–708 (2015).

    Article  CAS  PubMed  Google Scholar 

  62. Luo, J. et al. AtMYB12 regulates caffeoyl quinic acid and flavonol synthesis in tomato: expression in fruit results in very high levels of both types of polyphenol. Plant J. 56, 316–326 (2008).

    Article  CAS  PubMed  Google Scholar 

  63. Park, J. S. et al. Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa). Plant Cell Rep. 27, 985–994 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Outchkourov, N. S. et al. Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves. Front. Plant Sci. 5, 519–527 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Li, X.-W. et al. A R2R3-MYB transcription factor, GmMYB12B2, affects the expression levels of flavonoid biosynthesis genes encoding key enzymes in transgenic Arabidopsis plants. Gene 532, 72–79 (2013).

    Article  CAS  PubMed  Google Scholar 

  66. Yi, J. et al. A single-repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean. Plant J. 62, 1019–1034 (2010).

    CAS  PubMed  Google Scholar 

  67. Matsui, K., Umemura, Y. & Ohme-Takagi, M. AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. Plant J. 55, 954–967 (2008).

    Article  CAS  PubMed  Google Scholar 

  68. Zhu, H. F., Fitzsimmons, K., Khandelwal, A. & Kranz, R. G. CPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis. Mol. Plant 2, 790–802 (2009).

    Article  CAS  PubMed  Google Scholar 

  69. Aharoni, A. et al. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J. 28, 319–332 (2001).

    Article  CAS  PubMed  Google Scholar 

  70. Yu, Y., Zhang, H., Long, Y., Shu, Y. & Zhai, J. Plant Public RNA-seq Database: a comprehensive online database for expression analysis of ~45,000 plant public RNA-seq libraries. Plant Biotechnol. J. 20, 806–808 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Lyne, R. L., Mulheirn, L. J. & Keen, N. T. Novel pterocarpinoids from glycine species. Tetrahedron Lett. 22, 2483–2484 (1981).

    Article  CAS  Google Scholar 

  72. Wang, Z. P. et al. Egg cell-specific promoter-controlled CRISPR–Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144–155 (2015).

  73. Liu, Q. et al. De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories. Nat. Commun. 12, 6085 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Liu, C. J. et al. Structural basis for dual functionality of isoflavonoid O-methyltransferases in the evolution of plant defense responses. Plant Cell 18, 3656–3669 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Liu, C. J., Huhman, D., Sumner, L. W. & Dixon, R. A. Regiospecific hydroxylation of isoflavones by cytochrome p450 81E enzymes from Medicago truncatula. Plant J. 36, 471–484 (2003).

    Article  CAS  PubMed  Google Scholar 

  76. Deavours, B. E. et al. Functional analysis of members of the isoflavone and isoflavanone O-methyltransferase enzyme families from the model legume Medicago truncatula. Plant Mol. Biol. 62, 715–733 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Panabieres, F. et al. Phytophthora nicotianae diseases: worldwide new knowledge of a long-recognised pathogen. Phytopathol. Mediterr. 55, 20–40 (2016).

    CAS  Google Scholar 

  78. Tyler, B. M. Phytophthora sojae: root rot pathogen of soybean and model oomycete. Mol. Plant Pathol. 8, 1–8 (2006).

    Article  Google Scholar 

  79. Wang, Z. et al. Sustainable production of genistin from glycerol by constructing and optimizing Escherichia coli. Metab. Eng. 74, 206–219 (2022).

    Article  CAS  PubMed  Google Scholar 

  80. Liu, X., Zhang, P., Zhao, Q. & Huang, A. C. Making small molecules in plants: a chassis for synthetic biology-based production of plant natural products. J. Integr. Plant Biol. 65, 417–443 (2023).

    Article  CAS  PubMed  Google Scholar 

  81. Huang, F.-C. et al. Structural and functional analysis of UGT92G6 suggests an evolutionary link between mono- and disaccharide glycoside-forming transferases. Plant Cell Physiol. 59, 862–875 (2018).

    Article  CAS  Google Scholar 

  82. Concordet, J.-P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR–Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242–W245 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Kereszt, A. et al. Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nat. Protoc. 2, 948–952 (2007).

    Article  CAS  PubMed  Google Scholar 

  84. Ward, E. W. B., Lazarovits, G., Uwin, C. H. & Buzzell, R. I. Hypocotyl reactions and glyceollin in soybeans inoculated with zoospores of Phytophthora megasperma var. sojae. Phytopathology 69, 951–955 (1979).

    Article  Google Scholar 

  85. Liu, X., Li, L. & Zhao, G. R. Systems metabolic engineering of Escherichia coli coculture for de novo production of genistein. ACS Synth. Biol. 11, 1746–1757 (2022).

    Article  CAS  PubMed  Google Scholar 

  86. Meng, Y., Liu, X., Zhang, L. & Zhao, G. R. Modular engineering of Saccharomyces cerevisiae for de novo biosynthesis of genistein. Microorganisms 10, 1402–1417 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Wang, Y. et al. Systematic engineering of Saccharomyces cerevisiae for the de novo biosynthesis of genistein and glycosylation derivatives. J. Fungi 10, 176–188 (2024).

    Article  CAS  Google Scholar 

  88. Lu, C. et al. Heterologous biosynthesis of medicarpin using engineered Saccharomyces cerevisiae. Synth. Syst. Biotechnol. 8, 749–756 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are grateful to all the members of the A.C. Huang Lab for discussion and suggestions. We thank J. Zhai (Southern University of Science and Technology) for helping with accessing the soybean transcriptomics data and Z. Ma (Nanjing Agricultural University) and S. Cheng (Agriculture Genomics Institute at Shenzhen) for providing soybean cultivar ‘Williams 82’ seeds. We acknowledge the technical support from the computing cluster and mass spectrometry platform of SUSTech-PKU Institute of Plant and Food Science. This study was supported by the Shenzhen Science and Technology Program (grant no. 20231120191353002 to A.C.H.; KCXFZ20211020174802004 to A.C.H.; ZDSYS20230626091659010 to A.C.H.; JCYJ202408133000227 to Q.Z.) and in part by the National Science Foundation of China (grant no. 32370298 to A.C.H.), the Guangdong Basic and Applied Basic Research Foundation (grant no. 2023A1515012550 to A.C.H.) and the Macao Science and Technology Development Fund (0019/2022/AGJ to G.-Y.Z.).

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Author notes

  1. Yuqiong Hao

    Present address: Institute of Wheat Research, Shanxi Agricultural University, Linfen, People’s Republic of China

  2. These authors contributed equally: Jiali Xie, Jiayu Tian.

Authors and Affiliations

  1. Shenzhen Key Laboratory of Plant Genetic Engineering and Molecular Design, SUSTech–PKU Institute of Plant and Food Science, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, People’s Republic of China

    Jiali Xie, Jiayu Tian, Salman Khan, Feilong Chen, Jingwei Yu, Yuqiong Hao, Qian Zhou & Ancheng C. Huang

  2. State Key Laboratory of Quality Research in Chinese Medicine, Guangdong–Hong Kong–Macao Joint Laboratory of Respiratory Infectious Disease, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau, People’s Republic of China

    Hao-Ming Xiong & Guo-Yuan Zhu

  3. College of Plant Protection, Nanjing Agricultural University, Nanjing, People’s Republic of China

    Feng Zhang

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A.C.H. conceived, designed and supervised this study. J.X. designed and conducted the majority of the experiments together with J.T. S.K. performed compound purification. F.C. and G.-Y.Z. carried out NMR-based structural elucidation. J.Y. carried out qPCR and western blot analysis. H.-M.X. and G.-Y.Z. acquired the NMR data. Y.H. supervised the molecular cloning. F.Z. supervised the microbial infection experiments. Q.Z. supervised the bioinformatics analysis and antimicrobial assays. G.-Y.Z. supervised structural elucidation. J.X. and A.C.H. analyzed the results and wrote the paper with contributions from all authors.

Corresponding author

Correspondence to Ancheng C. Huang.

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Competing interests

A.C.H. and J.X. have filed three patents for CN jurisdiction on the development and application of a high-yield isoflavonoid biosynthesis platform (CN202411927003X), findings of GSs for production of glyceollin VII (CN2024119291642) and other glyceollins (CN2024119262974) reported in this article, respectively. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Identification of high turnover enzymes for debottlenecking for isoflavone biosynthesis in N. benthamiana.

a, MS2 spectra of (iso)flavonoids. RT, retention time. b-e, In vitro enzymatic assays with total protein extracts of N. benthamiana leaves (WT) and those expressing NbCHI (NbCHI) or GmCHI1B1 (GmCHI1B1), as illustrated by the comparative LC–MS EICs of extracts of reaction products with isoliquiritigenin (IL, 50 μM, b) or naringenin chalcone (NC, 50 μM, d) as the substrate and the abundance of IL and 2 detected in b (c) or NC and 1 detected in d (e). Incubation of substrates with protein extraction buffer without proteins was used as mock. f-i, Screening of CHS (f), CHR (g), CHI (h) and IFS (i) isozymes from different plant species for naringenin (1), isoliquiritigenin (IL), daidzein (3) and genistein (4) production, respectively. GP8, AtPAL2 + GmCHS8 + GmCHR5 + GmIFS1 + GmHID. GP9, AtPAL2 + GmCHS8 + GmCHR5 + GmCHI1B1 + GmHID; GP10, AtPAL2 + GmCHS8. Relative yield is compared with the highest yield in the group. j, Impact of GmCHS8-P2A-GmCHR5 and fusion protein GmCHS8-GmCHR5 on 3 and 4 yields. GP11, AtPAL2 + GmCHS8 + GmCHR5 + GmCHI1B1 + GmIFS1 + GmHID. GP12, AtPAL2 + GmCHI1B1 + GmIFS1 + GmHID. ns, no significance. k, Relative expression of GmCHS8 and GmCHR5 in N. benthamiana leaves expressing gene sets in j as determined by qPCR analysis. l, Relative protein abundance of GmCHS8 and GmCHR5 (relative to GAPDH) in N. benthamiana leaves expressing gene sets in j as assessed by western blots and representative western blots. m, Western blots for l, unprocessed images can be found in source data. The data represent the mean of n = 3 (c, e, f-j), n = 6 (k) or n = 5 (l) biologically independent samples. Statistical analysis was performed by using two-way ANOVA and P > 0.05 indicates no significant difference. Chromatograms shown in (b,d) are representative ones of independent analysis of biological replicates (n > 3) with the same patterns.

Source data

Extended Data Fig. 2 Construction of multigene vectors and their effects on daidzein and genistein production.

a, Processes for multigene vector construction. b-c, Effects of one single vector containing all bottleneck genes for daidzein (b) or genistein (c) biosynthesis versus combinations of individual genes or vectors. GP13, AtPAL2 + MGV2 + GmCHI1B1 + GmIFS1 + GmHID + AtMYB60. GP14, AtPAL2 + GmCHS8 + GmIFS1 + GmMYB12B2 + AtMYB60. All Data represent the mean of n = 3 biologically independent samples and error bars show standard deviation. Statistical analysis was performed by using two-way ANOVA. d, Fold changes of 3 and 4 at different engineering stages.

Source data

Extended Data Fig. 3 Identification of downstream biosynthetic intermediates.

a-i, MS2 spectra of I1 (a), I2 (b), I2-gly (c), I3 (d), I3-gly (e), I4 (f), I4-gly (g), I5-gly-a (h) and I5-gly-b (i).

Extended Data Fig. 4 Identification and characterization of glyceollin precursors 5 and 6.

a, MS2 spectra of 5-gly and 6-gly. b, MS2 spectra of 5 and 6. c, Comparative LC–MS EICs of 5/6, glycosylated 5/6 and glucosidase hydrolysis products of purified 5-gly and 6-gly standards.

Extended Data Fig. 5 Characterization of glyceollin precurosors 7-gly, 8-gly, 9-gly and 7-12 by MS2.

MS2 spectra of compounds 7-gly, 8-gly, 9-gly and 7-12.

Extended Data Fig. 6 Metabolite profiling of glyceollins in soybean tissues and GmGS hairy root mutant lines.

a, Comparative LC–MS EICs of extracts of engineered N. benthamiana leaves expressing genes for different glyceollin biosynthesis and soybean tissues. b, Comparison of glyceollin contents detected from engineered N. benthamiana leaves and soybean tissues. c, Schematic representation of a Cas9 expression vector containing a guide RNA. d, Sequence alignment of wild type and GmGS mutants (gs1a, gs2a, gs3 and gs3e/7) at the mutated regions. e, Contents of glyceollin I (7) in soybean hairy root mutant (gs1a-1 and gs1a-2) and control (EV) lines. f, Contents of glyceofuran (10), 13-epi-glyceollin III (11) and glyceollin VII (12) in soybean hairy root mutant (gs3e/7-1 and gs3e/7-2) and control (EV) lines. All data represent the mean of n = 4 biologically independent samples and error bars show standard deviation. Statistical analysis in e and f was performed by using one-way ANOVA and two-way ANOVA, respectively. Chromatograms shown in (a) are representative ones of independent analysis of biological replicates (n > 3) with the same patterns.

Source data

Extended Data Fig. 7 Biosynthesis of miscellaneous bioactive isoflavonoids in engineered N. benthamiana leaves.

a, MS2 spectra of daidzin, genistin, puerarin, formononetin, biochanin A and medicarpin. b-g, Yields of different isoflavonoids in N. benthamiana. GP15, MtHI4’OMT + MtI2’H + MsIFR + MsVR + MtPTS. All Data represent the mean of n = 3 biologically independent samples and error bars show standard deviation.

Source data

Extended Data Fig. 8 Antimicrobial activity tests of various isoflavonoids.

a. Phenotypes of oomycete pathogen (Phytophthora sojae and Phytophthora nicotianae) seed inoculants cultured on V8 media containing the various isoflavonoids at 20, 50, 100 mg/mL for 48 h. b. Inhibition rate of the designated isoflavonoids at different concentrations against the two pathogens. The experiment was repeated independently three times with similar results. The different symbols indicate different concentrations: dot, 20 μg/mL; square, 50 μg/mL; triangle, 100 μg/mL. Symbol colors refer to the different compounds tested as indicated in the key.

Source data

Extended Data Fig. 9 Comparison of isoflavonoid yields in plant (this study) and microbial chassis previously reported.

Listed are the highest yields currently reported from different engineered microbial hosts55,73,79,85,86,87,88.

Extended Data Fig. 10 Mass fragments detected for the compounds depicted in this study.

Confidence level: A, by comparing to standards; B, based on accurate mass and MS/MS fragmentation patterns.

Supplementary information

Supplementary Information

Supplementary Figs. 1–14, Tables 5–15 and references.

Reporting Summary

Supplementary Tables 1–4

Sequences and information of genes, primers and constructs.

Source data

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Xie, J., Tian, J., Khan, S. et al. Glyceollin biosynthesis in a plant chassis engineered for isoflavone production. Nat Chem Biol 21, 1497–1508 (2025). https://doi.org/10.1038/s41589-025-01914-3

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