Methanol-essential growth of Escherichia coli

Abstract

Methanol represents an attractive substrate for biotechnological applications. Utilization of reduced one-carbon compounds for growth is currently limited to methylotrophic organisms, and engineering synthetic methylotrophy remains a major challenge. Here we apply an in silico-guided multiple knockout approach to engineer a methanol-essential Escherichia coli strain, which contains the ribulose monophosphate cycle for methanol assimilation. Methanol conversion to biomass was stoichiometrically coupled to the metabolization of gluconate and the designed strain was subjected to laboratory evolution experiments. Evolved strains incorporate up to 24% methanol into core metabolites under a co-consumption regime and utilize methanol at rates comparable to natural methylotrophs. Genome sequencing reveals mutations in genes coding for glutathione-dependent formaldehyde oxidation (frmA), NAD(H) homeostasis/biosynthesis (nadR), phosphopentomutase (deoB), and gluconate metabolism (gntR). This study demonstrates a successful metabolic re-routing linked to a heterologous pathway to achieve methanol-dependent growth and represents a crucial step in generating a fully synthetic methylotrophic organism.

Engineering Artificial Fusion Proteins for Enhanced Methanol Bioconversion

“…To fully take advantage of the fusion proteins in vivo, Ru5P generation needs to be efficient enough as not to limit the pathway flux. Metabolic engineered of the pentose phosphate pathway holds promise to address this problem . Notably, recently reported methanol‐dependent synthetic methylotrophs co‐utilized methanol and another carbon source as an Ru5P supplier, such as xylose or gluconate .…”

“…Metabolic engineered of the pentose phosphate pathway holds promise to address this problem . Notably, recently reported methanol‐dependent synthetic methylotrophs co‐utilized methanol and another carbon source as an Ru5P supplier, such as xylose or gluconate . Applying the fusion proteins in methanol‐dependent strains may further improve the methanol bioconversion efficiency.…”

“…There are three distinct classes of Mdhs based on their electron acceptor: PQQ‐dependent, Nicotinamide adenine dinucleotide (NAD)‐dependent, or oxygen‐dependent. Since biosynthesis of most valuable chemicals requires electrons in the form of nicotinamide adenine dinucleotide phosphate (NAD(P)H), NAD‐dependent Mdh is now widely used in engineering methanol bioconversion pathway . Formaldehyde generated by methanol oxidation is highly toxic to cells and thus needs to be assimilated immediately.…”

Evolutionary engineering of Corynebacterium glutamicum

“…Interestingly, the authors describe the metY (G1256A) mutation; another mutation in the same gene ( metY (A165T)) was also found in the methanol tolerance study . An elegant approach to realize methanol‐essential growth was recently reported for E. coli . The assimilation of methanol and gluconate was stoichiometrically coupled in a strain that had the ribulose monophosphate cycle.…”

Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway inE. coli

“…Similar results have also been reported in other model organisms such as Corynebacterium glutamicum, Pseudomonas putida and Saccharomyces cerevisiae (as reviewed recently by (Heux S. et al, 2018)). Improvements in methanol assimilation have been achieved using different strategies such as (i) optimizing the cultivation medium (Gonzalez et al, 2018), (ii) lowering the thermodynamic and kinetic constraints associated with NAD-dependent methanol oxidation (Roth et al, 2019; Wu et al, 2016), (iii) improving formaldehyde assimilation (Price et al, 2016; Woolston et al, 2018), (iv) increasing carbon fluxes through the autocatalytic cycle (Bennett et al, 2018), and (v) coupling the activity of the RuMP cycle to the growth of the host microorganism and then using adaptive laboratory evolution (Chen et al, 2018; He et al, 2018; Meyer et al, 2018). However, none of these synthetic strains are able to grow on methanol alone.…”