A designed modular chemoenzymatic platform simplifies the manufacture of bioactive components from Coptidis Rhizoma

Abstract

Traditional Chinese medicines (TCM) represent a valuable resource for drug discovery, with numerous bioactive components identified with diverse pharmacological activities. However, the scarcity of these compounds, particularly those derived from plants, presents significant challenges for their applications. Here, we develop a modular and concise chemoenzymatic platform that features a streamlined enzymatic cascade coupled with a simple chemical module to facilitate the highly cost-effective and sustainable manufacture of bioactive components from Coptidis Rhizoma (Huanglian). After enzyme screening and engineering, as well as the implementation of a “plug-and-play” strategy, we efficiently produce various protoberberine alkaloids, including berberine (44% yield), demethyleneberberine (48% yield), jatrorrhizine (39% yield), berberrubine (31% yield), columbamine (42% yield), and palmatine (44% yield), exhibiting high robustness and industrial potential of this designed chemoenzymatic platform. This approach not only addresses challenges associated with heterologous production in engineered cells and low regioselectivity in chemical synthesis, but also establishes a paradigm for manufacturing other bioactive components derived from TCM, further facilitating the modernization of TCM.

Keywords

Chemoenzymatic synthesis / Berberine / Protoberberine alkaloid / Coptidis Rhizoma / Whole cell catalysis

References

[1] Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al.. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 2024, 630: 493-500.

[2] Amann M, Nagakura N, Zenk MH. (S)-tetrahydroprotoberberine oxidase the final enzyme in protoberberine biosynthesis. Tetrahedron Lett, 1984, 25: 953-4.

[3] Atanasov AG, Zotchev SB, Dirsch VM, Orhan IE, Banach M, Rollinger JM, et al.. Natural products in drug discovery: advances and opportunities. Nat Rev Drug Discov, 2021, 20: 200-16.

[4] Bauer W, Zenk H. Two methylenedioxy bridge forming cytochrome P-450 dependent enzymes are involved in (S)-stylopine biosynthesis. Phytochemistry, 1991, 30: 2953-61.

[5] Bennett MR, Thompson ML, Shepherd SA, Dunstan MS, Herbert AJ, Smith DRM, et al.. Structure and biocatalytic scope of coclaurine N-methyltransferase. Angew Chem Int Ed, 2018, 57: 10600-4.

[6] Chen S, Yu Y, Wang X, Guo Q, Liu W, Lu M, et al.. Analysis of a near telomere-to-telomere genome of Phellodendron amurense reveals insights into berberine biosynthesis. Nat Commun, 2025, 16: 11437.

[7] Clark JH, Holland HL, Miller JM. Hydrogen bonding in organic synthesis IV: a simple, high-yield method for the methylenation of catechols. Tetrahedron Lett, 1976, 17: 3361-4.

[8] Dong HR, Guo NX, Hu DC, Hong BK, Liao DH, Zhu HJ, et al.. Chemoenzymatic total synthesis of alchivemycin A. Nat Synth, 2024, 3: 1124-33.

[9] Fang S, Dong L, Liu L, Guo J, Zhao L, Zhang J, et al.. HERB: a high-throughput experiment- and reference-guided database of traditional Chinese medicine. Nucleic Acids Res, 2021, 49: D1197-206.

[10] Finnigan W, Thomas A, Cromar H, Gough B, Snajdrova R, Adams JP, et al.. Characterization of carboxylic acid reductases as enzymes in the toolbox for synthetic chemistry. ChemCatChem, 2017, 9: 1005-17.

[11] France SP, Lewis RD, Martinez CA. The evolving nature of biocatalysis in pharmaceutical research and development. JACS Au, 2023, 3: 715-35.

[12] Frenzel T, Zenk MH. S-adenosyl-L-methionine: 3′-hydroxy-N-methyl-(S)-coclaurine-4′-O-methyl transferase, a regio- and stereoselective enzyme of the (S)-reticuline pathway. Phytochemistry, 1990, 29: 3505-11.

[13] Fu Y, Zhu K, Liang F, Wang W, Yi S, Zeng H, et al.. Natural and engineered regioselective O-demethylation with enantioselective substrate recognition by Fe(II)/2-Oxoglutarate-Dependent Oxygenases in corydalmine biosynthesis. ACS Catal, 2026, 16: 8440-53.

[14] Galanie S, Smolke CD. Optimization of yeast-based production of medicinal protoberberine alkaloids. Microb Cell Fact, 2015, 14: 144.

[15] Gao Y, Li F, Luo Z, Deng Z, Zhang Y, Yuan Z, et al.. Modular assembly of an artificially concise biocatalytic cascade for the manufacture of phenethylisoquinoline alkaloids. Nat Commun, 2024, 15: 30.

[16] Gatland AE, Pilgrim BS, Procopiou PA, Donohoe TJ. Short and efficient syntheses of protoberberine alkaloids using palladium-catalyzed enolate arylation. Angew Chem Int Ed, 2014, 53: 14555-8.

[17] Grycova L, Dostal J, Marek R. Quaternary protoberberine alkaloids. Phytochemistry, 2007, 68: 150-75.

[18] Han J, Li S. De novo biosynthesis of berberine and halogenated benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Commun Chem, 2023, 6: 27.

[19] Honorio P, Sainimnuan S, Hannongbua S, Saparpakorn P. Binding interaction of protoberberine alkaloids against acetylcholinesterase (AChE) using molecular dynamics simulations and QM/MM calculations. Chem Biol Interact, 2021, 344: 109523.

[20] Iizuka N, Miyamoto K, Okita K, Tangoku A, Hayashi H, Yosino S, et al.. Inhibitory effect of Coptidis Rhizoma and berberine on the proliferation of human esophageal cancer cell lines. Cancer Lett, 2000, 148: 19-25.

[21] Jiang X, Zeng Z, Hua Y, Xu B, Shen Y, Xiong J, et al.. Merging C-H vinylation with switchable 6π-electrocyclizations for divergent heterocycle synthesis. J Am Chem Soc, 2020, 142: 15585-94.

[22] Jiao X, Fu X, Li Q, Bu J, Liu X, Savolainen O, et al.. De novo production of protoberberine and benzophenanthridine alkaloids through metabolic engineering of yeast. Nat Commun, 2024, 15: 8759.

[23] Kong X-P, Liu EYL, Chen Z-C, Xu ML, Yu AXD, Wu Q-Y, et al.. Synergistic Inhibition of acetylcholinesterase by alkaloids derived from Stephaniae Tetrandrae Radix, Coptidis Rhizoma and Phellodendri Chinensis Cortex. Molecules, 2019, 24: 4567.

[24] Leng L, Xu Z, Hong B, Zhao B, Tian Y, Wang C, et al.. Cepharanthine analogs mining and genomes of Stephania accelerate anti-coronavirus drug discovery. Nat Commun, 2024, 15: 1537.

[25] Li F, Yuan Z, Gao Y, Deng Z, Zhang Y, Luo Z, et al.. A concise enzyme cascade enables the manufacture of natural and halogenated protoberberine alkaloids. Nat Commun, 2025, 16: 1904.

[26] Li J, Fan G, He Y. Predicting the current and future distribution of three Coptis herbs in China under climate change conditions, using the MaxEnt model and chemical analysis. Sci Total Environ, 2020, 698: 134141.

[27] Liu H, Yuan Z, Gao Y, Li F, Deng Z, Zhang Y, et al.. A modular chemoenzymatic cascade simplifies divergent synthesis of natural and unnatural benzylisoquinoline alkaloids. Adv Sci, 2025, 13: e13926.

[28] Liu K, Jiang X. Modular and Divergent Syntheses of Protoberberine and Protonitidine Alkaloids. Org Lett, 2021, 23: 1327-32.

[29] Liu Q, Liu Y, Li G, Savolainen O, Chen Y, Nielsen J. De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories. Nat Commun, 2021, 12: 6085.

[30] Liu Y, Wang B, Shu S, Li Z, Song C, Liu D, et al.. Analysis of the Coptis chinensis genome reveals the diversification of protoberberine-type alkaloids. Nat Commun, 2021, 12: 3276.

[31] Li Y, Li J, Chen W-K, Li Y, Xu S, Li L, et al.. Tuning architectural organization of eukaryotic P450 system to boost bioproduction in Escherichia coli. Nat Commun, 2024, 15: 10009.

[32] Li Y, Zhao M, Tang R, Fang K, Zhang H, Kang X, et al.. Study on the quality of Corydalis Rhizoma in Zhejiang based on multidimensional evaluation method. J Ethnopharmacol, 2024, 328: 118047.

[33] Loeffler S, Deus-Neumann B, Zenk MH. S-adenosyl-L-methionine: (S)-coclaurine-N-methyltransferase from Tinospora cordifolia. Phytochemistry, 1995, 38: 1387-95.

[34] Loeffler S, Zenk MH. The hydroxylation step in the biosynthetic pathway leading from norcoclaurine to reticuline. Phytochemistry, 1990, 29: 3499-503.

[35] Minami H, Dubouzet E, Iwasa K, Sato F. Functional analysis of norcoclaurine synthase in Coptis japonica. J Biol Chem, 2007, 282: 6274-82.

[36] Min X, Zhu T, Hu X, Hou C, He J, Liu X. Transcriptome and metabolome analysis of isoquinoline alkaloid biosynthesis of Coptis chinensis in different years. Genes, 2023, 14: 2232.

[37] Montaño López J, Duran L, Avalos JL. Physiological limitations and opportunities in microbial metabolic engineering. Nat Rev Microbiol, 2022, 20: 35-48.

[38] Mori-Quiroz LM, Hedrick SL, De Los Santos AR, Clift MD. A unified strategy for the syntheses of the isoquinolinium alkaloids berberine, coptisine, and jatrorrhizine. Org Lett, 2018, 20: 4281-4.

[39] Muemmler S, Rueffer M, Nagakura N, Zenk MH. S-adenosyl-L-methionine: (S)-scoulerine 9-O-methyltransferase, a highly stereo- and regio-specific enzyme in tetrahydroprotoberberine biosynthesis. Plant Cell Rep, 1985, 4: 36-9.

[40] Narita Y, Inouye K. Degradation kinetics of chlorogenic acid at various pH values and effects of ascorbic acid and epigallocatechin gallate on its stability under alkaline conditions. J Agric Food Chem, 2013, 61: 966-72.

[41] Pasquet PL, Julien-David D, Zhao MJ, Villain-Gambier M, Trébouet D. Stability and preservation of phenolic compounds and related antioxidant capacity from agro-food matrix: Effect of pH and atmosphere. Food Biosci, 2024, 57: 103586.

[42] Pienkny S, Brandt W, Schmidt J, Kramell R, Ziegler J. Functional characterization of a novel benzylisoquinoline O-methyltransferase suggests its involvement in papaverine biosynthesis in opium poppy (Papaver somniferum L). Plant J, 2009, 60: 56-67.

[43] Plazas E, Avila MMC, Muñoz DR, Cuca SLE. Natural isoquinoline alkaloids: pharmacological features and multi-target potential for complex diseases. Pharmacol Res, 2022, 177: 106126.

[44] Pyne ME, Kevvai K, Grewal PS, Narcross L, Choi B, Bourgeois L, et al.. A yeast platform for high-level synthesis of tetrahydroisoquinoline alkaloids. Nat Commun, 2020, 11: 3337.

[45] Resch V, Lechner H, Schrittwieser JH, Wallner S, Gruber K, Macheroux P, et al.. Inverting the regioselectivity of the berberine bridge enzyme by employing customized fluorine-containing substrates. Chem Eur J, 2012, 18: 13173-9.

[46] Roddan R, Carter EM, Thair B, Hailes HC. Chemoenzymatic approaches to plant natural product inspired compounds. Nat Prod Rep, 2022, 39: 1375-82.

[47] Roddan R, Subrizi F, Broomfield J, Ward JM, Keep NH, Hailes HC. Chemoenzymatic cascades toward methylated tetrahydroprotoberberine and protoberberine alkaloids. Org Lett, 2021, 23: 6342-7.

[48] Rudroff F, Mihovilovic MD, Gröger H, Snajdrova R, Iding H, Bornscheuer UT. Opportunities and challenges for combining chemo- and biocatalysis. Nat Catal, 2018, 1: 12-22.

[49] Rueffer M, Amann M, Zenk MH. S-adenosyl-L-methionine: columbamine-O-methyl transferase, a compartmentalized enzyme in protoberberine biosynthesis. Plant Cell Rep, 1986, 5: 182-5.

[50] Rueffer M, Nagakura N, Zenk MH. A Highly Specific O-Methyltransferase for Nororientaline Synthesis Isolated from Argemone platyceras Cell Cultures. Planta Med, 1983, 49: 196-8.

[51] Rueffer M, Zenk MH. Berberine synthase, the methylenedioxy group forming enzyme inberberine synthesis. Tetrahedron Lett, 1985, 26: 201-2.

[52] Rueffer M, Zenk MH. Canadine synthase from Thalictrum tuberosum cell cultures catalyses the formation of the methylenedioxy bridge in berberine synthesis. Phytochemistry, 1994, 36: 1219-23.

[53] Schrittwieser JH, Groenendaal B, Resch V, Ghislieri D, Wallner S, Fischereder EM, et al.. Deracemization by simultaneous bio-oxidative kinetic resolution and stereoinversion. Angew Chem Int Ed, 2014, 53: 3731-4.

[54] Shah SBA, Kong L, Dong Y, Fu Y, Tian Y, Du Y, et al.. Selection and functional identification of berberine biosynthetic genes in Berberis amurensis. Med Plant Biol, 2025, 4: e024.

[55] Sheng X, Himo F. Enzymatic Pictet-Spengler reaction: computational study of the mechanism and enantioselectivity of morcoclaurine Synthase. J Am Chem Soc, 2019, 141: 11230-8.

[56] Steffens P, Nagakura N, Zenk MH. Purification and characterization of the berberine bridge enzyme from berberis beaniana cell cultures. Phytochemistry, 1985, 24: 2577-83.

[57] Sun Y, Chen C, Lin C, Zhang H, Lian J, Hong B. Elucidation and de novo reconstitution of glyceollin biosynthesis. Mol Plant, 2025, 18: 820-32.

[58] Sun Y, Chen Z, Wang G, Lv H, Mao Y, Ma K, et al.. De novo production of versatile oxidized kaurene diterpenes in Escherichia coli. Metab Eng, 2022, 73: 201-13.

[59] Tan H-L, Chan K-G, Pusparajah P, Duangjai A, Saokaew S, Mehmood Khan T, et al.. Rhizoma Coptidis: a potential cardiovascular protective agent. Front Pharmacol, 2016, 7: 362.

[60] Tian C, Zhang Q, Zhang Y, Yan Z, Wu Y, Liu H et al. Fine-Tuned transcription factor engineering and precursor rewiring drive enhanced production of berberine in Nicotiana benthamiana. Engineering. 2025. https://doi.org/10.1016/j.eng.2025.10.021

[61] Trong Le N, Huyen Thi Chau N, Quynh Dinh Nguyen P, Thuy Thi Tran L, Thanh Phung H, Thi Nguyen H. Green extraction of berberine from Coscinium fenestratum (Gaertn.) Colebr. using ultrasound-assisted aqueous solutions of organic acids, polyalcohols, and deep eutectic solvents. Sep Purif Technol. 2024;330:125541. https://doi.org/10.1016/j.seppur.2023.125541

[62] Valentic TR, Payne JT, Smolke CD. Structure-guided engineering of a scoulerine 9-O-methyltransferase enables the biosynthesis of tetrahydropalmatrubine and tetrahydropalmatine in yeast. ACS Catal, 2020, 10: 4497-509.

[63] Wang HW, Peng HX, Ding WW, Zhang ZY, Zheng YD, Chen SK, et al.. Optimization of ultrasound enzyme-assisted trehalose lipid extraction of Coptis alkaloids and evaluation of its anti-inflammatory effects. Ultrason Sonochem, 2025, 119: 107396.

[64] Wang J, Wang L, Lou G-H, Zeng H-R, Hu J, Huang Q-W, et al.. Coptidis Rhizoma: a comprehensive review of its traditional uses, botany, phytochemistry, pharmacology and toxicology. Pharm Biol, 2019, 57: 193-225.

[65] Wang J, Yin Y, Zhang Q, Zhang W-D, Li J. Artificial farnesol epoxidase enables a concise synthesis of meroterpenoids. Science. 2025;389:732–5. https://doi.org/10.1126/science.adt2096.

[66] Wang J, Zhao J, Yu Z, Wang S, Guo F, Yang J, et al.. Concise and modular chemoenzymatic total synthesis of bisbenzylisoquinoline alkaloids. Angew Chem Int Ed, 2025, 64: e202414340.

[67] WHO international standard. terminologies on traditional Chinese medicine, 2022. Geneva, World Health Organization

[68] Winkler A, Lyskowski A, Riedl S, Puhl M, Kutchan TM, Macheroux P, et al.. A concerted mechanism for berberine bridge enzyme. Nat Chem Biol, 2008, 4: 739-41.

[69] Wu L, Zhao B, Deng Z, Wang B, Yu Y. A biosynthetic network for protoberberine production in Coptis chinensis. Hortic Res, 2024, 11: uhad259.

[70] Xu D, Lin H, Tang Y, Huang L, Xu J, Nian S, et al.. Integration of full-length transcriptomics and targeted metabolomics to identify benzylisoquinoline alkaloid biosynthetic genes in Corydalis yanhusuo. Hortic Res, 2021, 8: 16.

[71] Xu H, Zhang Y, Wang P, Zhang J, Chen H, Zhang L, et al.. A comprehensive review of integrative pharmacology-based investigation: a paradigm shift in traditional Chinese medicine. Acta Pharm Sin B, 2021, 11: 1379-99.

[72] Xu Z, Tian Y, Wang J, Ma Y, Li Q, Zhou Y, et al.. Convergent evolution of berberine biosynthesis. Sci Adv, 2024, 10: eads3596.

[73] Yang L, Dai Y, Sun L, Wang Q, Ou Y, Deng Z, et al.. A chemoenzymatic strategy for efficient synthesis of aporphine alkaloids. ACS Catal, 2025, 15: 19918-25.

[74] Zhang Q, Wu Y-H, Huang X-S, Liu H-L, Wang Y. Design and optimization for efficient production of (S)-canadine in Escherichia coli. ACS Sustainable Chem Eng, 2024, 12: 6941-51.

[75] Zhang Z, Zhang Y, Zhang Z, Yao H, Liu H, Zhang B, et al.. Comparative Analysis of DNA Barcoding and HPLC Fingerprint to Trace Species of Phellodendri Cortex, an Important Traditional Chinese Medicine from Multiple Sources. Biol Pharm Bull, 2016, 39: 1325-30.

[76] Zhao W, Liu M, Shen C, Liu K, Liu H, Ou C, et al.. Biosynthesis of plant-specific alkaloids tetrahydroprotoberberines in engineered Escherichia coli. Green Chem, 2021, 23: 5944-55.

[77] Zhao YX, Liu P, Zhang YQ, Jiang H, Luan HF, Xu YM, et al.. Demethyleneberberine blocked the maturation of IL-1β in inflammation by inhibiting TLR4-mitochondria signaling. Int Immunopharmacol, 2022, 113: 109319.

Funding

National Natural Science Foundation of China(32501318)

the Fundamental Research Funds for the Central Universities(JUSRP124020)

the Natural Science Foundation of Jiangsu Province(BK20202002)

RIGHTS & PERMISSIONS

Jiangnan University