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Nature CommunicationsPlant BiologyPeer-reviewed journal article

Phylogenomics and metabolic engineering reveal a conserved gene cluster in Solanaceae plants for withanolide biosynthesis

Samuel Edward HakimiD, Nancy ChoudharyiD, Karan MalhotraiD, Peng JianiD, Arne BültemeieriD, Ahmed ArafaiD, Ronja FriedhoffiD, Maximilian BaueriD, Jessica Eikenberg, Claus‐Peter WitteiD, Marco HerdeiD, Philipp M. HeretschiD, Boas PuckeriD, Jakob FrankeiD

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Abstract

Withanolides are steroidal lactones from nightshade (Solanaceae) plants. Of the over 1,200 known representatives, many possess potent biological activities, but their drug potential has not been fully realised up until now. A central obstacle is the limited availability of minor withanolides, caused by a lack of knowledge about the underlying biosynthetic pathways. Here, we combine phylogenomics with metabolic engineering to overcome this limitation. By sequencing the genome of the medicinal plant and archetypical withanolide producer ashwagandha (Withania somnifera) and comparing the genome sequences of nine Solanaceae species, we discovered a conserved gene cluster for withanolide biosynthesis, consisting of two sub-gene clusters which differ in their expression patterns. To investigate the functions of the encoded enzymes, we established metabolic engineering platforms in yeast (Saccharomyces cerevisiae) and the model plant Nicotiana benthamiana. This allowed us to reconstitute the first three oxidative steps of withanolide biosynthesis, catalysed by the cytochrome P450 monooxygenases CYP87G1, CYP88C7, and CYP749B2, leading to the aglycone of the known compound withanoside V. Our work sets the basis for the biotechnological production of withanolides in heterologous hosts and will therefore help to fully harness the drug potential of these plant steroids in the future.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (28 Sept 2024) · CC BY 4.0

Comparing nine Solanaceae genomes reveals a conserved withanolide gene cluster; three of its P450s, expressed in yeast and N. benthamiana, oxidise 24-methyldesmosterol toward the withanoside V aglycone.

Only one withanolide-specific enzyme (24ISO) was known, which hampers engineering and production of the more than 1,200 known withanolides. The work places 24ISO in a conserved gene cluster and builds yeast and N. benthamiana platforms to test the enzymes encoded there.

Key results

  1. ShownA new Withania somnifera genome assembly from Oxford Nanopore reads is contiguous and largely complete, and it is the basis for the cluster analysis.

    102 contigs, N50 71 Mb, total size 2.8 Gbp; 35,118 predicted protein-coding genes; BUSCO (Solanales_odb10) 96.6% complete in predicted proteins. · Genome assembly of Withania somnifera

  2. ShownA syntenic gene cluster around 24ISO is present in withanolide producers and absent in non-producers. It is rich in P450, dioxygenase, reductase, acyltransferase and sulfotransferase genes, and it splits into two sub-clusters with different expression patterns.

    Synteny across Physalis floridana, P. grisea, P. pruinosa, W. somnifera, Datura stramonium and D. wrightii; absent in S. lycopersicum and S. tuberosum; no 24ISO orthologue in S. lycopersicum, S. tuberosum or N. tabacum; two expression groups in W. somnifera and D. stramonium RNA-seq. · Phylogenomics discovery of a putative…; Discussion

  3. ShownYeast engineered by deleting ERG4 and ERG5 and expressing P. peruviana 7RED and 24ISO produces the key intermediate 24-methyldesmosterol. Sterol-homeostasis engineering raised titres but impaired growth.

    Strain KMY23: 7.6 mg/g dry cell weight; KMY53 (ARE2 plus YEH2): 9.3; KMY54 (ARE2 plus YEH1): 13.5 mg/g DCW, with severely reduced final OD600; a 7RED-limited shunt product (compound 6) persisted. · Development of a yeast platform for production…

  4. ShownCo-expressing the full 15-gene phytosterol set, including tHMGR and 24ISO, raised 24-methyldesmosterol in N. benthamiana to 0.9 mg/g dry weight. This avoided the yeast 7RED shunt product but added background-epoxidation shunt products.

    0.9 mg/g dry weight with the 15-gene set; tHMGR plus 24ISO alone gave a peak below the limit of quantification; leave-one-out (three biological replicates) showed significant losses without tHMGR, SMO2-1 plus SMO2-2, DWARF5 or DWARF7. · Engineering of Nicotiana benthamiana for…; Figure legends

  5. SuggestedData support a three-step route in which P. pruinosa CYP87G1 hydroxylates C-22, CYP88C7 hydroxylates C-1 and CYP749B2 forms the lactone ring. The three together give a product matching the withanoside V aglycone.

    CYP87G1 product assigned as 22R by NMR (isolated yield 0.04 mg/g dry weight); a shunt product of CYP88C7 showed C-1 hydroxylation by NMR (0.01 mg/g); the aglycone arose only with all three CYPs, matching an authentic reference by retention time, high-resolution mass and MS/MS. · Elucidation of oxidative lactone formation in…

Methods, data and reproducibility

Nanopore sequencing and assembly of the W. somnifera genome, then microsynteny and phylogenetic comparison with other Solanaceae genomes around 24ISO. Candidate P450s were tested by pathway reconstitution in engineered yeast and transiently transformed N. benthamiana, with GC-MS, LC-MS and NMR to identify products.

  • W. somnifera genome (Oxford Nanopore) and RNA-seq · Withania somnifera · n = 102 contigs; RNA-seq sample number not stated · PRJEB64854 (genomic reads); ERR13615536 (RNA-seq)
  • Genomes of 5 other producers (P. floridana, P. grisea, P. pruinosa, D. stramonium, D. wrightii) and 3 non-producers (S. lycopersicum, S. tuberosum, N. tabacum) vs the new W. somnifera assembly · Solanaceae species · n = 9 species in the synteny comparison
  • Public paired-end RNA-seq from multiple tissues for expression analysis · Datura stramonium; Withania somnifera
  • Protein sequences for phylogenetic trees · Solanaceae species · n = 32 species
  • Engineered yeast strains and transient N. benthamiana expression with GC-MS and LC-MS metabolite profiling · Saccharomyces cerevisiae; Nicotiana benthamiana · n = three biological replicates in bar plots
Methods & tools
NextDenovo2, RagTag, GeMoMa, BUSCO, JCVI/MCscan, MAFFT, IQ-TREE, Kallisto, EDTA, GC-MS, LC-MS (Orbitrap), NMR
Software & environment
Versions given: NextDenovo2 v2.5.2, Guppy v6.4.6, GeMoMa v1.9, BUSCO v5.6.1, JCVI/MCscan v1.4.14, MAFFT v7.520, IQ-TREE v1.6.12, Kallisto v0.50.1, EDTA v2.2.1. No container or requirements file stated.
Reproducibility gaps
The GitHub repository covers only Circos plot scripts and commands; no code for assembly, expression or synteny workflows is listed. Cluster gene sequences are in a supplementary xlsx file.
Limitations (6)
  • Yeast titres remain low relative to need: a shunt product from limited 7RED activity persists, and the best-titre strain showed severe growth defects, so further strain improvement is needed. · stated by the authors
  • N. benthamiana needs 15 co-expressed genes for useful 24-methyldesmosterol levels and still produces shunt products from background epoxidation. · stated by the authors
  • The C-1 hydroxylated CYP88C7 product 14 could not be purified for NMR, so C-1 hydroxylation rests on mass-spectral fragmentation plus NMR of a related shunt product (15). · stated by the authors
  • Functional differences between the two sub-gene clusters, and their evolutionary and biological roles, are left for future work. · stated by the authors
  • The final aglycone was identified only by LC-MS match to a hydrolysed withanoside V reference and was detected in N. benthamiana only, not isolated or characterised by NMR; only three of the cluster's enzymes were tested. · noted in this summary
  • The characterised enzymes are P. pruinosa orthologues, tested only in heterologous hosts; activity of the W. somnifera copies and in planta function were not tested here. · noted in this summary

Read this if you work on plant specialised-metabolism gene clusters, steroid pathway engineering in yeast or N. benthamiana, or Solanaceae comparative genomics.

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