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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. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States, Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, United States, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
  2. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, 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:
Journal Article: Published Article
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Name: ACS Synthetic Biology Journal Volume: 8 Journal Issue: 4; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society
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. https://doi.org/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. https://doi.org/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},
url = {https://www.osti.gov/biblio/1501961}, journal = {ACS Synthetic Biology},
issn = {2161-5063},
number = 4,
volume = 8,
place = {United States},
year = {2019},
month = {3}
}

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

Citation Metrics:
Cited by: 8 works
Citation information provided by
Web of Science

Figures / Tables:

Figure 1 Figure 1: Phage assisted noncontinuous evolution of methanol dehydrogenase. (A) General procedure for PANCE. Starting from a saturated, overnight culture of host cells containing the accessory plasmid (AP) and mutagenesis Plasmid (MP), cultures are (1) diluted; (2) grown to logphase; (3) infected with selection phage (SP) and treated with desiredmore » inputs ( e.g., methanol, arabinose, ATc, glutathione); and (4) grown overnight and isolated the next day as a new population of SP; before (5) infecting a fresh log-phase host cell culture. In the selection circuit, black stars represent permissive and beneficial mutations, and red stars represent detrimental mutations. (B) The selection circuit for evolving methanol dehydrogenase genes in PANCE. Phage-associated genes are shown in blue, genes related to mdh activity are shown in orange, and genes relating to mutagenesis and drift regulation are shown in green. “ara” = arabinose. (C) Phage titers, dilution rates, and selection conditions for a single, 2 mL culture of a Selection Phage encoding B. methanolicus mdh2 evolved for 70 passages in total.« less

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Works referencing / citing this record:

Continuous evolution of base editors with expanded target compatibility and improved activity
journal, July 2019


Continuous evolution of SpCas9 variants compatible with non-G PAMs
journal, February 2020


Growth of E. coli on formate and methanol via the reductive glycine pathway
journal, February 2020


    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.