Scaffoldless engineered enzyme assembly for enhanced methanol utilization

103Citations
Citations of this article
140Readers
Mendeley users who have this article in their library.

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 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.

Cite

CITATION STYLE

APA

Price, J. V., Chen, L., Whitaker, W. B., Papoutsakis, E., & Chen, W. (2016). Scaffoldless engineered enzyme assembly for enhanced methanol utilization. Proceedings of the National Academy of Sciences of the United States of America, 113(45), 12691–12696. https://doi.org/10.1073/pnas.1601797113

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free