Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast

Bradley, Samuel A.; Lehka, Beata J.; Hansson, Frederik G.; Adhikari, Khem B.; Rago, Daniela; Rubaszka, Paulina; Haidar, Ahmad K.; Chen, Ling; Hansen, Lea Gram; Gudich, Olga; Giannakou, Konstantina; Lengger, Bettina; Gill, Ryan T.; Nakamura, Yoko; de Bernonville, Thomas Dugé; Koudounas, Konstantinos; Romero-Suarez, David; Ding, Ling; Qiao, Yijun; Frimurer, Thomas M.; Petersen, Anja A.; Besseau, Sébastien; Kumar, Sandeep; Gautron, Nicolas; Melin, Celine; Marc, Jillian; Jeanneau, Remi; O’Connor, Sarah E.; Courdavault, Vincent; Keasling, Jay D.; Zhang, Jie; Jensen, Michael Krough

doi:10.1038/s41589-023-01430-2

Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast

Journal Article · Mon Nov 06 00:00:00 EST 2023 · Nature Chemical Biology

DOI:https://doi.org/10.1038/s41589-023-01430-2· OSTI ID:2367187

Bradley, Samuel A. ^[1]; Lehka, Beata J. ^[1]; Hansson, Frederik G. ^[1]; Adhikari, Khem B. ^[1]; Rago, Daniela ^[1]; Rubaszka, Paulina ^[1]; Haidar, Ahmad K. ^[1]; Chen, Ling ^[1]; ^[1]; Gudich, Olga ^[1]; Giannakou, Konstantina ^[1]; ^[1]; Gill, Ryan T. ^[1]; Nakamura, Yoko ^[2]; ^[3]; ^[3]; ^[1]; Ding, Ling ^[1]; ^[1]; ^[4] more »

Technical Univ. of Denmark, Lyngby (Denmark)
Max Planck Institute for Chemical Ecology, Jena (Germany)
University of Tours (France)
Univ. of Copenhagen (Denmark)
Axyntis, Pithiviers (France)
Technical Univ. of Denmark, Lyngby (Denmark); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Joint BioEnergy Institute and Environmental Genomics and Systems Biology Division; Univ. of California, Berkeley, CA (United States); Shenzhen Institutes of Advance Technologies (China)

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker’s yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231; SC0018368

OSTI ID:: 2367187

Journal Information:: Nature Chemical Biology, Journal Name: Nature Chemical Biology Vol. 19; ISSN 1552-4450

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (65)

Regioselective Arene Halogenation using the FAD-Dependent Halogenase RebH Payne, James T.; Andorfer, Mary C.; Lewis, Jared C. Angewandte Chemie International Edition, Vol. 52, Issue 20 https://doi.org/10.1002/anie.201300762	journal	April 2013
Enantioselective Syntheses of Heteroyohimbine Natural Products: A Unified Approach through Cooperative Catalysis Younai, Ashkaan; Zeng, Bi‐Shun; Meltzer, Herbert Y. Angewandte Chemie International Edition, Vol. 54, Issue 23 https://doi.org/10.1002/anie.201502011	journal	April 2015
EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9 Jessop-Fabre, Mathew M.; Jakočiūnas, Tadas; Stovicek, Vratislav Biotechnology Journal, Vol. 11, Issue 8 https://doi.org/10.1002/biot.201600147	journal	June 2016
Construction of ajmalicine and sanguinarine de novo biosynthetic pathways using stable integration sites in yeast Liu, Tengfei; Gou, Yuanwei; Zhang, Bei Biotechnology and Bioengineering, Vol. 119, Issue 5 https://doi.org/10.1002/bit.28040	journal	January 2022
The Polar Hydrophobicity of Fluorinated Compounds Biffinger, Justin C.; Kim, Hong Woo; DiMagno, Stephen G. ChemBioChem, Vol. 5, Issue 5 https://doi.org/10.1002/cbic.200300910	journal	April 2004
Improving the Stability and Catalyst Lifetime of the Halogenase RebH By Directed Evolution Poor, Catherine B.; Andorfer, Mary C.; Lewis, Jared C. ChemBioChem, Vol. 15, Issue 9 https://doi.org/10.1002/cbic.201300780	journal	May 2014
Facile in Vitro Biocatalytic Production of Diverse Tryptamines McDonald, Allwin D.; Perkins, Lydia J.; Buller, Andrew R. ChemBioChem, Vol. 20, Issue 15 https://doi.org/10.1002/cbic.201900069	journal	June 2019
Modular Combination of Enzymatic Halogenation of Tryptophan with Suzuki-Miyaura Cross-Coupling Reactions Frese, Marcel; Schnepel, Christian; Minges, Hannah ChemCatChem, Vol. 8, Issue 10 https://doi.org/10.1002/cctc.201600317	journal	May 2016
Fermentation Titer Optimization and Impact on Energy and Water Consumption during Downstream Processing Pothakos, Vasileios; Debeer, Nadine; Debonne, Ignace Chemical Engineering & Technology, Vol. 41, Issue 12 https://doi.org/10.1002/ceat.201800279	journal	October 2018
Substrate specificity of strictosidine synthase McCoy, Elizabeth; Galan, M. Carmen; O’Connor, Sarah E. Bioorganic & Medicinal Chemistry Letters, Vol. 16, Issue 9 https://doi.org/10.1016/j.bmcl.2006.01.098	journal	May 2006
Engineering a Model Cell for Rational Tuning of GPCR Signaling Shaw, William M.; Yamauchi, Hitoshi; Mead, Jack Cell, Vol. 177, Issue 3 https://doi.org/10.1016/j.cell.2019.02.023	journal	April 2019
Redesign of a Central Enzyme in Alkaloid Biosynthesis Chen, Shi; Galan, M. Carmen; Coltharp, Carla Chemistry & Biology, Vol. 13, Issue 11 https://doi.org/10.1016/j.chembiol.2006.10.009	journal	November 2006
Unlocking the Diversity of Alkaloids in Catharanthus roseus: Nuclear Localization Suggests Metabolic Channeling in Secondary Metabolism Stavrinides, Anna; Tatsis, Evangelos C.; Foureau, Emilien Chemistry & Biology, Vol. 22, Issue 3 https://doi.org/10.1016/j.chembiol.2015.02.006	journal	March 2015
Structural characterization of monoterpene indole alkaloids in ethanolic extracts of Rauwolfia species by liquid chromatography with quadrupole time-of-flight mass spectrometry Kumar, Sunil; Singh, Awantika; Bajpai, Vikas Journal of Pharmaceutical Analysis, Vol. 6, Issue 6 https://doi.org/10.1016/j.jpha.2016.04.008	journal	December 2016
Improved protein glycosylation enabled heterologous biosynthesis of monoterpenoid indole alkaloids and their unnatural derivatives in yeast Shahsavarani, Mohammadamin; Utomo, Joseph Christian; Kumar, Rahul Metabolic Engineering Communications, Vol. 16 https://doi.org/10.1016/j.mec.2022.e00215	journal	June 2023
A dynamic regression analysis tool for quantitative assessment of bacterial growth written in Python Hoeflinger, Jennifer L.; Hoeflinger, Daniel E.; Miller, Michael J. Journal of Microbiological Methods, Vol. 132 https://doi.org/10.1016/j.mimet.2016.11.015	journal	January 2017
Anxiolytic properties of the antipsychotic alkaloid alstonine Costa-Campos, L.; Dassoler, S. C.; Rigo, A. P. Pharmacology Biochemistry and Behavior, Vol. 77, Issue 3 https://doi.org/10.1016/j.pbb.2003.12.002	journal	March 2004
Leveraging synthetic biology and metabolic engineering to overcome obstacles in plant pathway elucidation Kwan, Brooke D.; Seligmann, Benedikt; Nguyen, Trinh-Don Current Opinion in Plant Biology, Vol. 71 https://doi.org/10.1016/j.pbi.2022.102330	journal	February 2023
Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae Jakočiūnas, Tadas; Bonde, Ida; Herrgård, Markus Metabolic Engineering, Vol. 28 https://doi.org/10.1016/j.ymben.2015.01.008	journal	March 2015
Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives Milne, N.; Thomsen, P.; Mølgaard Knudsen, N. Metabolic Engineering, Vol. 60 https://doi.org/10.1016/j.ymben.2019.12.007	journal	July 2020
Tailored biosynthesis of gibberellin plant hormones in yeast Kildegaard, Kanchana R.; Arnesen, Jonathan A.; Adiego-Pérez, Belén Metabolic Engineering, Vol. 66 https://doi.org/10.1016/j.ymben.2021.03.010	journal	July 2021
Discovery of Highly Potent Serotonin 5-HT₂ Receptor Agonists Inspired by Heteroyohimbine Natural Products Orr, Meghan J.; Cao, Andrew B.; Wang, Charles Tiancheng ACS Medicinal Chemistry Letters, Vol. 13, Issue 4 https://doi.org/10.1021/acsmedchemlett.1c00694	journal	March 2022
Serotonin G Protein-Coupled Receptor-Based Biosensing Modalities in Yeast Lengger, Bettina; Hoch-Schneider, Emma E.; Jensen, Christina N. ACS Sensors, Vol. 7, Issue 5 https://doi.org/10.1021/acssensors.1c02061	journal	April 2022
A Reporter System for Cytosolic Protein Aggregates in Yeast Romero-Suarez, David; Wulff, Tune; Rong, Yixin ACS Synthetic Biology, Vol. 10, Issue 3 https://doi.org/10.1021/acssynbio.0c00476	journal	February 2021
Engineered Production of Strictosidine and Analogues in Yeast Misa, Joshua; Billingsley, John M.; Niwa, Kanji ACS Synthetic Biology, Vol. 11, Issue 4 https://doi.org/10.1021/acssynbio.2c00037	journal	March 2022
A Highly Characterized Synthetic Landing Pad System for Precise Multicopy Gene Integration in Yeast Bourgeois, Leanne; Pyne, Michael E.; Martin, Vincent J. J. ACS Synthetic Biology, Vol. 7, Issue 11 https://doi.org/10.1021/acssynbio.8b00339	journal	October 2018
Directed Biosynthesis of Alkaloid Analogs in the Medicinal Plant Catharanthus roseus McCoy, Elizabeth; O'Connor, Sarah E. Journal of the American Chemical Society, Vol. 128, Issue 44 https://doi.org/10.1021/ja066787w	journal	October 2006
Strictosidine Synthase: Mechanism of a Pictet−Spengler Catalyzing Enzyme Maresh, Justin J.; Giddings, Lesley-Ann; Friedrich, Anne Journal of the American Chemical Society, Vol. 130, Issue 2 https://doi.org/10.1021/ja077190z	journal	December 2007
Tandem Action of the O ₂ - and FADH ₂ -Dependent Halogenases KtzQ and KtzR Produce 6,7-Dichlorotryptophan for Kutzneride Assembly Heemstra, John R.; Walsh, Christopher T. Journal of the American Chemical Society, Vol. 130, Issue 43 https://doi.org/10.1021/ja806467a	journal	October 2008
Total Synthesis of Vinblastine, Vincristine, Related Natural Products, and Key Structural Analogues Ishikawa, Hayato; Colby, David A.; Seto, Shigeki Journal of the American Chemical Society, Vol. 131, Issue 13 https://doi.org/10.1021/ja809842b	journal	March 2009
10′-Fluorovinblastine and 10′-Fluorovincristine: Synthesis of a Key Series of Modified Vinca Alkaloids Gotoh, Hiroaki; Duncan, Katharine K.; Robertson, William M. ACS Medicinal Chemistry Letters, Vol. 2, Issue 12 https://doi.org/10.1021/ml200236a	journal	October 2011
Integrating carbon–halogen bond formation into medicinal plant metabolism Runguphan, Weerawat; Qu, Xudong; O’Connor, Sarah E. Nature, Vol. 468, Issue 7322 https://doi.org/10.1038/nature09524	journal	November 2010
StringTie enables improved reconstruction of a transcriptome from RNA-seq reads Pertea, Mihaela; Pertea, Geo M.; Antonescu, Corina M. Nature Biotechnology, Vol. 33, Issue 3 https://doi.org/10.1038/nbt.3122	journal	February 2015
Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity Stavrinides, Anna; Tatsis, Evangelos C.; Caputi, Lorenzo Nature Communications, Vol. 7, Issue 1 https://doi.org/10.1038/ncomms12116	journal	July 2016
Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method Gietz, R. Daniel; Schiestl, Robert H. Nature Protocols, Vol. 2, Issue 1 https://doi.org/10.1038/nprot.2007.15	journal	January 2007
De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis Haas, Brian J.; Papanicolaou, Alexie; Yassour, Moran Nature Protocols, Vol. 8, Issue 8 https://doi.org/10.1038/nprot.2013.084	journal	July 2013
De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories Liu, Quanli; Liu, Yi; Li, Gang Nature Communications, Vol. 12, Issue 1 https://doi.org/10.1038/s41467-021-26361-1	journal	October 2021
A GPCR-based yeast biosensor for biomedical, biotechnological, and point-of-use cannabinoid determination Miettinen, Karel; Leelahakorn, Nattawat; Almeida, Aldo Nature Communications, Vol. 13, Issue 1 https://doi.org/10.1038/s41467-022-31357-6	journal	June 2022
Complete biosynthesis of cannabinoids and their unnatural analogues in yeast Luo, Xiaozhou; Reiter, Michael A.; d’Espaux, Leo Nature, Vol. 567, Issue 7746 https://doi.org/10.1038/s41586-019-0978-9	journal	February 2019
A microbial supply chain for production of the anti-cancer drug vinblastine Zhang, Jie; Hansen, Lea G.; Gudich, Olga Nature, Vol. 609, Issue 7926 https://doi.org/10.1038/s41586-022-05157-3	journal	August 2022
Sarpagan bridge enzyme has substrate-controlled cyclization and aromatization modes Dang, Thu-Thuy T.; Franke, Jakob; Carqueijeiro, Ines Soares Teto Nature Chemical Biology, Vol. 14, Issue 8 https://doi.org/10.1038/s41589-018-0078-4	journal	June 2018
Chemistry and biology of monoterpene indole alkaloid biosynthesis O'Connor, Sarah E.; Maresh, Justin J. Natural Product Reports, Vol. 23, Issue 4 https://doi.org/10.1039/b512615k	journal	January 2006
Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids Gerasimenko, Irina; Sheludko, Yuri; Ma, Xueyan European Journal of Biochemistry, Vol. 269, Issue 8 https://doi.org/10.1046/j.1432-1033.2002.02878.x	journal	April 2002
Trastuzumab Deruxtecan in Previously Treated HER2-Positive Breast Cancer Modi, Shanu; Saura, Cristina; Yamashita, Toshinari New England Journal of Medicine, Vol. 382, Issue 7 https://doi.org/10.1056/NEJMoa1914510	journal	February 2020
Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis Yeh, E.; Garneau, S.; Walsh, C. T. Proceedings of the National Academy of Sciences, Vol. 102, Issue 11 https://doi.org/10.1073/pnas.0500755102	journal	March 2005
Molecular origins of fluorocarbon hydrophobicity Dalvi, V. H.; Rossky, P. J. Proceedings of the National Academy of Sciences, Vol. 107, Issue 31 https://doi.org/10.1073/pnas.0915169107	journal	July 2010
Misfolded proteins impose a dosage-dependent fitness cost and trigger a cytosolic unfolded protein response in yeast Geiler-Samerotte, K. A.; Dion, M. F.; Budnik, B. A. Proceedings of the National Academy of Sciences, Vol. 108, Issue 2 https://doi.org/10.1073/pnas.1017570108	journal	December 2010
Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin Westfall, P. J.; Pitera, D. J.; Lenihan, J. R. Proceedings of the National Academy of Sciences, Vol. 109, Issue 3, p. E111-E118 https://doi.org/10.1073/pnas.1110740109	journal	January 2012
Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast Qu, Yang; Easson, Michael L. A. E.; Froese, Jordan Proceedings of the National Academy of Sciences, Vol. 112, Issue 19 https://doi.org/10.1073/pnas.1501821112	journal	April 2015
DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery Kapolka, N. J.; Taghon, G. J.; Rowe, J. B. Proceedings of the National Academy of Sciences, Vol. 117, Issue 23 https://doi.org/10.1073/pnas.2000430117	journal	May 2020
Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway Jiang, Lily I.; Collins, Julie; Davis, Richard Journal of Biological Chemistry, Vol. 282, Issue 14 https://doi.org/10.1074/jbc.M609695200	journal	April 2007
Treatment of opioid use disorder with ibogaine: detoxification and drug use outcomes Brown, Thomas Kingsley; Alper, Kenneth The American Journal of Drug and Alcohol Abuse, Vol. 44, Issue 1 https://doi.org/10.1080/00952990.2017.1320802	journal	May 2017
Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences Li, W.; Godzik, A. Bioinformatics, Vol. 22, Issue 13 https://doi.org/10.1093/bioinformatics/btl158	journal	May 2006
fastp: an ultra-fast all-in-one FASTQ preprocessor Chen, Shifu; Zhou, Yanqing; Chen, Yaru Bioinformatics, Vol. 34, Issue 17 https://doi.org/10.1093/bioinformatics/bty560	journal	September 2018
The Alkaloid Alstonine: A Review of Its Pharmacological Properties Elisabetsky, E.; Costa-Campos, L. Evidence-Based Complementary and Alternative Medicine, Vol. 3, Issue 1 https://doi.org/10.1093/ecam/nek011	journal	January 2006
SeaView Version 4: A Multiplatform Graphical User Interface for Sequence Alignment and Phylogenetic Tree Building Gouy, M.; Guindon, S.; Gascuel, O. Molecular Biology and Evolution, Vol. 27, Issue 2 https://doi.org/10.1093/molbev/msp259	journal	October 2009
Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments Nour-Eldin, Hussam H.; Hansen, Bjarne G.; Nørholm, Morten H. H. Nucleic Acids Research, Vol. 34, Issue 18 https://doi.org/10.1093/nar/gkl635	journal	September 2006
Improved virus-induced gene silencing allows discovery of a serpentine synthase gene in Catharanthus roseus Yamamoto, Kotaro; Grzech, Dagny; Koudounas, Konstantinos Plant Physiology, Vol. 187, Issue 2 https://doi.org/10.1093/plphys/kiab285	journal	June 2021
Enhanced bioproduction of anticancer precursor vindoline by yeast cell factories Kulagina, Natalja; Guirimand, Grégory; Melin, Céline Microbial Biotechnology, Vol. 14, Issue 6 https://doi.org/10.1111/1751-7915.13898	journal	July 2021
How enzyme promiscuity and horizontal gene transfer contribute to metabolic innovation Glasner, Margaret E.; Truong, Dat P.; Morse, Benjamin C. The FEBS Journal, Vol. 287, Issue 7 https://doi.org/10.1111/febs.15185	journal	January 2020
Genome‐guided investigation of plant natural product biosynthesis Kellner, Franziska; Kim, Jeongwoon; Clavijo, Bernardo J. The Plant Journal, Vol. 82, Issue 4 https://doi.org/10.1111/tpj.12827	journal	April 2015
Complete biosynthesis of opioids in yeast Galanie, S.; Thodey, K.; Trenchard, I. J. Science, Vol. 349, Issue 6252 https://doi.org/10.1126/science.aac9373	journal	August 2015
BLAST+: architecture and applications Camacho, Christiam; Coulouris, George; Avagyan, Vahram BMC Bioinformatics, Vol. 10, Issue 1 https://doi.org/10.1186/1471-2105-10-421	journal	January 2009
Drug Shortages: The View Across an Ocean Shuman, Andrew; Unguru, Yoram The Oncologist, Vol. 25, Issue 4 https://doi.org/10.1634/theoncologist.2019-1010	journal	February 2020
Halogenating Enzymes for Active Agent Synthesis: First Steps Are Done and Many Have to Follow Fejzagić, Alexander Veljko; Gebauer, Jan; Huwa, Nikolai Molecules, Vol. 24, Issue 21 https://doi.org/10.3390/molecules24214008	journal	November 2019

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