Two-Tier Reconstitution: Nicotiana benthamiana and Yeast as Complementary but Non-Neutral Hosts for Plant P450 Clusters

Question: How have plant cytochrome P450 multienzyme clusters been functionally reconstituted in Nicotiana benthamiana and yeast for the discovery of specialized metabolic pathways?

Answer

Across these papers, reconstitution has settled into a two-tier division of labour rather than a contest between hosts: N. benthamiana agroinfiltration is the fast, permissive first pass in which an entire candidate cluster is tested for sufficiency in days, and yeast is the second, genetically clean tier in which step order, branch points and flux are resolved and production strains are built. The strongest recent work uses both on the same gene set — withanolides, Securinega alkaloids and the Taxol pathway are all dissected this way. Crucially, the literature no longer treats either host as a neutral vessel: the papers that succeed are those that engineer host context — electron-donating reductase partners, accessory organizing proteins, endogenous shunt chemistry, immunity and expression machinery — as part of the pathway rather than as background. On this evidence, the answer to "how" is not a protocol but a workflow in which host choice and host modification are the decisive experimental variables, and in which computational cluster prediction plus haplotype-aware candidate selection increasingly sets the ceiling on what reconstitution can find.

N. benthamiana agroinfiltration is the first-pass host because an entire cluster can be interrogated within days

Heterologous reconstruction in N. benthamiana was sufficient to show that a seven-gene quinoa cluster encoding five cytochrome P450s, a fatty acid hydroxylase-like enzyme and a 2-oxoglutarate-dependent dioxygenase converts lathosterol to 20-hydroxyecdysone [23]. In the Securinega alkaloid pathway, N. benthamiana transient expression was one of four converging lines of evidence — with transcriptomics, in vitro assays and yeast reconstitution — that three P450s and a reductase make menisdaurilide from L-tyrosine, and the same host supported de novo allosecurinine production at preparative scale [22]. Withanolide cluster enzymes were characterized in parallel by stepwise yeast reconstitution and by transient expression in N. benthamiana [3]. The complete nicotine pathway was reconstructed in planta alongside its in vitro reconstitution [5]. An optimized N. benthamiana transient system, tuned by agroinfiltration parameters and co-expression of rate-limiting carotenoid genes, was then used to assign a previously unknown function to sorghum SbCYP728B35 [6].

Permissiveness is also the weakness: N. benthamiana supplies enzymes and metabolites the pathway did not encode

Reconstitution of stemmadenine biosynthesis in N. benthamiana produced shunt products from the substrate promiscuity of Redox2 and, separately, showed that stemmadenine is oxidized by endogenous N. benthamiana enzymes to condylocarpine [17]. The same study found that the Tabernanthe iboga GS ortholog generates 19Z-geissoschizine, seeding a low-flux Z-series in heterologous systems that is invisible in the native plant [17]. Agroinfiltration itself perturbs the host: Agrobacterium strain GV3101 carrying pMP90 induces de novo glandular trichomes and acylsugar production via its trans-zeatin synthase, while AGL1 and LBA4404 do not, and the authors recommend testing several strains when establishing a transient assay [1]. Endogenous P450 and glycosyltransferase activity is severe enough as a competing sink that multiplex editing of 23 fruit-expressed terpene-related genes was undertaken to build a tomato chassis for heterologous terpenes [8].

Yeast reconstitution supplies the step order and the quantitative flux that agroinfiltration cannot

Stepwise pathway reconstitution in yeast identified three P450s (CYP87G1, CYP88C7, CYP749B2) plus a short-chain dehydrogenase that jointly produce a lactone-ring intermediate, and a fourth P450 with a sulfotransferase that installs the withanolide A-ring [3]. Yeast reconstitution of the bisbenzylisoquinoline pathway established P450-catalysed regioselective oxidative dimerization and macrocyclization, and produced both native bisBIAs and non-natural analogues [20]. The first yeast galantamine platform yielded 7.0 ± 0.6 mg/L at 7.6% molar yield from 250 µM 4OMe-norbelladine, a roughly 173-fold improvement over the parental strain [13]. Optimized yeast gave 385 ± 14 mg/L quillaic acid in flasks and 471 ± 20 mg/L in fed-batch, with selectivity rising from 32.6% to 65.1% of total triterpenoids [16]. Yeast also enables causal separation that plant hosts cannot: expressing a catalytically inactive soybean isoflavone synthase showed that GmIFS2-mediated protein-protein interactions, not catalysis, partition flux between deoxyisoflavonoid and isoflavonoid routes [10].

Electron transfer and accessory organization, not P450 dosage, set the ceiling on flux in both hosts

Systematic screening of cytochrome P450 reductase and cytochrome b5 partners for NtCYP96T6 identified a Narcissus pseudonarcissus CPR mined from transcriptome data that supported the highest pathway flux [13]. Combining CYP450 expression tuning with CPR selection and aligning expression to the ethanol phase produced an 85-fold titre increase for quillaic acid [16]. The Taxol pathway requires a dedicated accessory protein: FoTO1 dramatically improves yield and alters enzyme product profiles when the pathway is reconstituted in N. benthamiana, is also functional in yeast where it resolves a key bottleneck, and interacts with and organizes multiple pathway P450s, with mutagenesis of its catalytic residues abolishing activity in vitro but not in planta [11]. Protein-level organization is now directly assayable in the plant host: transient expression in N. benthamiana with co-immunoprecipitation and size-exclusion analysis recovers heteromultimeric pathway complexes [12].

Reconstitution yield is now engineered by modifying the host rather than the pathway

Silencing of EIN2, CERK1, NPR1 and ICS increased transient GFP expression 2-, 4-, 4- and 11-fold respectively, and the npr1a/npr1b double mutant likewise improved accumulation of a transiently expressed protein [19]. Rhizobium rhizogenes A4 and disarmed A4-derived strains outperformed all tested agrobacterial strains for transient expression in N. benthamiana, were less sensitive to plant age, and outperformed standard strains in tomato, pepper and eggplant [4]. Recruiting pathway enzymes to synthetic biomolecular condensates produced several-fold yield increases in one- and three-step pathways, driven largely by several-fold higher enzyme accumulation in the condensate [9]. A 79-nucleotide Arabidopsis CYSTM1 5'UTR raised transient and stable transgene expression up to 7-fold and exceeded the TMV omega sequence by 50% in N. benthamiana [2]. Co-expressing rate-limiting precursor genes in the N. benthamiana strigolactone platform more than doubled carlactone production [6].

Candidate selection, not heterologous expression, is becoming the rate-limiting step

plantiSMASH 2.0 now reports 30,423 putative plant biosynthetic gene clusters across 430 genomes with 12 supported cluster types, substrate prediction and transcription-factor binding site detection [14]. Functional characterization of 36 P450s from the GAS clade resolved four activity groups and six MIA scaffolds, with ancestral sequence reconstitution dating the emergence of eight additional activities [7]. Minimal active-site changes among three related Catharanthus roseus P450s redirect 19E-geissoschizine into four distinct alkaloid scaffolds [21]. Reanalysis of the Rauvolfia tetraphylla yohimbane pathway found that most candidate genes exist as three homeologous copies with distinct tissue-specific expression and preferential homeolog interactions, and that several published candidates are chimeric — a whole-genome-triplication and phasing artefact [18].

Conflicts and gaps

The clearest disagreement is mechanistic. FoTO1 catalytic residues are required in vitro but dispensable in planta, and the authors explicitly read this as a non-catalytic contribution in the plant host [11]; no retrieved paper resolves whether yeast and N. benthamiana are reporting different functions of the same protein or different sensitivities of the assay.

There is no head-to-head flux benchmark. N. benthamiana studies report qualitative detection, mg per plant, or µmol per g fresh weight, whereas yeast studies report mg/L and molar yield [6] [13]; even the two papers that use both hosts for one pathway report different endpoints [3] [22]. Claims about host superiority are therefore not empirically adjudicated.

Cluster boundaries are unstable. A sulfotransferase behaves as a core, not tailoring, withanolide enzyme [3], and a liverwort cluster is defined by a shared bidirectional promoter maintained by PRC2-mediated H3K27me3 rather than by adjacency alone [15]. If core enzymes routinely sit outside rule-based cluster calls, the 30,423-cluster plantiSMASH 2.0 set [14] may systematically under-call, and no retrieved paper quantifies that error rate.

Shunt-product attribution remains unresolved. Whether the condylocarpine and isositsirikine products seen in N. benthamiana are host artefacts or genuine pathway chemistry cannot be settled without ablating the plant's own P450 and glycosyltransferase repertoire [17]; the principle has been demonstrated only in tomato [8]. Agrobacterium strain-dependent trichome and acylsugar induction likewise confounds any trichome-localized pathway readout and is not routinely controlled [1].

What would change the answer

A standard dual-host benchmark — the same multi-P450 cluster, for example the quinoa 20E cluster [23] or the withanolide cluster [3], expressed in N. benthamiana and in isogenic S. cerevisiae with isotope-dilution LC-MS quantification and the same reductase/cytochrome b5 partner panel used for galantamine [13] — would convert host-choice folklore into measured flux ratios.

CRISPR knockout or knockdown of the endogenous N. benthamiana P450 and glycosyltransferase complement, built as the tomato multiplex chassis was [8] and tested against the stemmadenine system [17], would assign shunt products to host or pathway.

Haplotype-resolved, homeolog-aware assemblies with isoform-level expression, as argued for Rauvolfia [18], applied prospectively before reconstitution would test whether failed clusters are mis-annotated rather than non-functional.

Finally, experimental validation of a stratified random sample of plantiSMASH 2.0 predictions by standardized agroinfiltration [14], and a proteome-wide proximity-labelling and co-immunoprecipitation survey for FoTO1-like P450-organizing proteins [11], would establish how general the context-dependence documented in individual pathways actually is.

References

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[3] Reynolds, E., Trauger, M., Li, F.-S., Huang, J., Moss, T., Christ, B., Xu, M., Knoch, E., Weng, J.-K.. "Elucidation of gene clusters underlying withanolide biosynthesis in ashwagandha through yeast metabolic engineering". bioRxiv. 2024-12-25T00:00:00.000Z. URL: https://doi.org/10.1101/2024.12.24.630284

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[7] Besseau, S., Durand, M., Zamar, D.-L., Lezin, E., Meteignier, L.-V., Birer Williams, C., Dutilleul, C., Perrot, T., Lanoue, A., Oudin, A., Papon, N., Sun, C., St-Pierre, B., Courdavault, V.. "Evolution of CYP71D as a driving force of the diversification of monoterpene indole alkaloid biosynthesis". bioRxiv. 2025-01-11T00:00:00.000Z. URL: https://doi.org/10.1101/2025.01.09.632084

[8] Deans, N. C., Cody, J., Reist, L., Hamilton, J. P., Starker, C., Prichard, L., Wood, J. C., Vaillancourt, B., Hamberger, B., Voytas, D., Buell, C. R.. "Engineering carotenoid and steroidal glycoalkaloid depleted tomato fruit for heterologous production of high value terpenes". bioRxiv. 2026-05-15T00:00:00.000Z. URL: https://doi.org/10.64898/2026.05.13.724861

[9] Lindstrom Battle, A. L., Barrett, A. W., Fricker, M. D., Sweetlove, L. J.. "Localising enzymes to biomolecular condensates increases their accumulation and benefits engineered metabolic pathway performance in Nicotiana benthamiana". bioRxiv. 2025-01-29T00:00:00.000Z. URL: https://doi.org/10.1101/2025.01.29.635479

[10] Liu, C., Li, S., Han, J.. "Elucidating the metabolic roles of isoflavone synthase-mediated protein-protein interactions in yeast". bioRxiv. 2024-10-24T00:00:00.000Z. URL: https://doi.org/10.1101/2024.10.24.620109

[11] Wick, C., Somani, A., Liu, J., Karunadasa, S. S., Xu, S.-L., Fordyce, P. M., McClune, C. J., Sattely, E.. "FoTO1 orchestrates Taxol biosynthesis through catalytic and non-catalytic mechanisms". bioRxiv. 2026-03-24T00:00:00.000Z. URL: https://doi.org/10.64898/2026.03.21.713420

[12] Dwivedi, V., Okertchiri, E., Yokom, A., Schenck, C.. "A Metabolic Complex Involved in Tomato Specialized Metabolism". bioRxiv. 2025-02-17T00:00:00.000Z. URL: https://doi.org/10.1101/2025.02.17.638719

[13] Holtz, M., de Haan, S., Castellvi Domingo, C., Madsen, N. G., Salazar, M. D. F., Hansson, F. G., van Aalst, A. C. A., Arnesen, J. A., Crocoll, C., d'Oelsnitz, S., Jensen, M. K., Acevedo-Rocha, C. G.. "Biocatalytic Production of Galantamine in Yeast through Cytochrome P450 Optimization". bioRxiv. 2026-09-11T00:00:00.000Z. URL: https://doi.org/10.64898/2026.09.09.750345

[14] Del Pup, E., Owen, C., Luo, Z., Augustijn, H. E., Draisma, A., Polturak, G., Kautsar, S. A., Osbourn, A., van der Hooft, J. J. J., Medema, M. H.. "plantiSMASH 2.0: improvements to detection, annotation, and prioritization of plant biosynthetic gene clusters". bioRxiv. 2025-10-28T00:00:00.000Z. URL: https://doi.org/10.1101/2025.10.28.683968

[15] Wei, G., Kawaguchi, T., Romani, F., Flores-Sandoval, E., Xie, M., Chen, X., Koedaka, T., Iwasaki, K.-i., Nakanishi, M., Hemmi, H., Chen, J.-G., Bowman, J., Haseloff, J., Matsui, K., Chen, F.. "Neryl diphosphate-derived monoterpene biosynthesis via a biosynthetic gene cluster in the liverwort Marchantia polymorpha". bioRxiv. 2026-07-17T00:00:00.000Z. URL: https://doi.org/10.64898/2026.07.16.738752

[16] Han, Y., Coe, L., Voss, J. D., Ebert, B. E.. "Optimizing cytochrome P450 activity for quillaic acid biosynthesis in Saccharomyces cerevisiae". bioRxiv. 2025-11-11T00:00:00.000Z. URL: https://doi.org/10.1101/2025.11.09.685466

[17] Kamileen, M. O., Nakamura, Y., Sigmund, M., Keshan, R., Grabe, V., Heinicke, S., Kunert, M., Hong, B., Alam, R., Kang, G., Caputi, L., O'Connor, S. E.. "Conserved early steps of stemmadenine biosynthesis". bioRxiv. 2025-11-26T00:00:00.000Z. URL: https://doi.org/10.1101/2025.11.23.690000

[18] Dwivedi, M., Vijay, N.. "WGT-aware analysis reveals increased complexity in the yohimbane biosynthesis pathway of Rauvolfia tetraphylla". bioRxiv. 2026-04-03T00:00:00.000Z. URL: https://doi.org/10.64898/2026.02.22.707333

[19] Dodds, I. L., Watts, E. C., Schuster, M., Buscaill, P., Tumtas, Y., Holton, N. J., Song, S.-j., Stuttmann, J., Joosten, M. H. A. J., Bozkurt, T. O., van der Hoorn, R. A. L.. "Immunity gene silencing increases transient protein expression in Nicotiana benthamiana". bioRxiv. 2025-01-31T00:00:00.000Z. URL: https://doi.org/10.1101/2025.01.30.635616

[20] Li, Q., Li, X., Jiao, X., Cui, G., Tan, X., Ma, Y., Wang, Y., Zhao, Y., Wang, J., Xu, W., Chen, T., Hu, Y., Su, P., Zhang, Y., Nielsen, J., Chen, Y., Guo, J., Huang, L.. "Biosynthesis of bisbenzylisoquinoline alkaloids". bioRxiv. 2026-05-26T00:00:00.000Z. URL: https://doi.org/10.64898/2026.05.22.726869

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How this survey was made

This survey answers one question: “How have plant cytochrome P450 multienzyme clusters been functionally reconstituted in Nicotiana benthamiana and yeast for the discovery of specialized metabolic pathways?”

We keep our own collection of recent preprints and refresh it every day from bioRxiv, medRxiv, arXiv, PsyArXiv, SocArXiv and ChemRxiv. For each survey, we search that collection for recent work and keep the strongest matches: papers that use the same terms as the question, and papers on the same topic in different words.

The text was written from the titles and abstracts of those papers; the full texts were not read. Statements that draw on a particular paper are marked with a number in brackets. The reference list just above is the 23 papers whose work this text draws on, chosen from the 123 papers those searches returned; the rest of the shortlist is not printed. It is not a complete review of everything published on the question.

Treat this as a starting point and a reading list, not a peer-reviewed review. The searches behind this survey ran on October 22, 2026 and covered work from the previous two years.