(693b) Engineering E. coli to Consume Methanol

Due to the volatility of sugar prices, growing concerns in the “food vs fuel” debate, and the recent availability of natural gas, methanol has emerged as a promising feedstock for bio-based manufacturing of fuels and chemicals. Organisms that natively metabolize methanol are not generally amenable to metabolic engineering, owing to poorly developed genetic tools, and an incomplete understanding of their physiology, biochemistry and regulation. By contrast, E. coli, the workhorse of metabolic engineering, does not natively consume methanol. Here we present progress in engineering E. coli to grow and produce a variety of target molecules toward from methanol. We take a rational approach that combines methodologies of metabolic engineering and of chemical biology to develop new techniques to experimentally probe key limitations in the pathway. Our approach yielded two main targets: poor kinetics of the enzyme catalyzing the first step of the pathway, and "flux leakage" from the RuMP pathway that ablates the pathway driving force by leading to the build-up of formaldehyde. Successful experimental strategies to overcome these obstacles were developed. These results lay the foundation for the development of an E. coli strain that can be “dropped in” to existing bioprocesses to enable the utilization of the alternative substrate, and underscore the value of developing interdisciplinary methodologies for the rapid identification of pathway bottlenecks to speed up the design-build-test cycle in metabolic engineering.