(390d) Development of a Formaldehyde Biosensor and its Application to Engineering of Methanol Metabolism in E. coli | AIChE

(390d) Development of a Formaldehyde Biosensor and its Application to Engineering of Methanol Metabolism in E. coli


Roth, T., Harvard University
Liu, D. R., Harvard and MIT
Stephanopoulos, G., Massachusetts Institute of Technology
Formaldehyde is a prevalent environmental toxin, and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is therefore of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or the requirement for expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of E. coli, coupled to either luciferase or GFP. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 3 μM. We then used the sensor to benchmark the in vivo activity of several NAD-dependent methanol dehydrogenase (MDH) variants, the rate-limiting enzyme that catalyzes the first step of methanol assimilation. In support of MDH directed evolution using this assay, we examined the potential for cheaters in a mixed population, and developed a novel strategy to prevent cross-talk by using glutathione as a formaldehyde sink to minimize intercellular formaldehyde diffusion. We then applied phage-assisted continuous evolution (PACE) using the sensor architecture developed here to evolve high-activity variants of MDH. Finally, we show the utility of the reporter in balancing expression of MDH and the formaldehyde assimilation enzymes HPS and PHI in an engineered E. coli strain to minimize formaldehyde build-up while also reducing the burden of heterologous expression, and evaluating strains designed for increased RuMP pathway flux to enable high rates of formaldehyde assimilation. This biosensor offers a quick and simple method for sensitively detecting formaldehyde, and has the potential to be used as the basis for directed evolution of MDH and dynamic formaldehyde control strategies for establishing synthetic methylotrophy.