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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:
Sponsoring Org.:
USDOE
OSTI Identifier:
1329962
Grant/Contract Number:  
AR0000432
Resource Type:
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, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

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. doi:10.1073/pnas.1601797113.
Price, J. Vincent, Chen, Long, Whitaker, W. Brian, Papoutsakis, Eleftherios, and Chen, Wilfred. Mon . "Scaffoldless engineered enzyme assembly for enhanced methanol utilization". United States. doi: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},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 45,
volume = 113,
place = {United States},
year = {2016},
month = {10}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1073/pnas.1601797113

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Cited by: 5 works
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