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Title: Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2

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.

Authors:
 [1];  [2]; ORCiD logo [2]; ORCiD logo [1]
  1. Broad Inst. of MIT and Harvard, Cambridge, MA (United States); Harvard Univ., Cambridge, MA (United States)
  2. Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)
Publication Date:
Research Org.:
Harvard Univ., Cambridge, MA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1501961
Alternate Identifier(s):
OSTI ID: 1508841
Grant/Contract Number:  
AR0000433
Resource Type:
Published Article
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 8; Journal Issue: 4; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; directed evolution; methanol assimilation; methanol dehydrogenase; phage-assisted continuous evolution; phage-assisted noncontinuous evolution; synthetic methylotrophy

Citation Formats

Roth, Timothy B., Woolston, Benjamin M., Stephanopoulos, Gregory, and Liu, David R.. Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2. United States: N. p., 2019. Web. doi:10.1021/acssynbio.8b00481.
Roth, Timothy B., Woolston, Benjamin M., Stephanopoulos, Gregory, & Liu, David R.. Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2. United States. doi:10.1021/acssynbio.8b00481.
Roth, Timothy B., Woolston, Benjamin M., Stephanopoulos, Gregory, and Liu, David R.. Mon . "Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2". United States. doi:10.1021/acssynbio.8b00481.
@article{osti_1501961,
title = {Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2},
author = {Roth, Timothy B. and Woolston, Benjamin M. and Stephanopoulos, Gregory and Liu, David R.},
abstractNote = {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.},
doi = {10.1021/acssynbio.8b00481},
journal = {ACS Synthetic Biology},
number = 4,
volume = 8,
place = {United States},
year = {2019},
month = {3}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1021/acssynbio.8b00481

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