BEAD IT ! Design of a prototype flow-microreactor for synthetic biology in vitro

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Christian R. Boehm, Paul S. Freemont, Oscar Ces

University of Cambridge, United Kingdom

Synthetic biology promises to enable the production of important commodity chemicals, drugs, and fuels from cheap and renewable feedstock. While the biosynthesis of a large number of natural products has already been achieved in genetically engineered microorganisms, the potential of in vitro synthetic biology has barely been touched due to limited availability, stability, and cost of purified enzymes. However, as biocatalyst prices continue to fall on grounds of significant progress in industrial enzymology, the attractiveness of synthetic pathway biotransformation is increasing rapidly. As a reference platform for in vitro synthetic biology, we have developed a prototype flow-microreactor for enzymatic biosynthesis to enable the small-scale evaluation of novel synthetic pathways, compartmentalization and immobilization strategies, and reaction conditions. Hereby, we report the design, implementation, and computer-aided optimization of a synthetic three-step model pathway within our microfluidic platform. A packed-bed format was shown to be optimal for enzyme compartmentalization after experimental evaluation of several approaches. Within this microreactor, the immobilized pathway’s specific substrate conversion efficiency could be significantly improved by an optimized parameter set obtained via computational modelling. While in vivo systems will always be attractive due to the intrinsic regeneration of biocatalyst and cofactors, our microreactor design provides a platform to explore new synthetic biology solutions for industrial biosynthesis in vitro.