Engineered Enzymes that Retain and Regenerate their Cofactors Enable Continuous-Flow Biocatalysis

Carol J. Hartley, Charlotte C. Williams, Judith A. Scoble, Quentin I. Churches, Andrea North, Nigel G. French, Tom Nebl, Greg Coia, Andrew C. Warden, Greg Simpson, Andrew R. Frazer, Chantel Nixon Jensen, Nicholas J. Turner, Colin Scott

Preprint posted on 5 March 2019

Article now published in Nature Catalysis at

How far it’ll flow, how far it’ll go: the use of engineered enzymes in continuous-flow catalytic biosynthesis

Selected by Zhang-He Goh

Background of preprint

Despite researchers’ ability to immobilise enzymes for continuous-flow applications, most continuous-flow applications rely heavily on cofactor-independent enzymes. The difficulty in using cofactor-dependent enzymes is twofold: the cofactor needs to be recycled for synthetic feasibility on an industrial scale; yet the recycling of the cofactor needs it to be able to diffuse throughout the reactor. This precludes the use of cofactors (and thus cofactor-dependent enzymes) in continuous-flow reactors due to the current lack of an engineering workaround. Therefore, in this preprint, Hartley et al. provide a proof-of-concept for the incorporation of cofactor-dependent enzymes into biocatalytic continuous-flow reactors.

Key findings of preprint

The findings of this preprint by Hartley et al. can be divided into three parts: (A) design of the “nanomachine”, which refers to the authors’ biocatalyst that retains and recycles its cofactor; (B) assembly of nanomachine; and (C) characterisation of the nanomachine’s function.

(A) Design of nanomachine

Inspired by enzymes that retain their substrates via covalent attachment during the reaction cascade, Hartley et al. invented a nanomachine with three domains (Figure 1). The first, a cofactor-dependent catalyst, carries out the desired chemical reaction. The second domain helps to recycle that cofactor catalyst. The last domain is an esterase conjugation domain that allows the immobilisation of the whole nanomachine to a surface. These three domains were encoded by a single gene for production in E. coli with a short spacer of 2-20 amino acids between each domain. The cofactor was modified and conjugated to the spacer between the catalytic and cofactor recycling modules using a flexible maleimide-functionalised polyethylene glycol (PEG) linker. This PEG linker allowed the modified cofactor to move between sites, thus overcoming the problem of cofactor diffusion faced by incorporating cofactor-dependent enzymes in continuous-flow bioreactors.

Figure 1. The structure of the nanofactory in the preprint by Hartley et al.. Reproduced from the original preprint by Hartley et al. under a CC BY-NC-ND 4.0 licence.

(B) Assembly of nanomachine

Due to the challenge in synthesising the highly chiral D-fagomine using non-biological catalysts, Hartley et al. decided to work on this synthesis for their proof-of-concept in this preprint. For the key steps in the assembly of the nanomachine, Hartley et al. chose the respective enzymes for their compatibility in batch reactions, high catalytic rates, relatively simple quaternary structures, and thermostability (Table 1). For the conjugation module, the authors chose a serine hydrolase enzyme coupled with the suicide inhibitor trifluoroketone to catalyse the formation of a site-specific and stable covalent bond between the inhibitor and the catalytic serine residue.

Table 1. Key steps in D-fagomine synthesis, their accompanying enzymes, and their respective yields. Parts of the nanofactory are highlighted in orange.

In synthesising the nanofactory, the authors created two gene fusions: the first contained the genes for GlpKTk (N-terminus) and for AceKMs (C-terminus); while the second contained the genes for G3PDEc (N-terminus) and NOXCa (C-terminus). For each of these fusions, the authors fused E2Aa to the respective C-termini. Importantly, the authors verified that their fused proteins largely retained their original catalytic functions as measured by the affinity for the enzyme for its substrate (kM) and the catalytic efficiency of the enzyme (kcat). This led Hartley et al. to conclude that their technique may be applicable to other biocatalytic systems as well.

(C) Characterisation of nanofactory

In the third section of their preprint, Hartley et al. found that their nanomachine reactors were able to synthesise products in high yields (Table 1). This observation could be ascribed to their use of a continuous flow reaction, which allowed the authors to mitigate the two limiting factors in these reactions: (i) product inhibition and (ii) equilibrium control.

Hartley et al. also characterised the turnover number (also known as the catalytic efficiency of the enzyme, kcat) for the respective cofactors. Because the turnover number of the cofactors in both reactions was identified to be limited by the inactivation of the domain rather than losses of the cofactors themselves, Hartley et al. posit that this could be improved by enhancing the stability of the enzymes involved.

What I like about this preprint

I selected this preprint for two reasons. The first is the significance behind the work: Hartley et al. present a solution to the challenge of using cofactor-dependent enzymes in continuous flow synthesis. Specifically, the authors describe the invention of a nanofactory that consists of three linked modules, along with a conjugated modified cofactor that can be recycled throughout the synthesis.

Second, the work is also elegant in its own way—to paraphrase Moana, everything is by design, and every component in the nanofactory has a role in that nanofactory. The authors have documented their painstaking efforts in the supplementary section, which is certainly worth a read: just to name a few, these experiments include (i) transformation of the cells, (ii) synthesis and purification of the enzymes, and (iii) characterisation of enzymatic activity using various assays.

Future directions

The modular aspect of the system will probably lend itself to other applications, and future directions are likely to develop in the following three ways:

  • Application of the nanofactory’s modular approach to other synthetic challenges. The same principle behind the nanofactory may be adapted to link different combinations and permutations of other enzymes together. This would enable scientists and engineers to perform series of other stereoselective syntheses. While it is conceivable that this method may be applied to achieve a near-infinitesimal number of synthetic routes, such an ambition may be tempered by biological limitations. For example, as transformation efficiencies of coli decrease as the size of the plasmid to be inserted increases, there may be a practical maximum to the gene size which can be transformed into the E. coli.1 As a result, there may be an upper limit to the number of modules that can be incorporated into a single nanofactory produced by E. coli. Therefore, larger nanofactories may need be achieved through other biotechnological strategies and by using other producing organisms, such as Chinese Hamster Ovary (CHO) mammalian cells.
  • Optimisation of the nanofactory’s modular approach to boost yields. Hartley et al. point out that stability is likely the main factor that limits the catalytic efficiency, kcat, of the enzymes. Therefore, future work will involve the enhancement of the stability of these enzymes.
  • Optimisation of the individual enzymatic modules in the nanofactory. Due to the multifactorial nature of the conditions in the nanofactory, there is plenty of room for optimisation beyond the extensive work already undertaken by the preprint authors. Factors such as the peptide spacer of 2-20 amino acids between the modules, temperature, and flow rate are all examples of aspects that could be further optimised to boost yields of this nanofactory. This is especially the case for advances in biotechnology, in which a balance must be struck between considerations beyond the optimisation of the desired protein itself—these may include the growth rate of host cells that express the desired protein, as well as the expression levels of the desired protein in host cells.

All in all, the potential applications of such a technology are especially exciting. Watch how far it flows.


  1. Hanahan, D. Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 1983, 166, 557-580.

Tags: biocatalysis, catalysis, cofactor immobilisation, continuous flow biocatalysis, enzyme immobilisation, enzymes, nanofactory

Posted on: 22 March 2019 , updated on: 29 September 2019


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Author's response

Carol Hartley, Charlotte Williams, and Colin Scott shared

Carol Hartley (First Author)

Thank you for choosing our article for preLights and for the excellent Moana analogy – we are also excited to see “how far it’ll go, how it’ll flow” as a chemogenetically engineered solution for using cofactor-dependant enzymes in flow biocatalysis.

My co-authors and I were pleased with the demonstrated functionality of our meticulously designed Nanomachines and the success of the Nanofactory proof of concept for D-fagomine synthesis – combining our many different areas of expertise was necessary to ensure that the design principles were sound, and were soundly executed.  Functional expression of large multimeric fusion proteins in E.coli is not guaranteed but has been generally successful for several such Nanomachine proteins now, as has modification and bioconjugation of NAD, NADP and ADP cofactors to various nanomachines. We now working to extend the concept to other applications such as synthesis of conjugated chiral amines and stereo-inversion of amines and alcohols.

Further exploration and optimisation of design components is also underway including alternative cofactor modifications, different linker chemistries to attach the modified cofactors to the Nanomachine proteins, different spacer regions between the protein domains of the Nanomachine fusion proteins and engineering individual enzyme components for enhanced stability and efficiency.

In order to design fit-for-purpose catalysts for industry we are also exploring various other chemistries and alternative means of immobilising the Nanomachines for flow biocatalysis.

Apart from extending the nanofactory concept to other useful chemical syntheses, one can envisage the potential to assemble self-regulating in vitro metabolic networks using finely controlled interlinked nanomachine flow reactor systems for ex vivo synthetic biology and cell-free metabolic engineering applications, or to spatially array nanomachines to create sequentially ordered Nanofactories using techniques such as DNA origami.

Let’s see how far it’ll flow.

Charlotte Williams (Second Author)

We have explored methods to chemically functionalize a range of organic cofactors, small molecules that are required by a vast array of enzymes for a range of transformations. These transformations may be of biological significance, or can be tailor-made to produce complex, high value industrially or pharmaceutically relevant products.

Organic cofactors are highly phosphorylated, water-soluble organic small molecules that lend themselves to functionalisation due to a number of chemical reactive sites. It is important to understand the way cofactors bind to their corresponding enzymes to ensure that modification does not affect the cofactors ability to act as a catalyst for those dependant enzymes; protein crystallography, molecular modelling and SAR studies are techniques we can use to understand protein binding.

While many organic cofactors tend to possess a core ‘parent’ chemical structure, such as the nucleotide, adenosine monophosphate, synthetic methods used to chemically modify each molecule needs to be individually optimised, as well as the approach used to purify the functionalised, tethered cofactors.

We have modified ADP, ATP, NAD+, and NADP+, and moving forward we seek to understand the conjugation of other organic cofactors, such as, TPP, FAD, CoA and SAM amongst others. We also see value in exploring analogues (or fragments) of organic cofactors, which will also open up our understanding of the way enzymes utilise these molecules as catalysts.

There exists a number of ways to achieve chemical bioconjugation of the tethered cofactor to the enzymatic fusion protein. We have demonstrated the use of Michael addition chemistry, one of many chemical methods. The burgeoning area of orthogonal bioconjugation means that any protein can be adapted (or we can use the protein’s natural synthetic handles) for installation of a tethered cofactor, via a number of chemical, or enzymatic, coupling methods.

Our methods of conjugating a cofactor to its corresponding cofactor-dependent enzyme–cofactor-recycling enzyme fusion, with a flexible, length-optimised linker, can be applied to other biocatalytic systems that require these highly utilised, complex and valuable biocatalysts.

Colin Scott (Corresponding Author)

Thanks for selecting our work for preLights – we’re all very proud of this work, and it’s nice to see it picked up as a highlight.

The work described in the paper describes a complex project with lots of moving parts – we needed a really multidisciplinary team to make it work, and at we were fortunate to be able to assemble a really great team from CSIRO and the University of Manchester. As you note – every step and component has a purpose in the nanomachine, but they also have other potential applications too: the immobilisation domain could be used to immobilise other proteins to surfaces on magnetic beads for simple biocatalyst recovery in batch reactors, for example.

In the next steps we’ll probably look at other reactions to demonstrate that the technology is completely generalizable – we’re very happy to talk with pharmaceutical manufacturers about this, so that we’re doing something that will be useful to them! As you note in your highlight, we’d also like to explore some of the components and their parameters in more depth. We’re also thinking about ways in which we can simplify the construction of the machines and drive down the cost of their production – this will broaden the applications in which nanomachines can be used.

Thanks so much for selecting our work as one of your highlights, I’ll certainly be following preLights in future to see other highlights from the preprint archives!

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