Title: Development of a formaldehyde biosensor with application to synthetic methylotrophy

Abstract

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 require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of Escherichia coli . Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 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 order to use this biosensor to distinguish individuals in a mixed population of Mdh variants, we developed a strategy to prevent cross‐talk by using glutathione as a formaldehyde sink to minimize intercellular formaldehyde diffusion. Finally, we applied this biosensor to balance 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. This biosensor offers a quick and simple method for sensitively detecting formaldehyde,more » and has the potential to be used as the basis for directed evolution of Mdh and dynamic formaldehyde control strategies for establishing synthetic methylotrophy.« less

Authors:

Woolston, Benjamin M.  ^[1];

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Roth, Timothy ^[2]; Kohale, Ishwar ^[3]; Liu, David R. ^[2]; Stephanopoulos, Gregory ^[1]

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+ Show Author Affiliations

1. Department of Chemical Engineering MIT Cambridge MA 02139 USA
2. Department of Chemistry and Chemical Biology Harvard University Cambridge MA 02138 USA, The Broad Institute of MIT and Harvard Cambridge MA 02141 USA, Howard Hughes Medical Institute Harvard University Cambridge MA 02138 USA
3. Department of Biological Engineering MIT Cambridge 02139 USA

Publication Date:

2017-11-03

Sponsoring Org.:

USDOE

OSTI Identifier:

1407817

Resource Type:

Publisher's Accepted Manuscript

Journal Name:

Biotechnology and Bioengineering

Additional Journal Information:

Journal Name: Biotechnology and Bioengineering Journal Volume: 115 Journal Issue: 1; Journal ID: ISSN 0006-3592

Publisher:

Wiley Blackwell (John Wiley & Sons)

Country of Publication:

United States

Language:

English