Tools and strategies for constructing cell-free enzyme pathways

Kerstin Petroll, Dominik Kopp, Andrew Care, Peter L. Bergquist, Anwar Sunna*

*Corresponding author for this work

    Research output: Contribution to journalReview articlepeer-review

    27 Citations (Scopus)


    Single enzyme systems or engineered microbial hosts have been used for decades but the notion of assembling multiple enzymes into cell-free synthetic pathways is a relatively new development. The extensive possibilities that stem from this synthetic concept makes it a fast growing and potentially high impact field for biomanufacturing fine and platform chemicals, pharmaceuticals and biofuels. However, the translation of individual single enzymatic reactions into cell-free multi-enzyme pathways is not trivial. In reality, the kinetics of an enzyme pathway can be very inadequate and the production of multiple enzymes can impose a great burden on the economics of the process. We examine here strategies for designing synthetic pathways and draw attention to the requirements of substrates, enzymes and cofactor regeneration systems for improving the effectiveness and sustainability of cell-free biocatalysis. In addition, we comment on methods for the immobilisation of members of a multi-enzyme pathway to enhance the viability of the system. Finally, we focus on the recent development of integrative tools such as in silico pathway modelling and high throughput flux analysis with the aim of reinforcing their indispensable role in the future of cell-free biocatalytic pathways for biomanufacturing.

    Original languageEnglish
    Pages (from-to)91-108
    Number of pages18
    JournalBiotechnology Advances
    Issue number1
    Early online date3 Dec 2018
    Publication statusPublished - Jan 2019


    • Synthetic biology
    • In vitro biocatalysis
    • Cell-free enzyme pathways
    • Multi-enzyme cascades
    • Pathway design
    • Enzyme immobilisation
    • Kinetic modelling
    • Metabolite analysis


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