Metabolic engineering has matured as a powerful technology to design and construct synthetic pathways in living (micro)organisms. However, living cells equipped with such synthetic pathways often suffer from unexpected perturbations in the natural network of cell metabolisms and results in poor growth and production of target metabolites. To overcome this problem, our group has developed an alternative approach, in which enzyme cascades mimicking natural and synthetic metabolic pathways are constructed outside of the cells using recombinant thermophilic enzymes. Owing to their excellent thermal stability, thermophilic enzymes can be semi-purified through a simple heat-treatment of the cell lysate of recombinant mesophiles (e.g., Escherichia coli) and are readily available as building blocks for the construction of multi-step enzyme cascades.

By using this approach, we have constructed a variety of synthetic cascades, and demonstrated one-pot production of useful chemicals. We have also developed a series of supporting technologies to improve the feasibility of the cascades. In this lecture, we will present some representative studies of our project, and will discuss about its impact and remaining challenges.