Phage Assisted Evolutions for Engineering Biosynthetic Pathways

Abstract

Phage Assisted Continuous Evolution (PACE) and its derivative techniques are powerful tools for evolving improved and altered enzymatic activities. Prior to this work, no one had demonstrated the applicability of these techniques for metabolic pathway enzyme engineering. Here I demonstrate ways in which bacterial sensors of metabolically relevant phenotypes can be optimized and applied to the PACE system to evolve greater in vivo pathway activity based on individual improvements to target enzyme activities. First, I optimized a system for sensing polyhydroxyalkanoate inclusion bodies to evolve increased production of polyhydroxybutyrate (PHB) in E. coli. I demonstrated increased production either by evolving a single enzyme in a pathway or by evolving an entire transcript containing all three enzymes responsible for PHB production. I also engineered the E. coli formaldehyde repressor circuit to have greater sensitivity and used this sensor in a non-continuous variant of PACE to evolve a series of mutant Bacillus methanolicus methanol dehydrogenase genes in E. coli. I characterized this set of evolved dehydrogenases in vitro to show that they have significantly improved kinetic parameters, and, with collaborators in the Stephanopoulos lab, I also demonstrated that they enable us to incorporate roughly twice as much methanol through the ribulose monophosphate pathway in E. coli compared to the state-of-the-art methanol dehydrogenases under equivalent conditions. Together, the results of these projects demonstrate that in addition to its many previously reported capabilities, PACE can also be used to evolve a diverse set of metabolic enzyme activities.