Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2
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acssynbio.8b00481.pdf
Description
Published version
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2.11 MB
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Author(s)
Roth, Timothy B.
Woolston, Benjamin Michael
Stephanopoulos, Gregory
Liu, David R.
Date Issued
March 2019
Journal
ACS Synthetic Biology
Publisher
American Chemical Society (ACS)
Citation
Roth, Timothy B. et al., "Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2." ACS Synthetic Biology 8, 4 (April 2019): p. 796–806 doi. 10.1021/acssynbio.8b00481 ©2019 Authors
Version
Final published version
Abstract
Synthetic methylotrophy, the modification of organisms such as E. coli to grow on methanol, is a longstanding goal of metabolic engineering and synthetic biology. The poor kinetic properties of NAD-dependent methanol dehydrogenase, the first enzyme in most methanol assimilation pathways, limit pathway flux and present a formidable challenge to synthetic methylotrophy. To address this bottleneck, we used a formaldehyde biosensor to develop a phage-assisted noncontinuous evolution (PANCE) selection for variants of Bacillus methanolicus methanol dehydrogenase 2 (Bm Mdh2). Using this selection, we evolved Mdh2 variants with up to 3.5-fold improved Vmax. The mutations responsible for enhanced activity map to the predicted active site region homologous to that of type III iron-dependent alcohol dehydrogenases, suggesting a new critical region for future methanol dehydrogenase engineering strategies. Evolved Mdh2 variants enable twice as much 13C-methanol assimilation into central metabolites than previously reported state-of-the-art methanol dehydrogenases. This work provides improved Mdh2 variants and establishes a laboratory evolution approach for metabolic pathways in bacterial cells. ©2019 American Chemical Society.
MIT Department
Broad Institute of MIT and Harvard
Massachusetts Institute of Technology. Department of Chemical Engineering
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DOI of Published Version
https://doi.org/10.1021/acssynbio.8b00481
