Skip to main content

Thank you for visiting 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.

Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis


Biocatalysis is an attractive route for the synthesis of complex organic molecules, such as pharmaceuticals, due to the properties of enzymes (high specificity and high catalytic rate). Ideally, we would be able to use enzymes in continuous-flow reactors to benefit from the advantages of continuous-flow chemistry (flexibility, control, product stream purity, low capital cost and improved yields for some reactions). However, continuous-flow applications for biocatalysis face substantial technical obstacles, particularly for enzymes that require cofactors. In the work presented here we tackle two of these obstacles: the provision of cofactor and cofactor recycling in flow, and enzyme immobilization without loss of activity. This is achieved through the production of modular biocatalysts that retain and recycle their cofactors, and that allow orthogonal, site-specific covalent conjugation to a surface. This generalizable engineering approach allowed us to build a complex, multistep flow reactor that outperforms previously published systems for cofactor-dependent continuous-flow biocatalysis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Biocatalyst design.
Fig. 2: Reactor design for the conversion of glycerol to a chiral d-fagomine precursor.
Fig. 3: Synthesis of d-fagomine from glycerol.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request. The gene sequences for constructs reported in this work have been deposited at GenBank ( under the accession numbers: MK910748, MK910749, MK910750, MK910751, MK910752, MK910753, MK910754, MK910755, MK910756, MK910757 and Q07159.


  1. 1.

    Wells, A. & Meyer, H.-P. Biocatalysis as a strategic green technology for the chemical industry. ChemCatChem 6, 918–920 (2014).

    CAS  Article  Google Scholar 

  2. 2.

    Turner, N. J. & O’Reilly, E. Biocatalytic retrosynthesis. Nat. Chem. Biol. 9, 285–288 (2013).

    CAS  Article  Google Scholar 

  3. 3.

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

    CAS  Article  Google Scholar 

  4. 4.

    Tamborini, L., Fernandes, P., Paradisi, F. & Molinari, F. Flow bioreactors as complementary tools for biocatalytic process intensification. Trend Biotechnol. 36, 73–88 (2017).

    Article  Google Scholar 

  5. 5.

    Zhang, Y.-H. P., Sun, J. & Ma, Y. Biomanufacturing: history and perspective. J. Ind. Microbiol. Biotechnol. 44, 773–784 (2016).

    Article  Google Scholar 

  6. 6.

    Santacoloma, P. A. & Woodley, J. M. in Cascade Biocatalysis (eds Riva, S. & Fessner, W. -D.) 231–248 (Wiley-VCH Verlag GmbH & Co. KGaA, 2014).

  7. 7.

    Weiser, D. et al. i n Biocatalysis: An Industrial Perspective (eds Gonzalo G. & de María, P) 397–430 (Royal Society of Chemistry, 2018).

  8. 8.

    Thompson, M. P., Peñafiel, I., Cosgrove, S. C. & Turner, N. J. Biocatalysis using immobilized enzymes in continuous flow for the synthesis of fine chemicals. Org. Process Res. Dev. 23, 9–18 (2019).

    CAS  Article  Google Scholar 

  9. 9.

    Dudley, Q. M., Karim, A. S. & Jewett, M. C. Cell-free metabolic engineering: biomanufacturing beyond the cell. Biotechnol. J. 10, 69–82 (2015).

    CAS  Article  Google Scholar 

  10. 10.

    López-Gallego, F., Jackson, E. & Betancor, L. Heterogeneous systems biocatalysis: the path to the fabrication of self-sufficient artificial metabolic cells. Chem.: Eur. J. 23, 17841–17849 (2017).

    Article  Google Scholar 

  11. 11.

    Akio Nakamura, H. M., Itaru, Urabe & Hirosuke, Okada Properties of glucose-dehydrogenase-poly(ethyleneglycol)-NAD conjugate as an NADH-regeneration unit in enzyme reactors. J. Ferment. Technol. 66, 267–272 (1988).

    Article  Google Scholar 

  12. 12.

    Fu, J. et al. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm. Nat. Nanotechnol. 9, 531–536 (2014).

    CAS  Article  Google Scholar 

  13. 13.

    Krzek, M., van Beek, H. L., Permentier, H. P., Bischoff, R. & Fraaije, M. W. Covalent immobilization of a flavoprotein monooxygenase via its flavin cofactor. Enzym. Microb. Technol. 82, 138–143 (2016).

    CAS  Article  Google Scholar 

  14. 14.

    Susana, V. L., Benítez-Mateos, A. I. & Fernando, L. G. Co‐immobilized phosphorylated cofactors and enzymes as self‐sufficient heterogeneous biocatalysts for chemical processes. Angew. Chem. Int. Ed. 56, 771–775 (2017).

    Article  Google Scholar 

  15. 15.

    Keatinge-Clay, A. T. Stereocontrol within polyketide assembly lines. Nat. Prod. Rep. 33, 141–149 (2016).

    CAS  Article  Google Scholar 

  16. 16.

    Dutta, S. et al. Structure of a modular polyketide synthase. Nature 510, 512–517 (2014).

    CAS  Article  Google Scholar 

  17. 17.

    Nguyen, C. et al. Trapping the dynamic acyl carrier protein in fatty acid biosynthesis. Nature 505, 427–431 (2014).

    CAS  Article  Google Scholar 

  18. 18.

    Cronan, J. E., Zhao, X. & Jiang, Y. F. attachment and synthesis of lipoic acid in Escherichia coli. Adv. Microb. Physiol. 50, 103–146 (2005).

    CAS  Article  Google Scholar 

  19. 19.

    Davies, S. G., Fletcher, A. M., Kennedy, M. S., Roberts, P. M. & Thomson, J. E. Asymmetric synthesis of D-fagomine and its diastereoisomers. Tetrahedron 74, 7261–7271 (2018).

    CAS  Article  Google Scholar 

  20. 20.

    Castillo, J. A. et al. Fructose-6-phosphate aldolase in organic synthesis: preparation of D-fagomine, N-alkylated derivatives, and preliminary biological assays. Org. Let. 8, 6067–6070 (2006).

    CAS  Article  Google Scholar 

  21. 21.

    Hartley, C. J. et al. Sugar analog synthesis by in vitro biocatalytic cascade: a comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis. PLoS ONE 12, e0184183 (2017).

    Article  Google Scholar 

  22. 22.

    Minařik, A. et al. Ligand-Directed immobilization of proteins through an esterase 2 fusion tag studied by atomic force microscopy. ChemBioChem 9, 124–130 (2008).

    Article  Google Scholar 

  23. 23.

    Huang, Y., Humenik, M. & Sprinzl, M. Esterase 2 from Alicyclobacillus acidocaldarius as a reporter and affinity tag for expression and single step purification of polypeptides. Protein Expr. Purif. 54, 94–100 (2007).

    CAS  Article  Google Scholar 

  24. 24.

    Peschke, T. et al. Self-immobilizing fusion enzymes for compartmentalized biocatalysis. ACS Catal. 7, 7866–7872 (2017).

    CAS  Article  Google Scholar 

  25. 25.

    Wang, W., Liu, M., You, C., Li, Z. & Zhang, Y.-H. P. ATP-free biosynthesis of a high-energy phosphate metabolite fructose 1,6-diphosphate by in vitro metabolic engineering. Metabol. Eng. 42, 168–174 (2017).

    CAS  Article  Google Scholar 

  26. 26.

    Schumperli, M., Pellaux, R. & Panke, S. Chemical and enzymatic routes to dihydroxyacetone phosphate. Appl. Microbiol. Biotechnol. 75, 33–45 (2007).

    Article  Google Scholar 

  27. 27.

    Britton, J., Majumdar, S. & Weiss, G. A. Continuous flow biocatalysis. Chem. Soc. Rev. 47, 5891–5918 (2018).

    CAS  Article  Google Scholar 

  28. 28.

    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).

    CAS  Article  Google Scholar 

  29. 29.

    Jemli, S., Ayadi-Zouari, D., Hlima, H. B. & Bejar, S. Biocatalysts: application and engineering for industrial purposes. Crit. Rev. Biotechnol. 36, 246–258 (2016).

    CAS  Article  Google Scholar 

  30. 30.

    Modarres, H. P., Mofrad, M. R. & Sanati-Nezhad, A. Protein thermostability engineering. RSC Adv. 6, 115252–115270 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Babich, L., Hartog, A. F., van Hemert, L. J. C., Rutjes, F. P. J. T. & Wever, R. Synthesis of carbohydrates in a continuous flow reactor by immobilized phosphatase and aldolase. ChemSusChem 5, 2348–2353 (2012).

    CAS  Article  Google Scholar 

  32. 32.

    Nakamura, A., Minami, H., Urabe, I. & Okada, H. Properties of glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate as an NADH-regeneration unit in enzyme reactors. J. Ferment. Technol. 66, 267–272 (1988).

    CAS  Article  Google Scholar 

  33. 33.

    Schoevaart, R., van Rantwijk, F. & Sheldon, R. A. A four-step enzymatic cascade for the one-pot synthesis of non-natural carbohydrates from glycerol. J. Org. Chem. 65, 6940–6943 (2000).

    CAS  Article  Google Scholar 

  34. 34.

    Schoevaart, R., van Rantwijk, F. & Sheldon, R. A. Class I fructose-1,6-bisphosphate aldolases as catalysts for asymmetric aldol reactions. Tetrahedron 10, 705–711 (1999).

    CAS  Article  Google Scholar 

  35. 35.

    Babich, L. et al. Synthesis of non-natural carbohydrates from glycerol and aldehydes in a one-pot four-enzyme cascade reaction. Green. Chem. 13, 2895–2900 (2011).

    CAS  Article  Google Scholar 

  36. 36.

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

    Article  Google Scholar 

Download references


We acknowledge the Science and Industry Endowment Fund for funding this work. We thank M. Wilding (Australian National University) and J. Oakeshott (CSIRO) for their constructive comments during the preparation of this manuscript.

Author information




C.C.W., G.S., N.J.T. and C.S. obtained the funding for this work. C.J.H., C.S., C.C.W., N.J.T., J.A.S. and G.C. conceived and designed the study. C.J.H., J.A.S., C.C.W., N.G.F., Q.I.C., A.N., A.R.F., T.N. and C.N.J. performed the experiments. C.J.H., C.C.W., J.A.S., A.R.F., A.N., T.N., Q.I.C. and C.N.J. analysed the data. A.C.W. performed the computational modelling analysis. C.J.H., C.C.W., A.N., J.A.S., N.G.F., T.N. and C.S. wrote the paper.

Corresponding author

Correspondence to Colin Scott.

Ethics declarations

Competing interests

The authors have submitted a PCT Patent Application (WO 2017_011870_A1) based on the research results reported in this paper.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Tables 1–2 and Supplementary Figs. 1–8

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing