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 usingEscherichia colilactate 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.
authors: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716.
authors: J Vincent
authors: JV
authors: Price
authors: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716.
authors: Long
authors: L
authors: Chen
authors: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716.|The Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711.
authors: W Brian
authors: WB
authors: Whitaker
authors: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716.|The Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711.
authors: Eleftherios
authors: E
authors: Papoutsakis
authors: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716; wilfred@udel.edu.
authors: Wilfred
authors: W
authors: Chen
keywords: methane; methylotophs; scaffold; substrate channeling; supramolcular