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 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. methane | methylotophs | supramolcular | scaffold | substrate channeling

Engineering Artificial Fusion Proteins for Enhanced Methanol Bioconversion

“…By selecting Mdh, Hps, and Phi enzymes with high activities and employing the fusing engineering strategy, accelerated F6P formation from methanol was achieved in vitro. When purified enzymes were used at a concentration of 1 μ m , the highest F6P formation rate reached 1.45 μ m min −1 , which was above the previously engineered self‐assembly Mdh‐Hps‐Phi complex (67.7 μ m F6P produced in 14 h) . Although the fusion proteins worked well in vitro, its application in vivo still faced challenges.…”

“…Next, the formation efficiency of F6P was used to evaluate the performance of fusion proteins on methanol bioconversion (Figure A). A cascade reaction was set up for F6P formation from methanol and ribose‐5‐phosphate (R5P) and detection according to a procedure described previously . In the formation phase, in addition to Mdh, Hps and Phi, phosphoriboisomerase (Rpi) was required to convert R5P to the formaldehyde accepter Ru5P.…”

“…Price et al. used the decameric Mdh3 from Bacillus methanolicus MGA3, the artificial Hps‐Phi fusion from Mycobacterium gastri MB19, and the well‐known interaction pair SH3 and its ligand (sSH3lig), to construct Mdh3‐sSH3lig and SH3‐Hps‐Phi that can self‐assemble into a scaffoldless supramolecular enzyme complex . This resulted in significantly increased F6P formation efficiency from methanol in vitro.…”

Biological conversion of methanol by evolved Escherichia coli carrying a linear methanol assimilation pathway

“…On the contrary, the linear formaldehyde assimilation pathway used in this study only requires one enzyme FLS and directly produces C3 intermediate DHA, which could be a great advantage for pathway engineering. Previous and the present studies revealed that constructing synthetic methylotrophs was far more complicated than complementing metabolic pathways where several crucial factors need to be considered, such as how to keep the intracellular formaldehyde concentration below the toxicity threshold (Witthoff et al 2015) and how to balance the reducing equivalent generated by methanol oxidation (Price et al 2016). In this case, combining metabolic engineering and adaptive evolution could be an easy strategy to prepare a desirable mutant that assimilates methanol efficiently.…”

Improved methanol-derived lovastatin production through enhancement of the biosynthetic pathway and intracellular lovastatin efflux in methylotrophic yeast

“…Meanwhile, methanol possesses a higher degree of reduction than traditional sugars do (Whitaker et al 2015). Although efforts have been focused on engineering native or synthetic methylotrophs to convert methanol for the production of biochemical metabolites, the effects are still at the rudimentary level owing to the limited genetic tools for native methylotrophs and the weak methanol consumption abilities of the artificial modified methylotrophs (Whitaker et al 2015;Price et al 2016). Pichia pastoris is a natural methylotrophic yeast that can utilize methanol for growth to high cell densities (Yang and Zhang 2017).…”