Combining Rational and Evolutionary Approaches to Optimize Enzyme Activity in Saccharomyces cerevisiae

Metabolic engineering has become an increasingly important tool for the production of bulk and fine chemicals. New biosynthetic pathways can be built in a tractable production host using enzymes from a wide variety of organisms. However, these enzymes did not evolve to function in their new host, and as a result their activity may be unacceptably low. Additionally, the host has not adapted to support this new pathway, and its response to any new stresses imposed by the pathway may further limit productivity. I describe two methods for optimizing the host-enzyme interface, using an evolutionary approach to adapt an enzyme to its new host and a rational approach to modify the host in response. Using a synthetic RNA switch to screen for improvements in enzymatic activity in vivo, I increased the activity of a model enzyme more than 30-fold. I then used a systems-level analysis of the host to identify a stress, heme depletion, that the enzyme placed on its host. Alleviating that stress increased the activity of an optimized enzyme by a further 2.3-fold. These results highlight the advantages of combining systems and synthetic biology during the construction of a metabolic pathway. I also consider options for extending the uses of synthetic RNA switches both earlier and later in the pathway development process. An RNA switch could first be used in a functional screen for enzyme discovery and then be used to adapt the newly discovered enzyme to its production host. Finally, a variant of that switch could be used to dynamically regulate a biosynthetic pathway and improve the pathway reliability