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Title: Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Abstract

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assemblymore » was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Univ. of Delaware, Newark, DE (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
OSTI Identifier:
1329962
Alternate Identifier(s):
OSTI ID: 1742082
Grant/Contract Number:  
AR0000432
Resource Type:
Journal Article: Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 113 Journal Issue: 45; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 60 APPLIED LIFE SCIENCES; methane; methylotophs; supramolcular; scaffold; substrate channeling

Citation Formats

Price, J. Vincent, Chen, Long, Whitaker, W. Brian, Papoutsakis, Eleftherios, and Chen, Wilfred. Scaffoldless engineered enzyme assembly for enhanced methanol utilization. United States: N. p., 2016. Web. doi:10.1073/pnas.1601797113.
Price, J. Vincent, Chen, Long, Whitaker, W. Brian, Papoutsakis, Eleftherios, & Chen, Wilfred. Scaffoldless engineered enzyme assembly for enhanced methanol utilization. United States. https://doi.org/10.1073/pnas.1601797113
Price, J. Vincent, Chen, Long, Whitaker, W. Brian, Papoutsakis, Eleftherios, and Chen, Wilfred. 2016. "Scaffoldless engineered enzyme assembly for enhanced methanol utilization". United States. https://doi.org/10.1073/pnas.1601797113.
@article{osti_1329962,
title = {Scaffoldless engineered enzyme assembly for enhanced methanol utilization},
author = {Price, J. Vincent and Chen, Long and Whitaker, W. Brian and Papoutsakis, Eleftherios and Chen, Wilfred},
abstractNote = {Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.},
doi = {10.1073/pnas.1601797113},
url = {https://www.osti.gov/biblio/1329962}, journal = {Proceedings of the National Academy of Sciences of the United States of America},
issn = {0027-8424},
number = 45,
volume = 113,
place = {United States},
year = {2016},
month = {10}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at https://doi.org/10.1073/pnas.1601797113

Citation Metrics:
Cited by: 5 works
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Works referenced in this record:

Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization
journal, June 2015


Beyond Oil and Gas: The Methanol Economy
journal, April 2005


Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator frmR
journal, October 2004


Identification of a Magnesium-dependent NAD(P)(H)-binding Domain in the Nicotinoprotein Methanol Dehydrogenase from Bacillus methanolicus
journal, September 2002


Recent advances in the methanol synthesis via methane reforming processes
journal, January 2015


Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism
journal, January 2015


Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate
journal, August 2015


Highly Ordered Protein Nanorings Designed by Accurate Control of Glutathione S-Transferase Self-Assembly
journal, July 2013


Biomolecular scaffolds for enhanced signaling and catalytic efficiency
journal, August 2014


Engineering the spatial organization of metabolic enzymes: mimicking nature's synergy
journal, October 2008


Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria
journal, February 2009


The Molecular Basis of Substrate Channeling
journal, April 1999


Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC
journal, February 2007


Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli
journal, May 2010


Engineering Escherichia coli for methanol conversion
journal, March 2015


Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways
journal, February 2011


Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus
journal, February 2004


Synthetic scaffolds for pathway enhancement
journal, December 2015


DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency
journal, October 2011


Artificial Multienzyme Supramolecular Device: Highly Ordered Self-Assembly of Oligomeric Enzymes In Vitro and In Vivo
journal, October 2014


3-Hexulose phosphate synthase from a new facultative methylotroph, Mycobacterium gastri MB19.
journal, January 1988


Purification of recombinant proteins by fusion with thermally-responsive polypeptides
journal, November 1999


The Physiological Role of the Ribulose Monophosphate Pathway in Bacteria and Archaea
journal, January 2006


Synthetic protein scaffolds provide modular control over metabolic flux
journal, August 2009


Molecular Basis of Formaldehyde Detoxification: CHARACTERIZATION OF TWO S-FORMYLGLUTATHIONE HYDROLASES FROM ESCHERICHIA COLI, FrmB AND YeiG
journal, March 2006


Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli
journal, March 2011


Towards Oil Independence Through Renewable Methanol Chemistry
journal, December 2012


Building carbon–carbon bonds using a biocatalytic methanol condensation cycle
journal, October 2014


Bifunctional enzyme fusion of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase
journal, May 2007


Works referencing / citing this record:

Enzyme assembly guided by SPFH‐induced functional inclusion bodies for enhanced cascade biocatalysis
journal, February 2020


Computation-Guided Design of a Stimulus-Responsive Multienzyme Supramolecular Assembly
journal, September 2017


Engineering Artificial Fusion Proteins for Enhanced Methanol Bioconversion
journal, October 2018


Engineering substrate channeling in biosystems for improved efficiency: Approaches to engineering substrate channeling
journal, June 2018


Improving formaldehyde consumption drives methanol assimilation in engineered E. coli
journal, June 2018


Biological conversion of methanol by evolved Escherichia coli carrying a linear methanol assimilation pathway
journal, September 2017


Evaluation of synthetic formaldehyde and methanol assimilation pathways in Yarrowia lipolytica
journal, December 2019