Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Significance Methane, the major natural-gas component, can be converted industrially to syngas (a mixture of CO2, CO, and H2), which is used to produce methanol, an important industrial precursor for producing commodity and specialty chemicals. Unlike methanol’s chemical conversion, which requires high temperature and pressure, its biological conversion, proceeding through its oxidation to formaldehyde, takes place at ambient conditions but suffers from relative low rates. As metabolite biosynthesis by industrial organisms utilizes NADH as an electron donor, methanol oxidation must be carried out by NAD-dependent methanol dehydrogenases, which have poor thermodynamic characteristics at mesophilic conditions. To solve this problem, we developed an engineering strategy for supramolecular enzyme assembly to dramatically enhance the carbon flux from methanol to the key intermediate fructose-6-phosphate. 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.