Reconstitution of early paclitaxel biosynthetic network.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
15 Feb 2024
15 Feb 2024
Historique:
received:
05
10
2023
accepted:
25
01
2024
medline:
16
2
2024
pubmed:
16
2
2024
entrez:
15
2
2024
Statut:
epublish
Résumé
Paclitaxel is an anticancer therapeutic produced by the yew tree. Over the last two decades, a significant bottleneck in the reconstitution of early paclitaxel biosynthesis has been the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. Here, we structurally characterize four new products of T5αH, many of which appear to be over-oxidation of the primary mono-oxidized products. By tuning the promoter strength for T5αH expression in Nicotiana plants, we observe decreased levels of these proposed byproducts with a concomitant increase in the accumulation of taxadien-5α-ol, the paclitaxel precursor, by three-fold. This enables the reconstitution of a six step biosynthetic pathway, which we further show may function as a metabolic network. Our result demonstrates that six previously characterized Taxus genes can coordinatively produce key paclitaxel intermediates and serves as a crucial platform for the discovery of the remaining biosynthetic genes.
Identifiants
pubmed: 38360800
doi: 10.1038/s41467-024-45574-8
pii: 10.1038/s41467-024-45574-8
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1419Subventions
Organisme : U.S. Department of Health & Human Services | NIH | National Center for Complementary and Integrative Health (NCCIH)
ID : AT010593
Informations de copyright
© 2024. The Author(s).
Références
Gallego-Jara, J., Lozano-Terol, G., Sola-Martínez, R. A., Cánovas-Díaz, M. & de Diego Puente, T. A compressive review about Taxol®: history and future challenges. Molecules 25, 5986 (2020).
pubmed: 33348838
pmcid: 7767101
doi: 10.3390/molecules25245986
Wani, M. C. & Horwitz, S. B. Nature as a remarkable chemist: a personal story of the discovery and development of Taxol. Anticancer Drugs 25, 482–487 (2014).
pubmed: 24413390
pmcid: 3980006
doi: 10.1097/CAD.0000000000000063
Wall, M. E. & Wani, M. C. Camptothecin and taxol: from discovery to clinic. J. Ethnopharmacol. 51, 239–253 (1996). discussion 253–4.
pubmed: 9213622
doi: 10.1016/0378-8741(95)01367-9
Wang, Y.-F. et al. Natural taxanes: developments since 1828. Chem. Rev. 111, 7652–7709 (2011).
pubmed: 21970550
doi: 10.1021/cr100147u
Liu, W. C., Gong, T. & Zhu, P. Advances in exploring alternative Taxol sources. RSC Adv. 6, 48800–48809 (2016).
doi: 10.1039/C6RA06640B
Ro, D.-K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006).
pubmed: 16612385
doi: 10.1038/nature04640
Srinivasan, P. & Smolke, C. D. Biosynthesis of medicinal tropane alkaloids in yeast. Nature 585, 614–619 (2020).
pubmed: 32879484
pmcid: 7529995
doi: 10.1038/s41586-020-2650-9
Mutanda, I., Li, J., Xu, F. & Wang, Y. Recent advances in metabolic engineering, protein engineering, and transcriptome-guided insights toward synthetic production of Taxol. Front. Bioeng. Biotechnol. 9, 632269 (2021).
pubmed: 33614616
pmcid: 7892896
doi: 10.3389/fbioe.2021.632269
Ajikumar, P. K. et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330, 70–74 (2010).
pubmed: 20929806
pmcid: 3034138
doi: 10.1126/science.1191652
Yadav, V. G. Unraveling the multispecificity and catalytic promiscuity of taxadiene monooxygenase. J. Mol. Catal. B Enzym. 110, 154–164 (2014).
doi: 10.1016/j.molcatb.2014.10.004
Sagwan-Barkdoll, L. & Anterola, A. M. Taxadiene-5α-ol is a minor product of CYP725A4 when expressed in Escherichia coli. Biotechnol. Appl. Biochem. 65, 294–305 (2018).
pubmed: 28876471
doi: 10.1002/bab.1606
Nowrouzi, B., Lungang, L. & Rios-Solis, L. Exploring optimal Taxol® CYP725A4 activity in Saccharomyces cerevisiae. Microb. Cell Fact. 21, 197 (2022).
pubmed: 36123694
pmcid: 9484169
doi: 10.1186/s12934-022-01922-1
Walls, L. E. et al. Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies. Biotechnol. Bioeng. 118, 279–293 (2021).
pubmed: 32936453
doi: 10.1002/bit.27569
Li, J. et al. Chloroplastic metabolic engineering coupled with isoprenoid pool enhancement for committed taxanes biosynthesis in Nicotiana benthamiana. Nat. Commun. 10, 4850 (2019).
pubmed: 31649252
pmcid: 6813417
doi: 10.1038/s41467-019-12879-y
Rontein, D. et al. CYP725A4 from yew catalyzes complex structural rearrangement of taxa-4 (5), 11 (12)-diene into the cyclic ether 5 (12)-oxa-3 (11)-cyclotaxane. J. Biol. Chem. 283, 6067–6075 (2008).
pubmed: 18167342
doi: 10.1074/jbc.M708950200
Barton, N. A. et al. Accessing low-oxidation state taxanes: is taxadiene-4(5)-epoxide on the taxol biosynthetic pathway? Chem. Sci. 7, 3102–3107 (2016).
pubmed: 29997802
pmcid: 6005263
doi: 10.1039/C5SC03463A
Zhou, K., Qiao, K., Edgar, S. & Stephanopoulos, G. Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nat. Biotechnol. 33, 377–383 (2015).
pubmed: 25558867
pmcid: 4867547
doi: 10.1038/nbt.3095
Biggs, B. W. et al. Orthogonal assays clarify the oxidative biochemistry of taxol P450 CYP725A4. ACS Chem. Biol. 11, 1445–1451 (2016).
pubmed: 26930136
doi: 10.1021/acschembio.5b00968
Edgar, S. et al. Mechanistic insights into taxadiene epoxidation by taxadiene-5α-hydroxylase. ACS Chem. Biol. 11, 460–469 (2016).
pubmed: 26677870
doi: 10.1021/acschembio.5b00767
Wheeler, A. L. et al. Taxol biosynthesis: differential transformations of taxadien-5 alpha-ol and its acetate ester by cytochrome P450 hydroxylases from Taxus suspension cells. Arch. Biochem. Biophys. 390, 265–278 (2001).
pubmed: 11396929
doi: 10.1006/abbi.2001.2377
Biggs, B. W. et al. Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli. Proc. Natl Acad. Sci. USA 113, 3209–3214 (2016).
pubmed: 26951651
pmcid: 4812725
doi: 10.1073/pnas.1515826113
Walker, K., Schoendorf, A. & Croteau, R. Molecular cloning of a taxa-4(20),11(12)-dien-5α-ol-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli. Arch. Biochem. Biophys. 374, 371–380 (2000).
pubmed: 10666320
doi: 10.1006/abbi.1999.1609
Schoendorf, A., Rithner, C. D., Williams, R. M. & Croteau, R. B. Molecular cloning of a cytochrome P450 taxane 10β-hydroxylase cDNA from Taxus and functional expression in yeast. Proc. Natl Acad. Sci. USA 98, 1501–1506 (2001).
pubmed: 11171980
pmcid: 29286
doi: 10.1073/pnas.98.4.1501
Jennewein, S., Rithner, C. D., Williams, R. M. & Croteau, R. B. Taxol biosynthesis: taxane 13α-hydroxylase is a cytochrome P450-dependent monooxygenase. Proc. Natl Acad. Sci. USA 98, 13595–13600 (2001).
pubmed: 11707604
pmcid: 61086
doi: 10.1073/pnas.251539398
Walker, K. & Croteau, R. Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli. Proc. Natl Acad. Sci. USA 97, 583–587 (2000).
pubmed: 10639122
pmcid: 15373
doi: 10.1073/pnas.97.2.583
Jonguitud Borrego, N. Production of novel taxane intermediates of anticancer drug Taxol using microbial consortia. [Doctoral dissertation, The University of Edinburgh] Edinburgh Research Archive. (2023).
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).
pubmed: 19627561
doi: 10.1111/j.1467-7652.2009.00434.x
De La Peña, R. & Sattely, E. S. Rerouting plant terpene biosynthesis enables momilactone pathway elucidation. Nat. Chem. Biol. 17, 205–212 (2021).
pubmed: 33106662
doi: 10.1038/s41589-020-00669-3
Reider Apel, A. et al. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 45, 496–508 (2017).
pubmed: 27899650
doi: 10.1093/nar/gkw1023
Rubenstein, S. M., Vazquez, A., Sanz-Cervera, J. F. & Williams, R. M. Synthesis of stable and radioisotopomers of Taxa-4(5),11(12)-diene, Taxa-4(20), 11(12)-diene and Taxa-4(20), 11(12)-dien-5-α-ol, early intermediates in Taxol® biosynthesis. J. Label. Comp. Radiopharm. 43, 481–491 (2000).
doi: 10.1002/(SICI)1099-1344(200004)43:5<481::AID-JLCR334>3.0.CO;2-O
Chau, M., Walker, K., Long, R. & Croteau, R. Regioselectivity of taxoid-O-acetyltransferases: heterologous expression and characterization of a new taxadien-5α-ol-O-acetyltransferase. Arch. Biochem. Biophys. 430, 237–246 (2004).
pubmed: 15369823
doi: 10.1016/j.abb.2004.07.013
Edgar, S., Li, F. S., Qiao, K., Weng, J. K. & Stephanopoulos, G. Engineering of taxadiene synthase for improved selectivity and yield of a key Taxol biosynthetic intermediate. ACS Synth. Biol. 6, 201–205 (2017).
pubmed: 27794603
doi: 10.1021/acssynbio.6b00206
Lau, W. & Sattely, E. S. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone. Science 349, 1224–1228 (2015).
pubmed: 26359402
pmcid: 6861171
doi: 10.1126/science.aac7202
Sarrion-Perdigones, A. et al. GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162, 1618–1631 (2013).
pubmed: 23669743
pmcid: 3707536
doi: 10.1104/pp.113.217661
Ahn, T., Guengerich, F. P. & Yun, C. H. Membrane insertion of cytochrome P450 1A2 promoted by anionic phospholipids. Biochemistry 37, 12860–12866 (1998).
pubmed: 9737864
doi: 10.1021/bi980804f
Laursen, T. et al. Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum. Science 354, 890–893 (2016).
pubmed: 27856908
doi: 10.1126/science.aag2347
Menhard, B. & Zenk, M. H. Purification and characterization of acetyl coenzyme A: 10-hydroxytaxane O-acetyltransferase from cell suspension cultures of Taxus chinensis. Phytochemistry 50, 763–774 (1999).
pubmed: 10192963
doi: 10.1016/S0031-9422(98)00674-8
Schultz, B. J., Kim, S. Y., Lau, W. & Sattely, E. S. Total biosynthesis for milligram-scale production of etoposide intermediates in a plant chassis. J. Am. Chem. Soc. 141, 19231–19235 (2019).
pubmed: 31755709
pmcid: 7380830
doi: 10.1021/jacs.9b10717
Luo, X. et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature 567, 123–126 (2019).
pubmed: 30814733
doi: 10.1038/s41586-019-0978-9
Li, Z. et al. Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications. J. Biol. Chem. 295, 833–849 (2020).
pubmed: 31811088
doi: 10.1016/S0021-9258(17)49939-X
Hausjell, J., Halbwirth, H. & Spadiut, O. Recombinant production of eukaryotic cytochrome P450s in microbial cell factories. Biosci. Rep. 38, BSR20171290 (2018).
pubmed: 29436484
pmcid: 5835717
doi: 10.1042/BSR20171290
Shang, Y. & Huang, S. Engineering plant cytochrome P450s for enhanced synthesis of natural products: past achievements and future perspectives. Plant Commun. 1, 100012 (2020).
pubmed: 33404545
doi: 10.1016/j.xplc.2019.100012
McDaniel, R., Ebert-Khosla, S., Hopwood, D. A. & Khosla, C. Engineered biosynthesis of novel polyketides: actVII and actIV genes encode aromatase and cyclase enzymes, respectively. J. Am. Chem. Soc. 116, 10855–10859 (1994).
doi: 10.1021/ja00103a001
Paddon, C. J. et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528–532 (2013).
pubmed: 23575629
doi: 10.1038/nature12051
Michener, J. K., Nielsen, J. & Smolke, C. D. Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases. Proc. Natl Acad. Sci. USA 109, 19504–19509 (2012).
pubmed: 23129650
pmcid: 3511110
doi: 10.1073/pnas.1212287109
Geisler, K., Jensen, N. B., Yuen, M. M. S., Madilao, L. & Bohlmann, J. Modularity of conifer diterpene resin acid biosynthesis: P450 enzymes of different CYP720B clades use alternative substrates and converge on the same products. Plant Physiol. 171, 152–164 (2016).
pubmed: 26936895
pmcid: 4854711
doi: 10.1104/pp.16.00180
Forman, V. et al. A gene cluster in Ginkgo biloba encodes unique multifunctional cytochrome P450s that initiate ginkgolide biosynthesis. Nat. Commun. 13, 5143 (2022).
pubmed: 36050299
pmcid: 9436924
doi: 10.1038/s41467-022-32879-9
Galanie, S., Thodey, K., Trenchard, I. J., Filsinger Interrante, M. & Smolke, C. D. Complete biosynthesis of opioids in yeast. Science 349, 1095–1100 (2015).
pubmed: 26272907
pmcid: 4924617
doi: 10.1126/science.aac9373
Huang, A. C. et al. A specialized metabolic network selectively modulates Arabidopsis root microbiota. Science 364, eaau6389 (2019).
pubmed: 31073042
doi: 10.1126/science.aau6389
De La Peña, R. et al. Complex scaffold remodeling in plant triterpene biosynthesis. Science 379, 361–368 (2023).
pubmed: 36701471
doi: 10.1126/science.adf1017
Pateraki, I. et al. Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii. Elife 6, e23001 (2017).
pubmed: 28290983
pmcid: 5388535
doi: 10.7554/eLife.23001
Lanier, E. R., Andersen, T. B. & Hamberger, B. Plant terpene specialized metabolism: complex networks or simple linear pathways? Plant J. 114, 1178–1201 (2023).
pubmed: 36891828
doi: 10.1111/tpj.16177
Kaspera, R. & Croteau, R. Cytochrome P450 oxygenases of Taxol biosynthesis. Phytochem. Rev. 5, 433–444 (2006).
pubmed: 20622990
pmcid: 2901147
doi: 10.1007/s11101-006-9006-4
Williams, D. C. et al. Heterologous expression and characterization of a ‘Pseudomature’ form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction. Arch. Biochem. Biophys. 379, 137–146 (2000).
pubmed: 10864451
doi: 10.1006/abbi.2000.1865