Review article
Cell-free reactions in continuous manufacturing systems

https://doi.org/10.1016/j.cogsc.2020.100380Get rights and content

Highlights

  • Continuous manufacturing combined with cell-free enzyme transformations provide a green and sustainable route to specialty chemicals.

  • Enzyme transformations in continuous systems often have high space-time yields.

  • Protein immobilization is key when combining continuous manufacturing and cell-free technology.

  • Cofactor recycling is a major economic determinant of cell-free small molecule manufacturing.

Cell-free enzymatic transformations can overcome some of the limitations of whole-cell biocatalysis. While flow chemistry has matured into an established discipline in organic synthesis, its use in cell-free biocatalysis is just beginning to be explored. Here, we review examples of recent advancements between 2018 and 2020. We particularly focus on enzyme immobilization and cofactor recycling as these are two major research focuses in this field. This review also provides perspective on future directions.

Introduction

We define a cell-free reaction as one which uses cell-originated enzymes to produce chemicals in vitro. While whole-cell biosynthesis can perform the same function, drawbacks include impeded transport of substrates and products, chemical solubility inside the cell, carbon flux issues, the need to redesign the cell to make new products, and cell death due to product and substrate toxicity limit the scope for manufacturing of demanding molecules. In cell-free reactions, these problems can be avoided. Reaction concentrations can be tuned and toxicity is not an issue because there are no cells involved (Figure 1a). These benefits are driving cell-free approaches into industrial applications [1,2].

In continuous manufacturing (‘flow chemistry’), reactants are continually pumped through a reactor where starting material (substrate) is converted into product before leaving the reactor for collection (Figure 1b) [3,4]. Continuous manufacturing benefits include greater consistency, lower manufacturing footprint, and improved throughput. While translating traditional organic chemistry into continuous manufacturing has made significant advances, combining continuous manufacturing and cell-free transformations is less widely explored.

Section snippets

Challenges and opportunities in cell-free continuous manufacturing

In our opinion, combining cell-free biosynthesis with continuous manufacturing is limited in two ways. First, cofactors must be recycled or regenerated to reduce the cost of operating enzymatic pathways in continuous systems (this problem is also present in batch manufacturing methods). Second, more general protein immobilization techniques with better retention of enzyme activity are required. Through the examples below, we examine recent approaches addressing these limitations and provide

New directions

Recent advances in additive manufacturing techniques have the potential to greatly improve the speed and complexity with which continuous flow cell-free systems can be prototyped and studied. A 3D printing method to create a continuous flow reactor which used a phenacrylate decarboxylase to convert p-coumaric acid to 4-vinylphenol has been described [13]. The authors mixed a thermophilic enzyme (phenacrylate decarboxylase) with solubilized agarose at 42°C and extruded this ‘bioink’ mixture

Conclusion

The innovations highlighted above showcase key drivers for translating cell-free biocatalysis into industrial manufacturing. Cofactor recycling is a major determinant of success when translating research into commercial applications. For cell-free continuous systems to be broadly commercialized, a ‘plug and play’ cofactor recycling solution is needed. Additionally, most proof-of-concept studies focus on immobilization of simple homo-oligomeric enzymes, whereas real-world applications may

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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