Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Multistep enzyme cascades as a route towards green and sustainable pharmaceutical syntheses

Nature Chemistry volume 14, pages 489–499 (2022)Cite this article

Subjects

Abstract

Enzyme cascades are a powerful technology to develop environmentally friendly and cost-effective synthetic processes to manufacture drugs, as they couple different biotransformations in sequential reactions to synthesize the product. These biocatalytic tools can address two key parameters for the pharmaceutical industry: an improved selectivity of synthetic reactions and a reduction of potential hazards by using biocompatible catalysts, which can be produced from sustainable sources, which are biodegradable and, generally, non-toxic. Here we outline a broad variety of enzyme cascades used either in vivo (whole cells) or in vitro (purified enzymes) to specifically target pharmaceutically relevant molecules, from simple building blocks to complex drugs. We also discuss the advantages and requirements of multistep enzyme cascades and their combination with chemical catalysts through a series of reported examples. Finally, we examine the efficiency of enzyme cascades and how they can be further improved by enzyme engineering, process intensification in flow reactors and/or enzyme immobilization to meet all the industrial requirements.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Classification of enzyme cascades.
Fig. 2: Enzyme cascades that involve TAs for the synthesis of pharmaceutical building blocks.
Fig. 3: Parallel enzymatic cascades for the synthesis of pharmaceutical building blocks.
Fig. 4: Enzyme cascades for the synthesis of simple drugs.
Fig. 5: (Chemo)enzymatic synthesis of complex drugs.
Fig. 6: Strategies to improve pharmaceutical syntheses by enzyme cascades for integration at the industrial scale.

Similar content being viewed by others

References

  1. Pharmaceuticals market to reach USD1,310.0 billion in 2020; eruption of the COVID-19 pandemic to accelerate the demand for effective treatments and drugs worldwide GlobeNewswire https://go.nature.com/3LdrZYh (2020).

  2. Vincent Rajkumar, S. The high cost of prescription drugs: causes and solutions. Blood Cancer J. 10, 71 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Moors, E. H. M., Cohen, A. F. & Schellekens, H. Towards a sustainable system of drug development. Drug Discov. Today 19, 1711–1720 (2014).

    Article  PubMed  Google Scholar 

  4. Dunn, P. J., Wells, A. S. & Williams, M. T. Green Chemistry in the Pharmaceuticals Industry (Wiley, 2010).

  5. Jarvis, E. A. A. Green chemistry in United States science policy. Green Chem. Lett. Rev. 12, 161–167 (2019).

    Article  CAS  Google Scholar 

  6. Kumar, R., Martinez, C., Martin, V. & Wong, J. in Green Biocatalysis (ed. Patel, R. N.) 165–178 (John Wiley & Sons, Inc, 2016).

  7. Sheldon, R. A. in Green Biocatalysis (ed. Patel, R. N.) 1–15 (John Wiley & Sons, Inc, 2016).

  8. Truppo, M. D. Biocatalysis in the pharmaceutical industry: the need for speed. ACS Med. Chem. Lett. 8, 476–480 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Winkler, C. K., Schrittwieser, J. H. & Kroutil, W. Power of biocatalysis for organic synthesis. ACS Cent. Sci. 7, 55–71 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sheldon, R. A. & Woodley, J. M. Role of biocatalysis in sustainable chemistry. Chem. Rev. 118, 801–838 (2018).

    Article  CAS  PubMed  Google Scholar 

  11. Chapman, J., Ismail, A. E. & Dinu, C. Z. Industrial applications of enzymes: recent advances, techniques, and outlooks. Catalysts 8, 20–29 (2018).

    Article  CAS  Google Scholar 

  12. Mazurenko, S., Prokop, Z. & Damborsky, J. Machine learning in enzyme engineering. ACS Catal. 10, 1210–1223 (2020).

    Article  CAS  Google Scholar 

  13. Bunzel, H. A., Garrabou, X., Pott, M. & Hilvert, D. Speeding up enzyme discovery and engineering with ultrahigh-throughput methods. Curr. Opin. Struct. Biol. 48, 149–156 (2018).

    Article  CAS  PubMed  Google Scholar 

  14. Köhler, V. & Turner, N. J. Artificial concurrent catalytic processes involving enzymes. Chem. Commun. 51, 450–464 (2015).

    Article  Google Scholar 

  15. Zhang, Y. & Hess, H. Toward rational design of high-efficiency enzyme cascades. ACS Catal. 7, 6018–6027 (2017).

    Article  CAS  Google Scholar 

  16. Birmingham, W. R. & Turner, N. J. A single enzyme oxidative ‘cascade’ via a dual-functional galactose oxidase. ACS Catal. 8, 4025–4032 (2018).

    Article  CAS  Google Scholar 

  17. Lubberink, M. et al. Biocatalytic monoacylation of symmetrical diamines and its application to the synthesis of pharmaceutically relevant amides. ACS Catal. 10, 10005–10009 (2020).

    Article  CAS  Google Scholar 

  18. Roura Padrosa, D., Benítez-Mateos, A. I., Calvey, L. & Paradisi, F. Cell-free biocatalytic syntheses of l-pipecolic acid: a dual strategy approach and process intensification in flow. Green Chem. 22, 3229–3238 (2020).

    Article  Google Scholar 

  19. Lichman, B. R. et al. One-pot triangular chemoenzymatic cascades for the syntheses of chiral alkaloids from dopamine. Green Chem. 17, 852–855 (2015).

    Article  CAS  Google Scholar 

  20. Özgen, F. F., Runda, M. E. & Schmidt, S. Photo-biocatalytic cascades: combining chemical and enzymatic transformations fueled by light. ChemBioChem 22, 790–806 (2021).

    Article  PubMed  CAS  Google Scholar 

  21. Schmermund, L. et al. Photo-biocatalysis: biotransformations in the presence of light. ACS Catal. 9, 4115–4144 (2019).

    Article  CAS  Google Scholar 

  22. Cannalire, R. et al. Visible light photocatalysis in the late-stage functionalization of pharmaceutically relevant compounds. Chem. Soc. Rev. 50, 866–897 (2021).

    Article  Google Scholar 

  23. Sun, H., Zhang, H., Ang, E. L. & Zhao, H. Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates. Bioorganic Med. Chem. 26, 1275–1284 (2018).

    Article  CAS  Google Scholar 

  24. Fischer, T. & Pietruszka, J. In Natural Products via Enzymatic Reactions (ed. Piel, J.) 1–43 (Springer, 2010).

  25. Ramsden, J. I., Cosgrove, S. C. & Turner, N. J. Is it time for biocatalysis in fragment-based drug discovery? Chem. Sci. 11, 11104–11112 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kulesa, A., Kehe, J., Hurtado, J. E., Tawde, P. & Blainey, P. C. Combinatorial drug discovery in nanoliter droplets. Proc. Natl Acad. Sci. USA 115, 6685–6690 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Schwarz, J., Rosenthal, K., Snajdrova, R., Kittelmann, M. & Lütz, S. The development of biocatalysis as a tool for drug discovery. Chimia 74, 368–377 (2020).

    Article  CAS  PubMed  Google Scholar 

  28. Truppo, M. D., Rozzell, J. D., Moore, J. C. & Turner, N. J. Rapid screening and scale-up of transaminase catalysed reactions. Org. Biomol. Chem. 7, 395–398 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Slabu, I., Galman, J. L., Lloyd, R. C. & Turner, N. J. Discovery, engineering, and synthetic application of transaminase biocatalysts. ACS Catal. 7, 8263–8284 (2017).

    Article  CAS  Google Scholar 

  30. Kelly, S. A. et al. Application of ω-transaminases in the pharmaceutical industry. Chem. Rev. 118, 349–367 (2018).

    Article  CAS  PubMed  Google Scholar 

  31. Adams, J. P., Brown, M. J. B., Diaz-Rodriguez, A., Lloyd, R. C. & Roiban, G. D. Biocatalysis: a pharma perspective. Adv. Synth. Catal. 361, 2421–2432 (2019).

    CAS  Google Scholar 

  32. Ghislieri, D. & Turner, N. J. Biocatalytic approaches to the synthesis of enantiomerically pure chiral amines. Top. Catal. 57, 284–300 (2014).

    Article  CAS  Google Scholar 

  33. Kelly, S. A., Mix, S., Moody, T. S. & Gilmore, B. F. Transaminases for industrial biocatalysis: novel enzyme discovery. Appl. Microbiol. Biotechnol. 104, 4781–4794 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mathew, S. & Yun, H. ω-Transaminases for the production of optically pure amines and unnatural amino acids. ACS Catal. 2, 993–1001 (2012).

    Article  CAS  Google Scholar 

  35. Guo, F. & Berglund, P. Transaminase biocatalysis: optimization and application. Green Chem. 19, 333–360 (2017).

    Article  CAS  Google Scholar 

  36. Savile, C. K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010).

    Article  CAS  PubMed  Google Scholar 

  37. Cutlan, R., De Rose, S., Isupov, M. N., Littlechild, J. A. & Harmer, N. J. Using enzyme cascades in biocatalysis: highlight on transaminases and carboxylic acid reductases. Biochim. Biophys. Acta Proteins Proteomics 1868, 140322 (2020).

    Article  CAS  PubMed  Google Scholar 

  38. Aumala, V. et al. Biocatalytic production of amino carbohydrates through oxidoreductase and transaminase cascades. ChemSusChem 12, 848–857 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Koszelewski, D. et al. Formal asymmetric biocatalytic reductive amination. Angew. Chem. Int. Ed. 47, 9337–9340 (2008).

    Article  CAS  Google Scholar 

  40. Koszelewski, D., Lavandera, I., Clay, D., Rozzell, D. & Kroutil, W. Asymmetric synthesis of optically pure pharmacologically relevant amines employing ω-transaminases. Adv. Synth. Catal. 350, 2761–2766 (2008).

    Article  CAS  Google Scholar 

  41. Tauber, K. et al. Artificial multi-enzyme networks for the asymmetric amination of sec-alcohols. Chem. Eur. J. 19, 4030–4035 (2013).

    Article  CAS  PubMed  Google Scholar 

  42. Sattler, J. H. et al. Redox self-sufficient biocatalyst network for the amination of primary alcohols. Angew. Chem. Int. Ed. 51, 9156–9159 (2012).

    Article  CAS  Google Scholar 

  43. O’Reilly, E. et al. A regio- and stereoselective ω-transaminase/monoamine oxidase cascade for the synthesis of chiral 2,5-disubstituted pyrrolidines. Angew. Chem. Int. Ed. 126, 2479–2482 (2014).

    Article  Google Scholar 

  44. Zhang, J. et al. Cascade biocatalysis for regio- and stereoselective aminohydroxylation of styrenyl olefins to enantiopure arylglycinols. ACS Sustain. Chem. Eng. 8, 18277–18285 (2020).

    Article  CAS  Google Scholar 

  45. Sun, Z. B. et al. One pot asymmetric synthesis of (R)-phenylglycinol from racemic styrene oxide via cascade biocatalysis. ChemCatChem 11, 3802–3807 (2019).

    Article  CAS  Google Scholar 

  46. Gandomkar, S., Żądło-Dobrowolska, A. & Kroutil, W. Extending designed linear biocatalytic cascades for organic synthesis. ChemCatChem 11, 225–243 (2019).

    Article  CAS  PubMed  Google Scholar 

  47. Sheldon, R. A. & Pereira, P. C. Biocatalysis engineering: the big picture. Chem. Soc. Rev. 46, 2678–2691 (2017).

    Article  CAS  PubMed  Google Scholar 

  48. Walsh, C. T. & Wencewicz, T. A. Flavoenzymes: versatile catalysts in biosynthetic pathways. Nat. Prod. Rep. 30, 175–200 (2013).

    Article  CAS  PubMed  Google Scholar 

  49. Xu, F. Applications of oxidoreductases: recent progress. Ind. Biotechnol. 1, 38–50 (2005).

    Article  CAS  Google Scholar 

  50. Wang, X. et al. Cofactor NAD(P)H regeneration inspired by heterogeneous pathways. Chem 2, 621–654 (2017).

    Article  CAS  Google Scholar 

  51. Liu, W. & Wang, P. Cofactor regeneration for sustainable enzymatic biosynthesis. Biotechnol. Adv. 25, 369–384 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Rehn, G., Pedersen, A. T. & Woodley, J. M. Application of NAD(P)H oxidase for cofactor regeneration in dehydrogenase catalyzed oxidations. J. Mol. Catal. B: Enzym. 134, 331–339 (2016).

    Article  CAS  Google Scholar 

  53. Dall’Oglio, F. et al. Flow-based stereoselective reduction of ketones using an immobilized ketoreductase/glucose dehydrogenase mixed bed system. Catal. Commun. 93, 29–32 (2017).

    Article  CAS  Google Scholar 

  54. Liu, J., Pang, B. Q. W., Adams, J. P., Snajdrova, R. & Li, Z. Coupled Immobilized Amine Dehydrogenase and Glucose Dehydrogenase for Asymmetric Synthesis of Amines by Reductive Amination with Cofactor Recycling. ChemCatChem https://doi.org/10.1002/cctc.201601446 (2016).

  55. Velasco-Lozano, S., Benítez-Mateos, A. I. & López-Gallego, F. Co-immobilized phosphorylated cofactors and enzymes as self-sufficient heterogeneous biocatalysts for chemical processes. Angew. Chem. Int. Ed. 56, 771–775 (2017).

    Article  CAS  Google Scholar 

  56. Vrtis, J. M., White, A. K., Metcalf, W. W. & Van Der Donk, W. A. Phosphite dehydrogenase: a versatile cofactor-regeneration enzyme. Angew. Chem. Int. Ed. 41, 3257–3259 (2002).

    Article  CAS  Google Scholar 

  57. Shaw, N. M., Robins, K. T. & Kiener, A. Lonza: 20 years of biotransformations. Adv. Synth. Catal. 345, 425–435 (2003).

    Article  CAS  Google Scholar 

  58. Mitsukura, K. et al. Purification and characterization of a novel (R)-imine reductase from Streptomyces ssp. GF3587. Biosci. Biotechnol. Biochem. 75, 1778–1782 (2011).

    Article  CAS  PubMed  Google Scholar 

  59. Zhu, J. et al. Enantioselective synthesis of 1-aryl-substituted tetrahydroisoquinolines employing imine reductase. ACS Catal. 7, 7003–7007 (2017).

    Article  CAS  Google Scholar 

  60. Borlinghaus, N. & Nestl, B. M. Switching the cofactor specificity of an imine reductase. ChemCatChem 10, 183–187 (2018).

    Article  CAS  Google Scholar 

  61. Grogan, G. & Turner, N. J. InspIRED by nature: NADPH-dependent imine reductases (IREDs) as catalysts for the preparation of chiral amines. Chem. Eur. J. 22, 1900–1907 (2016).

    Article  CAS  PubMed  Google Scholar 

  62. Mangas-Sanchez, J. et al. Imine reductases (IREDs). Curr. Opin. Chem. Biol. 37, 19–25 (2017).

    Article  CAS  PubMed  Google Scholar 

  63. Ginesta, X., Pericàs, M. A. & Riera, A. Straightforward entry to the pipecolic acid nucleus. Enantioselective synthesis of baikiain. Tetrahedron Lett. 43, 779–782 (2002).

    Article  CAS  Google Scholar 

  64. Zhai, Y. & Chuang, S. S. C. Photocatalytic synthesis of pipecolic acid from lysine on TiO2: effects of the structure of catalysts and adsorbed species on chiral selectivity. Org. Process Res. Dev 22, 1636–1643 (2018).

    Article  CAS  Google Scholar 

  65. Bas, S., Kusy, R., Pasternak-Suder, M. & Nicolas, C. Total synthesis of pipecolic acid and 1-C-alkyl 1,5- iminopentitol derivatives by way of stereoselective aldol reactions from (S)-isoserinal. Org. Biomol. Chem. 16, 1118–1125 (2018).

    Article  CAS  PubMed  Google Scholar 

  66. Zhang, Y. H. et al. Stereocomplementary synthesis of pharmaceutically relevant chiral 2-aryl-substituted pyrrolidines using imine reductases. Org. Lett. 22, 3367–3372 (2020).

    Article  CAS  PubMed  Google Scholar 

  67. Sehl, T. et al. Two steps in one pot: enzyme cascade for the synthesis of nor(pseudo)ephedrine from inexpensive starting materials. Angew. Chem. Int. Ed. 52, 6772–6775 (2013).

    Article  CAS  Google Scholar 

  68. Sehl, T. et al. Efficient 2-step biocatalytic strategies for the synthesis of all nor(pseudo)ephedrine isomers. Green Chem. 16, 3341–3348 (2014).

    Article  CAS  Google Scholar 

  69. Sehl, T. et al. Asymmetric synthesis of (S)-phenylacetylcarbinol-closing a gap in C–C bond formation. Green Chem. 19, 380–384 (2017).

    Article  CAS  Google Scholar 

  70. Rother, D. et al. S-selective mixed carboligation by structure-based design of the pyruvate decarboxylase from Acetobacter pasteurianus. ChemCatChem 3, 1587–1596 (2011).

    Article  CAS  Google Scholar 

  71. Kelefiotis-Stratidakis, P., Tyrikos-Ergas, T. & Pavlidis, I. V. The challenge of using isopropylamine as an amine donor in transaminase catalysed reactions. Org. Biomol. Chem. 17, 1634–1642 (2019).

    Article  CAS  PubMed  Google Scholar 

  72. Richter, N. et al. A system for ω-transaminase mediated (R)-amination using l-alanine as an amine donor. Green Chem. 17, 2952–2958 (2015).

    Article  CAS  Google Scholar 

  73. Martinez, C. A. et al. Development of a chemoenzymatic manufacturing process for pregabalin. Org. Process Res. Dev. 12, 392–398 (2008).

    Article  CAS  Google Scholar 

  74. Chakrabarty, S., Romero, E. O., Pyser, J. B., Yazarians, J. A. & Narayan, A. R. H. Chemoenzymatic total synthesis of natural products. Acc. Chem. Res. https://doi.org/10.1021/acs.accounts.0c00810 (2021).

  75. Doyon, T. J. et al. Chemoenzymatic o-quinone methide formation. J. Am. Chem. Soc. 141, 20269–20277 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Mertens, M. A. S. et al. One-pot two-step chemoenzymatic cascade for the synthesis of a bis-benzofuran derivative. Eur. J. Org. Chem. 2019, 6341–6346 (2019).

    Article  CAS  Google Scholar 

  77. Erdmann, V. et al. Enzymatic and chemoenzymatic three-step cascades for the synthesis of stereochemically complementary trisubstituted tetrahydroisoquinolines. Angew. Chem. Int. Ed. 56, 12503–12507 (2017).

    Article  CAS  Google Scholar 

  78. Rabuffetti, M. et al. Synthesis of ribavirin, tecadenoson, and cladribine by enzymatic transglycosylation. Catalysts 9, 355 (2019).

  79. Birmingham, W. R. et al. Bioretrosynthetic construction of a didanosine biosynthetic pathway. Nat. Chem. Biol. 10, 392–399 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Fehlau, M. et al. Modular enzymatic cascade synthesis of nucleotides using a (d)ATP regeneration system. Front. Bioeng. Biotechnol. 8, 854 (2020).

  81. Huffman, M. A. et al. Design of an in vitro biocatalytic cascade for the manufacture of islatravir. Science 368, eabc1954 (2020).

    Article  CAS  Google Scholar 

  82. Wu, B. et al. Merging biocatalysis, flow, and surfactant chemistry: innovative synthesis of an FXI (Factor XI) inhibitor. Org. Process Res. Dev. https://doi.org/10.1021/acs.oprd.0c00412 (2020).

  83. Schober, M. et al. Chiral synthesis of LSD1 inhibitor GSK2879552 enabled by directed evolution of an imine reductase. Nat. Catal. 2, 909–915 (2019).

    Article  CAS  Google Scholar 

  84. Contente, M. L. & Paradisi, F. Self-sustaining closed-loop multienzyme-mediated conversion of amines into alcohols in continuous reactions. Nat. Catal. 1, 452–459 (2018).

    Article  CAS  Google Scholar 

  85. Hartley, C. J. et al. Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis. Nat. Catal. 2, 1006–1015 (2019).

    Article  CAS  Google Scholar 

  86. Al-Shameri, A. et al. Powering artificial enzymatic cascades with electrical energy. Angew. Chem. Int. Ed. 59, 10929–10933 (2020).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank SNSF (200021_192274, F.P.) and the University of Bern ‘SELF’ Postdoctoral Fellowship (SELF19-03 BIORPHANDRUG, A.I.B.-M.) for financial support.

Author information

Authors and Affiliations

  1. Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland

    Ana I. Benítez-Mateos, David Roura Padrosa & Francesca Paradisi

Authors
  1. Ana I. Benítez-Mateos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Roura Padrosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francesca Paradisi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.P. conceived the article and A.I.B.-M., D.R.P. and F.P. contributed to the discussions and wrote the manuscript. A.I.B.-M. and D.R.P. made equal contributions to the manuscript.

Corresponding author

Correspondence to Francesca Paradisi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemistry thanks Nicholas Turner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Benítez-Mateos, A.I., Roura Padrosa, D. & Paradisi, F. Multistep enzyme cascades as a route towards green and sustainable pharmaceutical syntheses. Nat. Chem. 14, 489–499 (2022). https://doi.org/10.1038/s41557-022-00931-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-022-00931-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research