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.

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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.


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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.

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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.

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Correspondence to Colin Scott.

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

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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).

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